Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Diversification and domain evolution of molluskan metallothioneins: a mini review

Diversification and domain evolution of molluskan metallothioneins: a mini review Background: Metallothionein (MT) is a multifunctional protein playing important roles in homeostatic regulation and detoxification of metals. Mollusk species have been considered as useful sentinel platforms for MT-based biomarker approaches, and they have been reported to display an extraordinary structural diversity of MT proteins. However, potential diversity of molluskan MTs has not been fully explored and recent updates have suggested the need of revision of evolutionary hypothesis for molluskan MTs. Results: Based on bioinformatic analysis and phylogenetic evidences, novel divergence mechanisms and paths were hypothesized in both gastropod and bivalve MT groups. Our analyses are suggestive of the taxon- or lineage- specific domain multiplication/duplication from the ancestral or prototypic MT. Diversification and selection of molluskan MTs might be driven by the needs for acquiring metal selectiveness, specialized novel function, and improved capacity of metal detoxification under environmentally stressed conditions. Conclusion: The structural diversity and variations of molluskan MTs are significantly larger than previously understood. Undoubtedly, molluskan MTs have undergone dynamic divergent processes in their evolutionary histories, giving rise to the great diversity of domain structures in extant MT isoforms. Novel evolutionary paths for molluskan MTs newly proposed in this review could shed additional light onto the revision of the hypothesis for evolutionary differentiation of MTs in the molluskan lineage. Keywords: Metallothionein, Phylum Mollusca, Structural diversity, Domain evolution Background Benthic mollusks have been proposed as the useful Metallothionein (MT) is a metal-binding protein identified sentinel platforms for biomarker approaches to aquatic in almost living organisms. This housekeeping protein is a and marine environments, in relation with their high multifunctional, non-enzymatic effector playing important bioaccumulation capacity of chemical elements from both roles in various criteria of physiology of organisms under water and sediment (Amiard et al. 2006; Geffard et al. basal and stressed conditions (Carpenè et al. 2007; Mao et 2007; Le et al. 2016). Further, their sedentary nature also al. 2012). Owing to the high inducibility of its expression makes it possible that biomagnification effects of the upon metal exposure, MT has been long given attention as pollution could be effectively visualized without a signifi- one of core suits for biomarkers to address risks associated cant consideration of complex migratory factors in the with metal-related environmental problems (Sarkar et al. interpretation of bioaccumulation data (Gupta and Singh 2006). Besides its fundamental roles in the homeostatic 2011). Accordingly, the exploitation of genetic determi- regulation of essential metals and detoxification of trace nants of molluskan MTs has been a progressively growing metals, MT plays important roles in host protective path- domain in the field of MT researches. To date, a number ways under environmentally or physiologically perturbed of previous literatures have claimed that molluskan conditions (Inoue et al. 2009; Chiaverini and Ley 2010; species should represent a great structural diversity of MT Lynes et al. 2014). proteins (Jenny et al. 2004; Jenny et al. 2006; Leignel and Laulier 2006). Moreover, some mollusk species particu- larly including American oyster Crassostrea virginica have * Correspondence: yoonknam@pknu.ac.kr shown extraordinarily large-sized MT isoforms compris- Department of Marine Bio-Materials and Aquaculture, Pukyong National ing of over 100 amino acid (aa) residues (Jenny et al. 2004; University, Busan 48513, South Korea © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 2 of 18 Jenny et al. 2006; Tanguy and Moraga 2001), which have reported yet from classes such as Aplacophora, Mono- not been usually observable in vertebrate orthologs placophora, Polyplacophora and Scaphopoda. No cloned (Blindauer and Leszczyszyn 2010; Serén et al. 2014). Based MT sequence is available in the class Cephalopoda, bar- on this, it has been widely proposed that the mollusks ring couples of uncharacterized sequences predicted might have undergone evolutionary unique history in the from the octopus (Octopus bimaculoides) genome scaf- divergence of MT proteins (Serén et al. 2014; Isani and fold. Hence, it is evident that genetic determinants of Carpenè 2014; Wang et al. 2014; Jenny et al. 2016). MT have been yet narrowly explored in the phylum Undoubtedly, structural diversifications would confer Mollusca. functional variations on these molluskan MTs, leading Many gastropod and bivalve species possess multiple species-specific adaptation to environmental changes. paralogue MT isoforms. American oyster Crassostrea virgi- Even yet fully elucidated, the prevalence of diversified nica (Ostreidae; Bivalvia) is the top species showing the isoforms are also likely in relation with significant variabil- greatest number of coexisting MT sequences in a single ities and inconsistencies in the responses of molluskan MTs species (n = 17; non-redundant paralogue sequences at aa to metal or other stress exposures (in both laboratories and levels) (Jenny et al. 2004; Jenny et al. 2016); this isoform fields) (Amiard et al. 2006; Le et al. 2016). number is also the highest among all metazoans. Currently, Evolution of molluskan MTs has been proposed to be in the Bivalvia class, 34 species belonging to four subclasses fundamentally based on the duplication events of their (Pteriomorphia, Anomalodesmata, Heteroconchia, and metal-binding domains. Domain duplication(s) from a Palaeoheterodonta) have been recorded to reveal MT common ancestral MT (a singular domain MT) might isoform(s) under GenBank accession codes. However, the have given rise to multi-domain structured MTs (Jenny species distribution for GenBank-deposited MT sequences et al. 2004; Palacios et al. 2011). The prototypic MT has in bivalves is highly skewed, in which only a few popularly been supposed to go through further diversification into studied species have dominated MT sequences. More than various distinguished isoforms in different taxa. This 50% of the total MT sequences have been from only the evolutionary theory has been comprehensively addressed top five species belonging to either Mytilidae (mussels) or with several well-known molluskan models such as Ostreidae (oysters). Meanwhile, in the Gastropoda class oysters, mussels and air-breathing snails (Leignel and (the largest and primitive class in the phylum Mollusca), Laulier 2006; Jenny et al. 2016; Palacios et al. 2011; only less than 20 non-redundant full-length MT sequences Aceto et al. 2011; Leung et al. 2014). However, in contrast have been exploited from twelve different species to rich information on these popularly studied species, belonging to one of seven families. These gastropod divergent processes of MTs in other mollusk species (in species include air-breathing pulmonate snails belong- both gastropods and bivalves) have been quite limitedly ing to the Heterobranchia (species from four families, explored yet. As the research on molluskan MTs pro- Planorbidae, Physidae, Helicidae, and Bradybaenidae), aba- gresses, it is becoming increasingly evident that structural lones (Haliotidae; Vetigastropoda), a limpet (Fissurellidae; variations and deviations of MT domains in this phylum Vetigastropoda), and a periwinkle (Littorinidae; Caenogas- may be significantly larger than previously understood. tropoda). The species information and the accession code Recently, there have been considerable efforts on the iso- for each MT sequence used in this review are referred to lation of novel MTs or MT-like proteins from different in Additional file 1: Table S1. molluskan taxa. Now it is not difficult to find certain A number of previous publications have reportedly in- molluskan MT isoforms of which domain structures are dicated that most vertebrate MTs including mammalian hardly assigned into one of traditionally described categor- and teleostean MTs may exhibit well-conserved primary ies. Hence, with this viewpoint, we aimed to review the structures. They are typically characterized by common structural diversity of molluskan MT domains with an features in polypeptide length (60~65 aa), calculated mo- angle to shed additional light onto the evolutionary path lecular weight (6~7 kDa), proportion of cysteine (Cys) of MT domains in the molluskan lineage. residues (30~35%; arranged typically as Cys-X-Cys, Cys- X-X-Cys and Cys-Cys where the X is any non-Cys aa), Status of MT isoform sequence data in public and theoretical pI values (8.0~8.5) (Wang et al. 2014; database Capasso et al. 2003; Cho et al. 2005; Cho et al. 2008; Currently the number of publically released MT gene Cho et al. 2009) (Additional file 1: Figure S1A). Such a sequences (both genomic and mRNA sequences; https:// structural homology among vertebrate MTs may also attri- www.ncbi.nlm.nih.gov/genbank/) from the mollusk spe- bute to a considerable degree of functional orthology be- cies has exceeded over 100 excluding the partial se- tween vertebrate species. However, it may not apply to the quences or untrimmed ESTs. Almost all molluskan MT mollusk species. Remarkably variable or deviated scores in genes have been obtained from bivalves and gastropods, theprimarystructurehavebeenfrequentlyobservablein whereas no characterized MT sequence has been molluskan MTs (Jenny et al. 2004; Tanguy and Moraga Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 3 of 18 2001; Baršytė et al. 1999; Tanguy et al. 2001) (Additional file the numbers of taxa available for MTs from Gastropoda 1: Figure S1B). First, molluskan MTs reveal often significant class were currently limited, theoretical pI values esti- variations in polypeptide lengths among paralogue and mated from the category group Vetigastropoda (pI orthologue isoforms. Median and mean values for the num- 5.16~6.00) were relatively lower than those from Hetero- ber of aa residues estimated from all the mollusk MT poly- branchia (pI 6.10~8.28). Possibly, MT proteins with peptide sequences addressed in this study (n = 109) are 73.0 largely different pI values might exhibit differential (7.2 kDa) and 80.2 (7.8 kDa) aa, respectively. Overall, these capability to bind or interact with charged molecules in values are higher than that estimated with representative the host protective process. In addition, contents of vertebrate MT orthologs. More noticeably in molluskan some positively charged aa (e.g., Lys) may influence group, considerable numbers of MTs represent extraordin- importantly the metal-binding function of MT protein. arily short or long lengths apparently deviated from the These aa residues (particularly at positions in vicinity to mean (and median) value above. Within the Bivalvia class, Cys) are thought to be involved in the stabilization of the length of MT polypeptides ranges from 43 aa (4.2 kDa) the interaction between MTs and metal ions through the to 204 aa (20.3 kDa); noticeably both are recorded as para- electrostatic interactions to bridge the protonated basic logs from a single species C. virginica (Ostreidae) (Gen- residues and the negatively charged metal-thiolate com- Bank accession numbers, AY331700/AY331701 and plex (Pedersen et al. 1994). Thereby, such a wide range AY331706, respectively). Other oyster species also dis- of pI values may be potentially suggestive of, at least in play the significant variations in the sizes of MT iso- part, dynamic diversification and subfunctionalization forms. However, the mussel species belonging to the among molluskan MT isoforms, although the biological order Mytiloida represents relatively a uniform feature and/or functional implications of such a wide range of in the range of the MT protein lengths (66~75 aa). Be- pI values should be explored in the future. sidesthe twomainbivalve taxa,itisworthytonoteun- Third, molluskan taxa are found to have a tendency of usually long MT isoforms reported from Argopecten relatively lower Cys content (mean ± s.d. = 28.1 ± 1.8%) in irradians (Pectinidae; EF093795, and EU734181) and their MT proteins than are mammalian (32.4 ± 1.3%) or Pisidium coreanum (Sphaeriidae; GQ268325). teleostean (33.3 ± 0.3%) groups. Also, the intra- and inter- On the other hand, mean and median values for MT species variations in Cys contents of MTs are larger in polypeptide lengths available for gastropod MTs were 67.5 mollusks (especially in bivalves; ranging 21.2 ~ 31.9%) than and 65.0 aa, respectively. Currently, the shortest gastropod in mammals (26.2~34.4%; 31.1~34.4% if MT-IIIs excluded) MT sequence is found to be Physa acuta (Physidae; and fish (32.8~35.0%) (Additional file 1: Figure S1). Metal GU259686) MT consisting of 59 aa, whereas the longest binding property and capacity of molluskan MTs have been one is the MT isoform with 100 aa reported in a periwinkle studied in only a few species (Palacios et al. 2011). How- species Littorina littorea (Littorinidae; AY034179). Except ever, Cys residues are known to be essential for the affinity these shortest and longest sequences, the polypeptide to bind metal ions where the number of metal ions bound lengths of other gastropod MTs are found to fall in the by the MT may be fundamentally determined by the num- range from 64 to 70 aa. Similarly with bivalves, multiple ber of Cys residues (Amiard et al. 2006; Jenny et al. 2016; isoform MTs (n = 2~4) have been reported in pulmonate Vergani et al. 2005). Hence, it is able to suggest that snails belonging to Helicidae or Planorbidae, while marine molluskan MTs may display relatively larger variations in gastropods including abalones have shown only a single the metal binding capacity among isoforms than vertebrate MT isoform; yet still unclear if they have additional MT orthologue groups. isoform gene(s) or not (Additional file 1: Table S1; Taken together, all the three parameters above mentioned Additional file 1: Figure S1). are undoubtedly indicative of large structural variations and Second, molluskan group shows a wider range of divergence of MT proteins among molluskan species. Based distribution for theoretical isoelectric point (pI) values on this overview, taxa (or lineage)-specific patterns for across MT isoforms as compared to mammalian and structural diversification of MTs are described more details teleostean MT groups (Additional file 1: Figure S1). In in following sections, with a particular attention on the mammals, MT-IIIs are only the isoform family (but arrangement of Cys motifs. excluding murine MT-IIIs) to show a significantly acidic pI values (4.79~4.82), while most other isoforms belong- Structural diversity of MT isoforms in major ing to MT-I, −II or -IV class typically represent pI values molluskan taxa higher than at least 7.5. Most teleostean MTs show pI Nomenclature and classification of MTs used in this values ranging from 7.8 to 8.4. On the other hand, bi- paper were referred to GenBank (NCBI) based on the valve MTs display pI values ranging 4.31 to 8.56 depend- definition of each sequence. If the MT sequence was ing on isoforms. In the Gastropoda group, the pI values published in scientific paper(s), its classification was of the MT proteins range from 5.16 to 8.28. Although checked again. Within a given species, the redundant Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 4 of 18 MT sequences at aa level were not included in analyses MT is proven to show a conserved pattern for Cys (Additional file 1: Table S1). Classification of putative arrangements in N- and C-terminal parts (i.e., nine Cys domain structure in each MT sequence was based on respectively in putative N- and C-terminal domain re- the number and arrangement pattern of Cys motifs as gions). Considering the Cys motif patterns, the N- and C- described previously (Jenny et al. 2016), since there have terminal parts of the L. littorea MT could be designated been no empirical studies on three-dimensional struc- β -and β -domains, respectively. Further, a closer examin- 2 1 tures of molluskan MT proteins. In general, domain ation on the intervening region (comprised by 32 aa) be- structure in MT is designated α and β (Braun et al. tween the β - (N-terminal) and β - (C-terminal) domains 2 1 1986; Binz and Kagi 1999). Usually, the α-domain con- has indicated that the 32-aa internal segment has been po- tains eleven to twelve Cys residues, binds four divalent tentially a duplicated copy of the N-terminal β -domain. It metal ions and confers structural stability on the MT conserves clearly 9 Cys residues and shows a considerably polypeptides. On the other hand, the β-domain, binds high sequence identity (75%) to the N-terminal (β -do- three divalent metal cations through the nine Cys resi- main) domain. Based on our peer review, the structure of dues and participates in metal exchange reactions via L. littorea MT could be considered as a novel shape of glutathione-shuttling with metal-requiring apoproteins gastropod MT characterized by β β β -domain form. 2 2 1 (Jiang et al. 1998; Jiang et al. 2000). Hence, this newly proposed structure suggests that domain duplication event might have served as a driving Gastropoda MTs force to figure the large MT in certain gastropod taxa. In a total, nineteen non-redundant MTs (from twelve Another example for the domain duplication in gastropod species belonging to seven families) including a putative MT is the 124-aa B. glabrata MT (XP_013080485). In that MT predicted in the unplaced genomic scaffold se- MT polypeptide sequence, three putative domain regions quence (Biomphalaria glabrata; Planorbidae) were sharing a considerable sequence similarity in one another analyzed with sequence alignments. Among the nineteen could be identified, and each of the three putative domains sequences, 17 sequences with 59 to 70 aa residues are maybedesignated β -structure based on their Cys arrange- found to be fairly aligned in the multiple sequence align- ment patterns (Additional file 1: Figure S2). As numbered ment trials. In spite of substantial differences in non-Cys from theN-terminal, thefirst β -domain and the second β - 2 2 residues among taxa, they share a conserved pattern of domain share the conserved Cys motif frame (except one Cys motifs. Eighteen Cys in these gastropoda MTs are additional Cys in the second β-domain). They also show the arranged as [Cys-X-X-X-Cys→ (Cys-X-Cys) → Cys]→ [( high sequence homology (76.5%) each other. The putative Cys-X-Cys) → Cys→ (Cys-X-Cys) ] (Additional file 1: third β -domain linked to the second β -domain is found to 2 2 2 2 Figure S2). It indicates that gastropoda MTs represent display 66.7% of sequence homology to the first and second the protein structure comprising of two distinguished β- β -domains. Within this context, the B. glabrata MT could domain forms (i.e., β β -form) designated by the recent be proposed to possess at least three duplicated β -domains 2 1 2 suggestion to propose the presence of two hypothetical tandemly arrayed in a tail-to-head fashion. On the other ancestral β-domains (Jenny et al. 2016). The β -domain hand, the remaining C-terminal part (23-aa) following the structure at the N-terminal of gastropod MT protein is β β β -domain region is found to contain five Cys (three 2 2 2 similarly observed in C. virginica MT-IIIs as well as in singlet Cys and a Cys-Cys doublet motif). Unlike the L. the β-domains of vertebrate (mammals and fish) MTs. littorea MT above, the C-terminal region of this B. glabrata On the other hand, the C-terminal β -domain of the MT display no typical shape to be categorized into one of gastropod MT is commonly found in various molluskan known domain structures (Additional file 1: Figure S2). MTs. According to the β β -structural scheme, the Currently, the origin of this C-terminal region has been 2 1 shortest P. acuta MT (comprised by 59 aa) is thought to unknown. Further validation of scaffold genomic sequences have lost two Cys in its β -domain. In that alignment, along with mining of similarly organized MTs from MTs from Vetigastropoda species possess more aa resi- other Heterobranchia genomes would be needed to dues (5 or 8 aa) in the intervening region between the get a deeper insight into the mechanism responsible β - and β -domains than those from Heterobranchia for the formation of the array of three β -domain 2 1 2 species (2 aa). region in this gastropod MT. This pulmonate snail Besides the common β β -shape, gastropod group species has already been knowntoexpress functionally 2 1 represents two significantly lengthy MT polypeptides. diversified (i.e., different metal selectiveness) MT iso- One is 100-aa MT from Littorina littorea (Caenogastro- forms [(i.e., Cd-MT (GQ205374), Cu-MT (GQ205373) poda; AY034179) (English and Storey 2003) and the and intermediate Cd/Cu-MT (GQ205375)] (Berger et other is 124-aa MT (XP_013080485; deduced from the al. 1997). Hence, it would also be valuable to examine unplaced genomic scaffold) from B. glabrata (Additional the expression patterns of this newly identified MT file 1: Figure S2). Based on manual alignment, L. littorea regarding its potential differentiation in physiological Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 5 of 18 function and/or metal responsiveness in comparison with IID/-IIE/-IIF and MT-IIG/-IIH display tandem arrays previously characterized paralogs from this species. comprised of three and four α-domain copies, respect- ively (Jenny et al. 2004). Between and among duplicated Bivalve-ostreidae MTs (repetitive) copies, a few aa substitutions have been Structural diversity of MT families in Ostreidae has been found. Collectively, the divergent process of C. virginica comprehensively described with C. virginica model MT-II family has occurred through the loss of β -do- (Jenny et al. 2004; Jenny et al. 2006). Currently, seven- main (e.g., MT-IIA/-IIB; α-domain-structure) followed teen C. virginica MT sequences available in GenBank by further divergence into various subisoforms having could be classified into one of four MT isoform families differential numbers of duplicated α-domains (e.g., MT- (MT-I, −II, −III, and IV). The MT families consist of 2 IIC to MT-IIH; α -domain structure) (Jenny et (n = 2 ~ 4) (MT-IA and MT-IB), 8 (MT-IIA, MT-IIB, MT-IIC, MT- al. 2016). IID, MT-IIE, MT-IIF, MT-IIG, and MT-IIH), 3 (MT- However, this divergence pattern has not been always IIIA, MT-IIIB, and MT-IIIC), and 3 (MT-IVA, MT-IVB, a common finding in the Ostreidae lineage (Additional and MT-IVC) subisoforms, respectively. In addition to file 1: Figure S3A). Rather than the loss of β-domain, C. these 16 sequences, one MT sequence named MTA gigas MT-II family has represented the tandem duplica- (GenBank accession no. AF506977) is independently tion of 32-aa β -domain with retaining the α-domain, recorded in this oyster species (see Additional file 1: giving rise to the αβ β -domain structure (Tanguy and 1 1 Table S1). Of the 16 isoforms, the prototypical MT Moraga 2001). Further, unlike in C. virginica, there has structure (corresponding to MT-I form; MT-IA and been no variation in repeat numbers among C. gigas MT-IB) is 75-aa MT possessing 21 Cys residues (28% of MT-II subisoforms. MT (AF349907) from another Cys content). The MTA isoform can also be classified to Crassostrea species, Portuguese oyster (C. angulata)has the same prototype (i.e., MT-I) with an addition of Asp exhibited the duplication of only a short, partial β -do- in the region between 16th and 17th Cys residues. They main fragment (7-aa region), giving rise to a non- share a high sequence homology one another in a proto- canonical αβ β -structure with truncated C-terminus. 1 1P typic αβ -domain-structure (Jenny et al. 2004). This αβ - Because there has been no other publically released MT 1 1 structure is well conserved also in other oyster species, paralog from C. angulata, it has been yet unclear if this including C. gigas (Pacific oyster; AJ242657), C. ariaken- oyster species may possess any paralog copies represent- sis (the Suminoe or Asian oyster; DQ342281) and C. ing the complete αβ β -domain structure or not. Beyond 1 1 rivularis (the Jinjiang oyster; JN225502). The same the Crassostrea genus, our survey against GenBank has domain structure is also relevant with isoforms from identified that Alectryonella plicatula MT (KP875559; non-Crassostrea species Ostrea edulis (Tanguy et al. 107-aa) should also display the typical αβ β -domain 1 1 2003). However, relative large substitutions of non-Cys structure. Moreover, the A. plicatula MT shows very aa (also the replacement of three Cys with other aa in high sequence homology to its Crassostrea orthologs the O. edulis MTb isoform) have been found in the O. (MT-IIs), indicating the common origin of this multi-do- edulis MTs compared to MT-I orthologs from Crassos- main structure. Currently, the known αβ β -structured 1 1 trea species (Additional file 1: Figure S3A). MT subisoforms in Ostreidae (except for the C. angulata C. virginica MT-II family includes eight subisoforms MT with a truncated C-terminus) share the same N- (MT-IIA to MT-IIH), and this MT-II family has been terminal (Met-Ser-Asp-Pro) and C-terminal (Cys-Lys-Lys) known to be classified into two subgroups. First, the motif residues (Additional file 1: Figure S3A). MT-IIA/-IIB group possesses a sole α-domain of which On the other hand, the C. virginica MT-III group consists sequence is highly conserved with that of the prototypic of three homogenous subisoforms MT-IIIA/-IIIB/-IIIC. αβ -MT-I. The loss of functional β -domain has been They share each other high sequence identity including 18 1 1 proposed to occur due to the point mutation in the Cys residues, and the distribution pattern of the 18 Cys linker region (i.e., non-sense mutation resulting in a stop have been proposed as the array of two β-domains as codon). Consequently, MTs belonging to this group re- [(Cys-X-Cys) → Cys] × 2 (i.e., β β -MT) (Additional 4 2 2 veal noticeably short polypeptide length (43 aa) (Jenny file 1: Figure S3B). The arrangement pattern of nine et al. 2004). Second, on the contrary, the remaining six Cys is obviously similar with the β -domain of the C. virginica MT-II isoforms are lengthy polypeptides gastropod β β -MTs (Jenny et al. 2006; Jenny et al. 1 2 (94-aa MT-IIC, 149-aa MT-IID/-IIE, 145-aa MT-IIF, 2016). The same β β -domain structure has also been 2 2 204-aa MT-IIG, and 200-aa MT-IIH). They have tan- found in C. gigas MT-III (JF781299); however unlikely demly duplicated copies of α-domain where the numbers in C. virginica, multiple MT-III subisoforms have not of repetitive duplications are variable among isoforms. been characterized in C. gigas (Cong et al. 2012). The C. In MT-IIC, only one duplication event is predicted (i.e., gigas MT-III shows a series of substitutions of non-Cys aa two tandem duplicate copies), while subisoforms MT- from the C. virginica MT-III isoforms. The overall Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 6 of 18 sequence identity of MT-IIIs between the two Crassostrea diverged from the prototypic MT-I through the non- species is about 70% (Additional file 1: Figure S3B). Mean- Cys aa changes. This isoform is likely to show, at least while, Crassostrea MT-III isoforms reveal considerably in part, certain functional orthology to the C. virginica low pI values (4.38~4.77 for C. virginica and 4.31 for C. MT-III, although they have different domain structures. gigas) as compared to other MT isoforms showing pI Finally, the MT-IV isoforms (MT-IVA, MT-IVB and values > 7.5. With the viewpoint of the low pI value, C. vir- MT-IVC; 83 aa in length) from C. virginica have been ginica MT-III may resemble mammalian MT-III family. proposed as variant forms of αβ -MT. This isoform Most known mammalian MT-III isoforms except murine group has been supposed to have experienced a series MT-IIIs reveal acidic pI ranges (4.79~4.82) with acidic 6- of aa substitutions including Cys residues, giving rise amino-acid insert in the C-terminal region. Synthesis of to 25 Cys with the formation of a Cys-Cys doublet (in the mammalian MT-III is not inducible by heavy metals and α-domain) and three Cys-Cys-Cys triplet motifs (in the localized predominantly in the central nervous system β -domain). The C-terminal residue of the C. virginica (Faller 2010). Unique roles of mammalian MT-III differing MT-IV isoform (glutamine-alanine-threonine) is also not- from other MT isoforms have been characterized as the ably different from those of other paralog isoforms. neuronal growth-inhibitory factor to inhibit neuronal out- Thereby, the proposed designation of domain structure growth (Wang et al. 2006). Specific roles of bivalve MT-III for MT-IVs could be α′β ′-form. In addition to C. virgi- differing other MT family groups have not been yet exten- nica,two Crassostrea species (C. gigas and C. ariakensis) sively addressed. However, the expression study with C. possess MT-IVs of which primary domain structures are virginica MT-III has indicated that the C. virginica MT-III fairly conserved with that of C. virginica MT-IV. However, showed quite a low basal level of expression in adult tis- C. gigas MT-IV (87 aa; AM265551) and C. ariakensis MT- sues (i.e., only actively expressed in early larvae). Further, IV (86 aa; JF919323) represents one additional Cys residue C. virginica MT-III represented only a moderate respon- at C-terminal region (Additional file 1: Figure S3B). siveness to heavy metal exposures in both larvae and Besides the above C. gigas MT-IV clone (AM265551), gen- adults (Jenny et al. 2006). However, on the contrary, the ome sequencing of C. gigas (Zhang et al. 2012) represents C. gigas MT-III has been reported to be significantly in- two unplaced genomic scaffolds [scaffold852 (JH816574) duced by zinc (as a main regulator for zinc homeostasis), and scaffold1297 (JH818394)] each containing the puta- and it may be a participating member for cadmium de- tive gene encoding MT (EKC32371 and EKC28510, re- toxification in the adult tissues (Cong et al. 2012). spectively, in the two scaffolds). The deduced sequences Thereby, it suggests the functional differentiation/diver- of these MTs are 128 and 137 aa in length, respectively. gence of MT-III isoforms during speciation events in the Both MTs are predicted to possess unusual N-terminal Crassostrea lineage. Within the context of this hypothesis, regions (41-aa for EKC32371 and 50-aa for EKC28510). it is worthy to have an attention on another C. gigas MT However, immediately following the N-terminal region, isoform named mt3 under the accession number the two putative MTs represent the 87-aa-structure that is AJ295157. This C. gigas mt3 isoform is not a true MT-III apparently homologous to the C. gigas MT-IV isoform family member with the β β -domain structure. Rather (AM265551). When the unusual N-terminal regions are 2 2 than, mt3 should be considered as a MT-I member be- excluded from these two MT-IV-like sequences, the cause it represents the αβ -domain structure (see three MT sequences (one characterized MT-IV and Additional file 1: Figure S3A). However, due to the two in-scaffold sequences) reveal only one aa substitu- significant non-Cys aa substitutions, C. gigas mt3 tion, although the scaffold sequences should be further displays only 67% sequence identity to its paralogue validated in future. MT-I isoform. Moreover, the substitutions include the change of three Lys residues to uncharged aa as well Bivalve-mytilidae MTs as the replacement of uncharged aa with negatively In Mytilidae, 33 full-length, non-redundant MT aa se- charged aa. Such aa substitutions might give rise to quences were retrieved from five taxa (12 sequences lower pI value (5.98) of mt3 than those of its paralo- form Mytilus edulis, 6 from M. galloprovincialis, 2 from gue MT-I members. A previous study has reported Mytilus sp., 11 from Perna viridis and 2 from Bathymo- that mt3 should have the extremely low basal expres- diolus azoricus). Mytilid MTs are a structurally more sion level with only moderate or minute responsive- homogeneous group as compared to Ostrea MT group. ness to metal exposure (Marie et al. 2006). The mt3 They represent 66-aa to 75-aa polypeptides containing has been suggested to have probably no significant 19 to 23 Cys residues, and display typically the αβ -do- physiological functions under metal exposure and to main structure (Additional file 1: Figure S4). be expressed only in particular developmental stages In the mytilid mussels, two types of MT isoforms, (Marie et al. 2006). Hence, taken together, it could be MT10 and MT20 have been described previously (Aceto hypothesized that C. gigas mt3 might have been et al. 2011; Leung et al. 2014). These two MT types Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 7 of 18 differ in mass and Cys arrangement. The monomeric (alignment position; Ser in P. viridis MTs and MT20s vs. form MT10 (~10 kDa) represents generally 73-aa poly- Gly in other mytilid MT10s), 39th (Gly vs. Lys), and 73rd peptides including 21 Cys residues mainly arranged as (Ser vs. Pro). Finally, the third group is comprised of six nine Cys-X-Cys motifs. On the other hand, the dimeric MT sequences from three Mytilus species. They are 72-aa form MT20 (~20 kDa) is typically 72-aa polypeptides polypeptide containing 23 Cys residues (i.e., MT20 type). containing 23 Cys residues. Unlike MT10, the MT20 iso- Only one exception is the substitution of Cys to Arg in the forms show a Cys-Cys doublet in the α-domain. MT10 M. edulis MT-20 clone. From the alignment, two MT20- and MT20 have been reported to be functionally differ- specific residues could be found at positions 24th (Lys entiated. MT10 has been found to be more abundant in MT20s vs. Glu in all MT10s) and 68th (Asn vs. Thr) than MT20, and hence it could be considered as a main (Additional file 1: Figure S4). In particular, the change player for the regulation of homeostasis under basal from negatively charged aa (e.g., Glu) to Lys at position conditions (Leung et al. 2014; Lemoine et al. 2000). 24th is likely related with the MT20-specific formation Under metal-exposed conditions, MT10 and MT20 have of Cys-Cys doublet motif, since the positively charged aa been known to display differential responses and binding (e.g., Lys) at vicinity to Cys motif is considered to play ability to essential and non-essential metals. MT10 could important roles in the stabilization of the metal binding be actively inducible by various metals while MT20 be reaction in most MT proteins (Pedersen et al. 1994). more preferentially associated with non-essential metals such as Cd and/or Hg (Raspor et al. 2004; Dondero et al. Bivalve-other taxa 2005; Vergani et al. 2007). Besides the two main bivalve taxa (Ostreidae and Mytilidae), From multiple sequence alignment, mytilid MTs could 27 non-redundant, full-length MT isoforms have been be categorized into three main groups (Additional file 1: exploited from 21 bivalve species belonging to one of six Figure S4). The first group consists of three MT10B orders Pterioida, Arcoida and Pectinoida (subclass sequences (two from M. edulis and one from M. gallopro- Pteriomorphia), Pholadomyoida (subclass Anomalodes- vincialis; AJ577126, AJ577127 and DQ848984, respect- mata), Veneroida (subclass Heteroconchia), and Union- ively). They represent 66-aa polypeptides containing 19 oida (belonging to Palaeoheterodonta). From the multiple Cys residues with the deletion of one Cys-X-Cys motif in sequence alignment, most of them are found to represent the α-domain. All the three MT sequences are originated αβ -domain-structure with a conserved 21-Cys-frame from intronless MT genes (Leignel et al. 2005; Yang et al. (Additional file 1: Figure S5A). However, several variant 2014). The second group contains twenty-four MT10 isoforms are also found to show modification(s) of Cys isoform sequences. They reveal 72 to 75 aa in lengths and motifs in one or two positions, giving rise to the gener- possess 21 Cys residues in conserved positions (Lemoine et ation of Cys-Cys doublet, substitution, insertion or dele- al. 2000; Khoo and Patel 1999). There are only two tion. Several variant isoforms are found to retain the total exceptions; one is the replacement of a Cys with Arg in P. number of Cys residues (i.e., 21 Cys) while others show viridis MT-IA (JN596471) and the other is an insertion of changes of the total number of Cys residues. MT isoforms an additional Cys in P. viridis MT (AF036904). Within the from Pinctada maxima (pearl oyster; FJ389580) (Tang et second group, theMTs are likelytobesub-grouped al. 2009) and Laternula elliptica (Atlantic clam; according to known taxonomic appraisal at genus level DQ832722/DQ832723) display the insertion of an add- (i.e., Bathymodiolus, Mytilus and Perna). All the P. viridis itional Cys in the third Cys-X-Cys motif, resulting in the MT-II isoforms (named MT-IIA, −IIB, −IIC and -IID) are Cys-Cys-Cys motif at that position. On the contrary, an found to possess 72 aa, as similarly with the mytilid MT20 MT isoform from Hyriopsis schlegelii (freshwater pearl isoforms. Nevertheless, based on their Cys motifs frame, mussel; MT2; KJ019821) has lost one Cys residue at the these P. viridis MT-IIs (JN596477 to JN596480) have been second Cys-X-Cys motif along with considerable alter- proposed as MT10 members. Previous phylogenetic ana- ations in non-Cys aa residues. Unlike its paralog (H. schle- lysis has claimed an early divergence of P. viridis MTs from gelii MT1; KJ019820), the H. schlegelii MT2 has been the main mytilid MT10/MT20 groups (Leung et al. 2014). proposed as a genetically separated isoform (Wang et al. When aligned with other mytilid orthologs, P. viridis 2016). A recent study has indicated that these two H. MT10-I and/or MT10-II represent several residues distinct schlegelii MT paralogs might have been subfunctionalized from other mytilid MT isoforms. They include positions as evidenced by clearly distinct tissue expression pat- 11th (alignment position; Gln/Lys in P. viridis MTs vs. Asn terns (i.e., constitutive expression of H. schlegelii MT1 in all other mytilid MT10 and MT20 isoforms), 62nd (Gln vs. gonad-specific or predominant expression of H. vs. Gly/Asp) and 74th (Ser vs. Gly). Further, P. viridis schlegelii MT2) (Wang et al. 2016). Another example MT10 isoforms (both MT10-I and MT10-II) are found to for the large difference between paralog MT isoforms is share the same aa in several positions with mytilid MT20 P. martensi MT isoforms. Even though the P. martensi isoforms. These could be exemplified by positions 34th MT1 (KC197172.1) represents the common αβ -shape, 1 Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 8 of 18 its paralog MT2 (KC832833.1) exhibits an apparently domains indicate that they share little sequence non-canonical pattern of Cys arrangement (20 Cys). similarity one another except conserved Cys residues On the other hand, two MT isoforms from Veneroida (Additional file 1: Figure S5B). Within this scheme, the A. are found to have noticeably less number of Cys residues irradians MT2 could be treated as a paralog having the than others: one is the duck clam Mactra veneriformis less number of β -domains (i.e., putatively designated (Mactridae) MT with 18 Cys (59-aa; Cys content = β β β -like structure). The A. irradians MT2 also reveals 2 2 1 30.5%; FJ611963) (Fang et al. 2010; Fang et al. 2013) and some non-canonical attributes including the lack of one the other is Venus clam Cyclina sinensis (Veneridae) Cys residue and the formation of Cys-Cys doublet in the MT with 16 Cys (74-aa; Cys content = 21.6%; N-terminal β -like domain. These two A. irradians MT HM246244) (Lü et al. 2012). The M. veneriformis MT isoforms are found to share only a little sequence hom- seems to have lost an internal fragment near N-terminal ology, indicating that they may be quite distantly related region (possibly corresponding to the α-domain) con- paralogs (Wang et al. 2009). taining three Cys residues (likely a Cys-X-Cys motif and On the other hand, the 105-aa P. coreanum (Sphaeriidae) one conserved Cys). The C. sinensis MT lacks a Cys-X- MT (GQ268325; 31 Cys) (Baek et al. 2009) is found to Cys motif in α-domain and additionally three Cys resi- show the domain multiplication to resemble the αβ β - 1 1 dues (Cys-X-Cys motif and one Cys residue) probably in structure, of which Cys arrangement is similar with that of the β-domain. C. gigas MT-II. However, unlike C. gigas MT-II to show Importantly, three MT isoforms display large polypep- thetandemarrayoftwo homogenous β -domains, P. tide sizes comprising of more than two putative domains coreanum MT contains the two heterogeneous β -domains (Additional file 1: Figure S5B). Of the three MTs, two with no apparent sequence homology between the two MT sequences (MT1 and MT2) are from the bay scallop domains. The P. coreanum MT lacks a common triplet Argopecten irradians (Pectinidae) and remaining one linker sequence (Lys-Val-Lys/Val) between α- and first β - isoform is from the fingernail clam Pisidium coreanum domain. The array of two heterogeneous β -domains (Sphaeriidae). These sequences have been reported linked to N-terminal α-domain observed in P. coreanum earlier but their domain structures have never been ad- MT could be the novel structure of bivalve MT proteins dressed clearly. Even though A. irradians MT1 (145-aa; (Additional file 1: Figure S5B). 40 Cys; EF093795; (Liu et al. 2006)) and MT2 (110-aa; 28 Cys; EU734181; (Wang et al. 2009)) exhibit essential features of mollusk MTs (i.e., the presence of character- Domain evolution in molluskan MTs istic Cys-X-Cys motifs), their overall structures are more Current theory for domain evolution of molluskan MTs or less complicated and difficult to be simply categorized Currently, the proposed hypothesis for the evolution of into one of currently known shapes of bivalve MTs. molluskan MT has been based on the domain duplica- However, in a broad sense, these isoforms may bear a tion event(s) from an ancestral single domain-structured resemblance to the MT isoforms with multi-β-domain- MT, in which the β-domain has been considered as the structure. For both A. irradians MT isoforms, the C- ancestral shape (Cols et al. 1999). After an early duplica- terminal region may be considered the β -domain posses- tion event of the ancestral β-domain, the resultant ββ- sing nine Cys as (Cys-X-Cys) → Cys→ (Cys-X-Cys) .In domain MT has undergone divergent processes, given 2 2 addition to the C-terminal β -domain, A. irradians MT1 rise to the αβ-structure in certain taxa (Jenny et al. 2016; potentially exhibits three tandemly arrayed β -like do- Braun et al. 1986; Cols et al. 1999). Difference in the mains. However, each β -like domain in the A. irradians metal-binding properties between the α-domain and the MT1 displays some non-canonical arrangement of Cys. β-domain makes the two domains to represent differen- First, two Cys of the first Cys-X-Cys motif in each domain tiated roles in the cellular physiology. Generally, α- is separated further by intervening 2~4 aa residues (i.e., domain plays a more prevalent role in Zn homeostasis similar with the pattern found in N-terminal regions of and detoxifying sequestration of toxic metals (e.g., Cd) gastropod β -domains). Second, Cys-Cys doublet motifs whereas the β-domain is primarily responsible for the rather than a canonical Cys-X-Cys motif are present in the homeostatic regulation of essential metals (e.g., Cu) first and third β -like domains. Third, an additional Cys- (Jenny et al. 2004; Cols et al. 1999; Nielson and Winge X-Cys motif exists in the flanking regions between the first 1984; Xiong et al. 1998). Consequently, the multi- and second β -like domains as well as between the second domain MT in specific taxa acquiring both α- and β- and third β -like domains. Nevertheless, the overall shape domains was able to perform the dual functions; the of A. irradians MT1 may be designated β β β β -like detoxification of toxic metals by the α-domain and the 2 2 2 1 structure, although this novel proposal should be further homeostasis of physiologically relevant metals by the β- challenged with empirical structural analyses. Sequence domain (Jenny et al. 2016; Cols et al. 1999; Nielson and comparisons among/between these successive β -like Winge 1984; Nielson and Winge 1983). 2 Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 9 of 18 The latest phylogenetic work has proposed that two domains. Based on this assumption, the unusual C- distinct ancestral β-domains (designated β and β terminal part of B. glabrata MT might be a reminiscent, 1 2 domains) might have existed and given rise to the struc- partial segment (designated β here) originated from 1P tural diversity of all molluskan MTs (Jenny et al. 2016). the early β -domain. Hence, the tandem duplications of In that literature, they have hypothesized separate paths β -domain accompanied with partial loss of the C- of the evolution of the two ancestral MTs in the major terminal β -domain may be a plausible mechanism to taxa within the mollusk phylum. The two β-domains ap- produce the current β β β β structure in this pulmon- 2 2 2 1P pear to have diverged into two structurally different MT ate species. We performed additional analyses on the isoform types (i.e., αβ -MT and the β β -MT) in bivalves duplicated β -domains of this large MT (first and second 1 2 2 2 whereas in gastropods, the two ancestral β-domains β - domains used for analysis) in order to hypothesize a form a single structural β β -MT isoform. With C. virgi- plausible reason responsible for the happening of this 2 1 nica model, the structural diversity of bivalve MT iso- evolutionary episode. For this, β -domains of previously forms has been highlighted to demonstrate evolutionary known MTs from pulmonate species (i.e., Cd-MT, Cu- paths from not only αβ -domain but also β β -domain MT and intermediate Cd/Cu-MT) were included in 1 2 2 (Jenny et al. 2016). On the contrary, in the gastropoda analyses together with the β -domains of this large B. lineage, the β β -domain structure has been proposed as glabrata MT. From the sequence alignment, the dupli- 2 1 a typical appearance common to most extant gastropod cated β -domains of the B. glabrata β β β β -MT 2 2 2 2 1P MTs. Instead of a series of domain duplications seen in revealed a more sequence similarity to Cd-MT than to bivalve MTs, gastropod MTs appears to have diverged to Cu-MT and Cd/Cu-MT. Phylogenetic analysis of β -do- functionally differentiated isoforms (i.e., Cd-MT, Cu-MT mains from B. glabrata paralogs also showed a close or intermediate Cd/Cu-MT) through the composition relationship between the β β β β -MT and Cd-MT 2 2 2 1P changes of non-Cys aa residues (Jenny et al. 2016; (Additional file 1: Figure S6). Although the tree topology Palacios et al. 2011; Cols et al. 1999). However, our bio- on the affiliation was not statistically supported, it could be informatic analyses in this study suggest that the current enough to hypothesize that the emergence of β β β β - 2 2 2 1P theory on MT domain evolution in the phylum Mollusca MT in B. glabrata might have been an evolutionary could be revised based on newly recognized evidences. process toward the need of more specificity for detoxifica- Novel hypothetical paths and additional insights into the tion of non-essential metals (i.e., primarily Cd). This domain evolution of molluskan MTs are proposed in hypothesis is congruent with the evolutionary theory of following sections. multi-domain MTs in bivalves. In bivalves, development of Cd-preferring MT has been proposed to be based on the Novel evidences for domain duplication in gastropod MTs conversion of a Cu-preferring β-domain to the Cd- The non-canonical domain structure of large MTs from preferring α-domain by the acquisition of additional Cys, two gastropod species (L. littorea and B. glabrata) may followed by subsequent domain duplications (Jenny et al. be considered as novel shapes. Phylogenetic analysis of 2004; Jenny et al. 2006). On the other hand, in pulmonate gastropoda MT domains (β or β ) has generated the gastropods, it has been widely proposed insofar that fur- 1 2 two major clades separated depending on the types of β- ther domain duplication from the prototypic β β -MT 2 1 domains (β or β ) (Fig. 1). The gastropod β -clade has form has unlikely happened. Instead, specific MT isoforms 1 2 2 been proven to contain all previously proposed gastro- with different metal selectiveness in pulmonate gastropods poda β -domains together with the putative β -domains have been achieved mainly through the composition 2 2 of the large MTs proposed in this study. For the large B. changes of non-Cys aa (Jenny et al. 2016; Palacios et al. glabrata MT, three β-domains (the first to third domains 2011). However, from the new evidence in this study, numbered from the N-terminal) are closely clustered domain duplication giving rise to large MTs should be con- together and placed in the major clade comprising the sidered as one of the important mechanisms permitting gastropod β -domains. This result suggests obviously pulmonate MTs to achieve more specificity for their that they are duplicated copies of β -domains that might cognate heavy metals. Taking into account that β β β β - 2 2 2 2 1P have evolved through the tandem duplication events. On MT-originated β -domains display much closer relation- the other hand, the C-terminal region containing only ship among themselves than with the Cd-MT-originated five Cys is not clustered with any typically known β or β -domain in the phylogenetic analysis, it is likely that the 1 2 β -domain sequences. Although we did not provide clear divergence to the β β β β -MT in the B. glabrata genome 2 2 2 2 1P evidence for the origin of this C-terminal region, the might have occurred through a separate path independent most likely scenario is that the originally existed β -do- of the pathway for generating the Cd-MT (Fig. 2). Hence, main at C-terminal in the ancestral β β -MT might have exposure experiments to examine metal selectiveness or 2 1 undergone certain recombination(s) including the loss of binding property of β β β β -MT domains would be 2 2 2 1P some parts during duplication events of neighboring β helpful to test this hypothesis. 2 Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 10 of 18 Fig. 1 Neighbor-joining tree showing the phylogenetic relationship among gastropoda MT β-domains (DM) analyzed with MEGA7software(ver. 7.0.21) (Kumar et al. 2016). Based on the bootstrap tests (1000 replicates), only the clades supported by higher than 50% (condensed tree cutoff value = 50%) are visualized. For MT abbreviations and species, refer to Additional file 1: Table S1. In the NJ tree, two distinct clades, respectively, comprising of β -and β - 1 2 domains indicate the presence of two ancestral β-domains in the gastropod lineage (Jenny et al. 2016). Typical arrangement patterns of Cys motifs for gastropod β -and β -domains are noted at the right side of the tree. Most gastropod MTs are structured as N-terminal β -domain linked to C-terminal 1 2 2 β -domain. However, noticeably, some gastropod MTs are proposed to possess more than two β-domains (likely caused by tandem duplication) based on newly recognized evidences from B. glabrata MT (β β β β -like shape) and L. littorea MT (β β β -structure). Evidence for the β -domain duplication 2 2 2 1P 2 2 1 2 from the sequence alignment analysis can also be referred to in Additional file 1: Figure S2 The other evidence for the β -domain duplication in successive β -domains from the N-terminal that is linked 2 2 the gastropod MTs is observable in the periwinkle (L. lit- to the C-terminal β -domain (i.e., β β β -MT). Like the 1 2 2 1 torea; Caenogastropoda; Hypsogastropoda) MT. In the B. glabrata β β β β -MT above, the first and second β 2 2 2 1P 2 molecular phylogenic tree, a subclade consisting of two domains in the L. littorea MT share high sequence simi- closely affiliated L. littorea β-domains (first and second larity including the conserved Cys motifs, suggesting domains numbered from the N-terminal) was placed that they might have evolved from a tail-to-head tandem within the gastropod β -clade, whereas the third β- duplication event (Additional file 1: Figure S2). Further domain of the L. littorea MT was positioned in the efforts to exploit potential paralog isoforms from this gastropod β -clade (Fig. 1). Based on the phylogenetic species or closely related species are needed to separation between first, second, and third β-domains hypothesize potential factor(s) to drive the domain indicates that the L. littorea MT is comprised of two duplication in L. littorea MT. Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 11 of 18 Fig. 2 (See legend on next page.) Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 12 of 18 (See figure on previous page.) Fig. 2 A schematic representation to suggest the revised hypothesis including the newly proposed evolutionary paths (thickened arrows) for the divergence of MTs in the molluskan lineage. For MT abbreviations and species, refer to Additional file 1: Table S1. Ancestral β-domain-MT has been duplicated to early β β - (eventually to αβ ), β β - and β β -structured MTs and then further diverged into various shapes of extant MTs in 1 1 1 2 2 2 1 the bivalve and gastropoda classes through domain duplication and/or aa substitutions in species/lineage-specific fashion. For MT isoforms with more than two metal-binding domains, the presumed duplicated domains are underlined with either a solid line (for homologous duplication giving rise to tandem array of domains with high sequence similarity) or dashed line (for heterologous duplication resulting in domains sharing little sequence similarity). Representative species examples for each MT type are noted below the schematic representation Postmortem studies have claimed that most gastropod MT would be responsive to not only various heavy MTs would be very conservative in the Cys arrangements metals including Cu, Cd, and Zn but also non-metal as a β β -domain structure (Jenny et al. 2016; Berger et al. stimulating stress treatments such as induced hypoxia, 2 1 1997; Dallinger et al. 1993). On the contrary, substitu- immune challenge and heat shock (Lee and Nam 2016; tions/replacements of non-Cys aa residues while retaining Guo et al. 2013). Based on this observation, the MT, at the β β -frame have been thought as the major process least in this vetigastropod species, is thought to have 2 1 for the evolutionary divergences of MTs in this primitive evolved to play readily multifunctional roles in diverse class Gastropoda. The Cu homeostatic requirements pathways involved in stress physiology. (thought to be mainly operated by β-domains) from the use of hemocyanin as a respiratory pigment in these Updates in ostreidae and mytilidae MTs gastropods, which is not present in oysters, has also been The evolutionary theory of MT based on domain dupli- proposed as one of plausible factors responsible for the cation has been the most comprehensively highlighted in lack of divergently duplicated domains in gastropod MTs the Crassostrea species, particularly in C. virginica.Two (Jenny et al. 2016; Berger et al. 1997; Perez-Rafael et al. ancestral β-domains (i.e., β and β ) appear to have di- 1 2 2012; Perez-Rafael et al. 1844). However, the present study verged to produce two different structural MT isoforms, claims that the formation of large MT with more than two i.e., αβ -MT and β β -MT in the oysters belonging to 1 2 2 domains should not be a bivalve-exclusive episode. It Ostreidae (Jenny et al. 2004; Jenny et al. 2006). The might have been an important path allowing some ancestral β -domain appears to have duplicated to pro- gastropod MTs to better modulate metal specificity in duce a two-domain-structured MT that ultimately led to response to variations occurred in their habitat environ- the evolution of the αβ -structured MTs, which is ments (Fig. 2). For both examples (B. glabrata MT and L. observable in the Crassostrea species MT-Is and MT- littorea MT), the target domain that has undergone dupli- IVs. On the other hand, the Crassostrea species MT-IIIs cation is the β -domain. Hence, novel or specified func- reveal the typical β β -domain structure, which might 2 2 tions (e.g., detoxification of non-essential metals) could be have been a descendant shape resulted from the duplica- assigned to duplicated β -domains while original and tion event of a single ancestral β -domain (Fig. 2). In the fundamental roles (e.g., Cu homeostatic regulation) are reconstructed phylogenetic tree, Ostreidae β-domain retained in the β -domain. Extra metal-binding residues sequences are placed on one of two main clades (i.e., offered by duplicated β -domains may also be potentially either β -or β -domain clade), although several sub- 1 2 advantageous to strengthen further capacity of both metal clades within the β -clade are not supported by high reservation and resistance to excessive metal ions. Taken confidence values (Additional file 1: Figure S7). Crassos- together, hypothesis for evolutionary mechanism to mold trea MT-IVs are closely related each other and distin- gastropod MTs should be revised taking into account the guished from MT-I/-II isoforms within the β -clade, inclusion of this novel path featured by the duplication of suggesting the early divergence between MT-IV and β -domain. MT-I/-II families from the prototypic αβ -MT form. On the other hand, unlike in Heterobranchia and Also within the β -clade, it is notable that the C. virgi- Caenogastropoda, the clear sign for taxa-specific domain nica MT isoforms have a tendency to be separately multiplication has not been yet identified in Vetigastro- clustered from MTs from other Crassostrea species poda species (abalone and limpet) (Lee and Nam 2016; (such as C. gigas, C. ariakensis, C. rivularis, and C. angu- Lieb 2003). Although the characterization of MT in this lata) as seen in both MT-I and MT-IV groups. It may taxonomic group has been very limited, vetigastropod suggest the divergence of these MT families during spe- species have reportedly shown only a single MT isoform ciation in the genus Crassostrea to make C. virginica to (i.e., β β -MT) within a given species (Fig. 2). Currently, be distinguished from other Crassostrea species (Jenny 2 1 it is unclear if vetigastropods possess functionally or et al. 2016) (Additional file 1: Figure S7 and Additional structurally diverged paralogs. However, a recent study file 1: Figure S8). The most apparent difference between has reported that the abalone (Haliotis discus hannai) C. virginica and other Crassostrea species is found in the Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 13 of 18 MT-II family. C. virginica MT-IIs have been reported to essential metals than MT10 (Leung et al. 2014; Lemoine et the most recently evolved isoforms, which have formed al. 2000; Vergani et al. 2007). Conversely, in some environ- through the loss of β-domain, giving rise to the sole α- mental situations, the early αβ -MT might have diverged domain structured MTs (Additional file 1: Figure S8). into functionally differentiated isoforms in the Mytilidae: However, this divergence pattern in C. virginica is clearly MT10 to execute primarily the homeostatic regulation of contrasted by the duplication of β -domain in C. gigas physiologically relevant metals and MT20 to function in MT-IIs (i.e., αβ β -structured MT), suggesting the differ- the detoxification of trace metals (Fig. 2). 1 1 ent evolutionary paths of the MT-II between the two closely related species belonging to the same genus Novel paths for domain duplication in bivalve MTs (Jenny et al. 2004; Tanguy and Moraga 2001). Such a Exploitation of genetic determinants for MTs from other solely α-domain-based structure has been found only in bivalve taxa has often showed the species (or lineage)- C. virginica insofar, whereas αβ β -domain structure has specific variations in MT structure. However, currently 1 1 also been observed similarly in other Crassostrea species limited volume of knowledge on these MTs still hurdles and non-Crassostrea species (Additional file 1: Figure S7). to hypothesize the evolutionary mechanism of non- Meanwhile, the β -domain clade comprising of C. virgi- canonical MT forms in detail. Reconstruction of molecu- nica and C. gigas MT-III isoforms shows a monophyletic lar phylogenetic trees in this study displays two main topology, indicating the divergence from a common an- clades: one is a large clade comprising of β -domains cestral β β -MT origin. However, within the β -clade, two from various taxa and the other is a small clade consist- 2 2 2 subclades are characterized by the first and second β -do- ing of five presumed β -domain sequences deciphered 2 2 mains rather than by species. All the N-terminally present from two A. irradians (Pectinidae) paralogue MTs β -domains are clustered together in a subclade while all (Fig. 3). Within a former β -clade, paralogue isoforms 2 1 the C-terminally present β -domains in other subclade from a given single species (e.g., R. philippinarum MT1/ (see also (Jenny et al. 2016)). This finding may be indica- MT2 and L. elliptica MT10a/10b) formed subclades tive of that the first and second β -domains of the Crassos- supported by high bootstrap values. Similarly, several trea MT-IIIs might appear to have originated before the subclades consisted of orthologs from closely related separation of the two oyster species (Fig. 2). species belonging to the same genus ([e.g., MTs from Mytilidae species represent uniformly the αβ -domain genus Meretrix (Chang et al. 2007; Wang et al. 2010; structure (known as the prototypic shape of bivalve MT). Jiang et al. 2016) and genus Cerastoderma (Desclaux- Some mytilid species have been reported to possess the Marchand et al. 2007; Ladhar-Chaabouni et al. 2009; intronless, relatively short MTs in their genomes. The pres- Paul-Pont et al. 2012)). Collectively, it suggests that they ence of intronless MT genes has been proposed as the might have evolved from recent divergence at species or organisms’ strategic means for the efficient response to genus levels. In contrast, some paralogue MT isoforms changes of cellular metal circumstances through the rapid are distantly placed in the phylogenetic tree, although transcription of MT genes (Leignel et al. 2005). Molecular they are placed in the same β -clade. Such a distant rela- phylogenetic analysis of Mytilidae MT domains generated tionship is found in the genus Hyriopsis where MT1 and trees consistently comprising of three main clades; clades MT2 paralogs are not affiliated depending upon species. for mytilid MT10s, P. viridis MTs and mytilid MT20s Although nomenclatures MT1 and MT2 are not estab- (Additional file 1: Figure S9). In congruent with the previ- lished clearly in this two species, an isoform of H. ous phylogenetic results using the entire MT polypeptide cumingii MT (GQ184290) is closely related with H. region, present phylogenetic analyses using separate do- schlegelii MT1 ortholog (KJ019820), rather than its para- mains also suggest the early divergence of P. viridis MTs logue isoform (FJ861993). This finding may indicate that from the other mytilid orthologs (Leung et al. 2014). In the the divergence between MT1 and MT2 might have Mytilidae lineage, a critical event before speciation is the occurred earlier than the speciation of the two Hyriopsis divergence of MT10 and MT20 forms (Aceto et al. 2011) species (Yang et al. 2014; Wang et al. 2016). (Fig. 2). As compared to MT10, MT20s are characterized From the present molecular phylogenetic analysis, novel by the acquisition of additional Cys residues (i.e., a Cys-Cys paths of MT evolution through domain duplication giving doublet) in their α-domains (Leignel and Laulier 2006). It rise to large-sized MTs with more than two metal-binding could be thought as a process to prepare the paralogue domains could be proposed (Fig. 3). Evidences come from MT varieties with better execution in the detoxification of two bivalve species: one is P. coreanum (Sphaeriidae) non-essential metals, since more Cys residues are generally (Baek et al. 2009) and the other is A. irradians (Pectinidae) taken into account for enhanced capability for sequestrat- (Liu et al. 2006; Wang et al. 2009). In the P. coreanum,the ing the toxic metals (i.e., metal tolerance). This hypothesis two putative β-domains are placed in the β -clade. Within could be supported by the fact that MT20 would be more the β -clade, the second (numbered from the N-terminal) preferentially associated or exclusive reacted with non- β -domain of P. coreanum MT is found to form a 1 Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 14 of 18 Fig. 3 Molecular phylogenetic tree (neighbor-joining tree drawn with MEGA7 (Kumar et al. 2016)) to propose the duplication of β - (for P. coreanum MT) and β -domain (A. irradians MT1 and MT2), giving rise to MTs with more than two metal-binding domains. Clades are visualized with the condensed tree cutoff value of 50% based on 1000 replicate bootstrap tests. For MT abbreviations and species, refer to Additional file 1: Table S1. Detailed domain structures of these multi-domain MTs are provided in Additional file 1: Figure S5B subclade with two β -domains from Arcidae species Ostreidae and Sphaeriidae (Fig. 2). In Ostreidae, the β - 1 1 [Scapharca broughtonii (FJ154101) and Tegillarca granosa domain seems to have evolved from a relatively recent (AY568678)]. Considering the N-terminally present puta- gene duplication, resulting in a tandem array of the two tive α-domain, this P. coreanum MT could be designated homologous β -domains. On the contrary, the newly αβ β -structure. In the phylum Mollusca, the αβ β -do- proposed P. coreanum αβ β -MT shows no apparent se- 1 1 1 1 1 1 main structure (i.e., duplication of β -domain from the quence homology between the two β -domains. Possibly, 1 1 anticipated prototypic αβ -MT) has been previously the multiplication of β -domain in Sphaeriidae might have 1 1 reported to be the Crassostrea-specific event (Tanguy and been an earlier divergent event. Currently, the evolution- Moraga 2001). However, we have already proposed above ary route for the acquisition of additional β -domain in P. that this event has also been true for non-Crassostrea oys- coreanum MT is open to hypothesize. One possible sce- ter (i.e., A. plicatula; Ostreidae). Further, the present P. nario is the duplication of β -domain from the prototypic coreanum (Sphaeriidae) MT could indicate that this dupli- αβ -MT, followed by further divergence in non-Cys resi- cation process would not be limited to the Ostreidae. dues. Alternatively, the other possibility is that the more However, the evolutionary scheme for the domain dupli- ancestral β β -MT (not yet reported in extant bivalve 1 1 cation may be different between the two families MTs) might have acquired additional α-domain through Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 15 of 18 the duplication to β β β -MT followed by the conversion event. Although need to be further challenged, the ances- 1 1 1 of one of β -domains to α-domain (Fig. 2). Molecular tral β β -MT in the phylum Mollusca perhaps might have 1 2 1 phylogeny of bivalve α-domains also shows that P. corea- gone through two separate paths (Fig. 2). Namely, one num α-domain is independently placed without being might have been a preservative path from the ancestral affiliated with any other bivalve α-domain ortholog in a β β -MT to produce the conserved β β -structured MTs 2 1 2 1 subclade (Additional file 1: Figure S8). Hence, further which seen in most extant gastropod MTs, although some efforts to exploit paralogue isoforms from this species (P. exceptional gastropod species represent additional coreanum) and/or similarly structured orthologs from its lineage-specific, recent duplication(s) of homologous β - closely related species should be needed to get a deeper domain. On the other hand, the other path might involve insight into the mechanism responsible for the emergence earlier duplication of β -domain from the ancestral β β - 2 2 1 of αβ β -MT in Sphaeriidae. MT, giving rise to multiple β -like domains linked to the 1 1 2 From the same molecular phylogenic tree, putative C-terminal β -domain in certain bivalve taxa. Based on domains from the two A. irradians MT isoforms (Liu et this, the extant shapes of A. irradians MT paralogs may al. 2006; Wang et al. 2009) are positioned in either the reflect the consequences of differential rounds of β -do- main β -clade or a small clade consisting of only A. irra- main duplication from an ancestral β β -MT. However, 1 2 1 dians MT domains (Fig. 3). Two C-terminal domains re- further validation is needed to test whether this divergent spectively from A. irradians MT1 and MT2 are placed process might have occurred before or after speciation in the main β -clade. On the other hand, the small clade events in the Pectinoida lineage. are exclusively comprised of three putative β -like do- What are the functional or physiological implications mains from the MT1 (first to third domains predicted of domain duplications (or multiplication) in molluskan from the N-terminal side) and two from MT2 (first and MTs? The evolution of larger MT proteins has been second domains). A. irradians MTs represent non- proposed as a strategic means that might likely be canonical shape that does not perfectly match the advantageous for benthic organisms that are believed to known typical β-domain structure. Nevertheless, if the experience a greater exposure to metals due to their eco- Cys distribution pattern is fundamentally considered, the logical niche (Jenny et al. 2004; Jenny et al. 2006; Tanguy multiplied domains in A. irradians MTs could be classified and Moraga 2001; Tschuschke et al. 2002). Although the as β -like structure (possibly designated β ′-domain). paucity or limitation of the functional studies on such 2 2 Hence, the overall domain structures of A. irradians MT1 large MTs hurdles to hypothesize comprehensively the and MT2 could be designated β ′β ′β ′β -MT and relevant mechanism(s) in detail, there have been some 2 2 2 1 β ′β ′β -MT, respectively. In bivalve class, the MT pro- hypothetical evidences or suggestive assumptions. First, 2 2 1 teins possessing multiple β-domains without α-domain based on the heterologous expression assay by using the have been reported only in the Crassostrea β β -MT-IIIs, recombinant microbial systems, a couple of noteworthy 2 2 and this structure has been considered to have developed experiments have shown that large-sized multi-domain- from the early duplication of the ancestral β -MT. How- structured MT proteins would be able to confer greater ever, A. irradians MTs represent a C-terminal β -domain Cd resistance of the hosts (Tanguy et al. 2001; as well as the multiple β ′-domains (Fig. 2). Two plausible, Tschuschke et al. 2002). Second, several independent but untested, hypotheses may be possible regarding the previous studies have claimed that amplification and/or evolution of such unusual domain structure in A. irra- tandem duplications of MT genes might have been an dians MTs. One is the acquisition of C-terminal β -do- advantageous process to attain the strengthened ability main in the ancestrally duplicated β β -MT (i.e., giving of metal tolerance (Cho et al. 2009; Beach and Palmiter 2 2 rise to β β β -MT) followed by further divergent process 1981; Maroni et al. 1987; Mehra et al. 1990; Stephan et 2 2 1 resulting in current shapes of A. irradians MT isoforms. al. 1994). Because the number of Cys residues in MT The other, more plausible, hypothetical path is the multi- proteins has been taken into account as a fundamental plication of β -domain from the prototypic β β -MT factor to determine the number of metal ions bound or 2 2 1 structure that is seen in most gastropod MTs (Jenny et al. reserved by the MTs (Amiard et al. 2006), more Cys 2016; Palacios et al. 2011). As described above, the present attained by domain duplications might be beneficial in study has already noted that the multiplication of β -do- the conference of metal resistance in a broad sense. main (i.e., β β -structure) might have been one of diver- Third, domain multiplication(s) accompanied with signifi- 2n 1 gence mechanisms in certain gastropod taxa (Fig. 2). cant substitutions/replacements of non-Cys residues could However, unlike the tandem duplication of homologous offer a chance to confer some novel functions on large β -domain in gastropod MTs, A. irradians MTs represent MTs. Because aa replacements on non-Cys residues in little sequence similarity among β ′-domains, suggesting MT have been known to represent significant effects on that the multiplication of these β ′-domains in each A. metal-binding specificity and kinetic reactivity (Palacios irradians MT isoform may not be the recent duplication et al. 2011; Pedersen et al. 1994; Kurasaki et al. 1997; Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 16 of 18 Munoz et al. 2000), such a divergent pattern (domain Received: 23 January 2017 Accepted: 25 May 2017 duplication together with significant aa substitutions) might also increase the kinds of metals reacted by these References MT proteins. Taken together, the ancestral or prototypic Aceto S, Formisano G, Carella F, Vico GD, Gaudio L. The metallothionein genes of MT protein has diverged into various isoforms with a Mytilus galloprovincialis: genomic organization, tissue expression and great structural diversity in the phylum Mollusca. Struc- evolution. Mar Genom. 2011;4:61–8. Amiard JC, Amiard-Triquet C, Barka S, Pellerin J, Rainbowd PS. Metallothioneins in tural diversifications driven by both domain duplication aquatic invertebrates: their role in metal detoxification and their use as and aa replacements might have led certain subfunctiona- biomarkers. Aquat Toxicol. 2006;76:160–202. lization and/or neofunctionalization of MT proteins in an Baek MK, Lee JS, Kang SW, Lee JB, Kang HJ, et al. Phylogenetic analysis based on metallothionein gene sequence of an indigenous species Pisidium isoform-dependent fashion (Tanguy and Moraga 2001). (Neopisidium) coreanum in Korea. Kor J Malacol. 2009;25:153–60. Baršytė D, White KN, Lovejoy DA. Cloning and characterization of metallothionein cDNAs in the mussel Mytilus edulis L. digestive gland. Comp Biochem Physiol Conclusions C Pharmacol Toxicol Endocrinol. 1999;122:287–96. Phylum Mollusca represents a great structural diversity Beach LR, Palmiter RD. Amplification of the metallothionein-I gene in cadmium- of MT, a core suite playing key roles in both homeostatic resistant mouse cells. Proc Natl Acad Sci. 1981;78:2110–4. Berger B, Dallinger R, Gehrig P, Hunziker PE. Primary structure of a copper- regulation of essential metals and detoxification of trace binding metallothionein from mantle tissue of the terrestrial gastropod Helix metals in living organisms. The structural diversity of pomatia L. Biochem J. 1997;328:219–24. molluskan MTs have been achieved essentially through Binz PA, Kagi JHR. Metallothionein: molecular evolution and classification. In: Klaassen C, editor. Metallothionein. Basel: Birkhäuser; 1999. p. 7–13. the domain duplication events from an ancestral, singu- Blindauer CA, Leszczyszyn OI. Metallothioneins: unparalleled diversity in structures lar domain-MT. Domain duplication have been followed and functions for metal ion homeostasis and more. Nat Prod Rep. 2010;27: by further diversification and selection toward needs for 720–41. Braun W, Wagner G, Worgotter E, Vasak M, Kagi JH, Wuthrich K. Polypeptide fold acquiring metal selectiveness, specialized novel function, in the two metal clusters of metallothionein- 2 by nuclear magnetic and improved capacity of metal homeostasis/detoxifica- resonance in solution. J Mol Biol. 1986;187:125–9. tion. With this viewpoint, novel paths for domain diver- Capasso C, Carginale V, Scudiero R, Crescenzi O, Spadaccini R, Temussi PA, Parisi E. Phylogenetic divergence of fish and mammalian metallothionein: gences of some gastropod and bivalve MT families relationships with structural diversification and organismal temperature. J proposed in this review could shed new light onto the Mol Evol. 2003;57:S250–7. revision and update of the hypothesis for evolutionary Carpenè E, Andreani G, Isani G. Metallothionein functions and structural characteristics. J Trace Elem Med Biol. 2007;21:35–9. differentiation of MTs in the molluskan lineage. Chang YT, Jong KJ, Liao BK, Wu SM. Cloning and expression of metallothionein cDNA in the hard clam (Meretrix lusoria) upon cadmium exposure. Additional file Aquaculture. 2007;262:504–13. Chiaverini N, Ley MD. Protective effect of metallothionein on oxidative stress- induced DNA damage. Free Radic Res. 2010;44:605–13. Additional file 1: Supplementary figures and tables. (PDF 433 kb) Cho YS, Choi BN, Ha EM, Kim KH, Kim SK, Kim DS, Nam YK. Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and Acknowledgements expression analysis. Mar Biotechnol. 2005;7:350–62. Not applicable. Cho YS, Lee SY, Kim K-Y, Bang IC, Kim DS, Nam YK. Gene structure and expression of metallothionein during metal exposures in Hemibarbus Funding mylodon. Ecotoxicol Environ Saf. 2008;71:125–37. This study was supported by the grant from the Golden Seed Project (GSP), Cho YS, Lee SY, Kim KY, Nam YK. Two metallothionein genes from mud loach Ministry of Oceans and Fisheries, Republic of Korea. Misgurnus mizolepis (Teleostei: Cypriniformes): gene structure, genomic organization, and mRNA expression analysis. Comp Biochem Physiol B Availability of data and materials Biochem Mol Biol. 2009;153:317–26. Not applicable. Cols N, Romero-Isart N, Bofill R, Capdevila M, Gonzalez-Duarte P, Gonzalez-Duarte R, Atrian S. In vivo copper- and cadmium-binding ability of mammalian metallothionein beta domain. Protein Eng. 1999;12:265–9. Authors’ contributions Cong M, Wu H, Liu X, Zhao J, Wang X, Lv J, Hou L. Effects of heavy metals on the YKN designed this study, carried out bioinformatic analysis and drafted the expression of a zinc-inducible metallothionein-III gene and antioxidant manuscript. EJK carried out the management of sequence data, reference enzyme activities in Crassostrea gigas. Ecotoxicology. 2012;21:1928–36. analyses, and polishing of the manuscript. Both authors read and approved Dallinger R, Berger B, Hunziker PE, Birchler N, Hauer CR, Kägi JHR. Purification and the final manuscript. primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A. Eur J Biochem. 1993;216:739–46. Competing interests Desclaux-Marchand C, Paul-Pont I, Gonzaleza P, Baudrimont M, Montaudouin X. The authors declare that they have no competing interests. Metallothionein gene identification and expression in the cockle (Cerastoderma edule) under parasitism (trematodes) and cadmium Consent for publication contaminations. Aquat Living Resour. 2007;20:43–9. Not applicable. Dondero F, Piacentini L, Banni M, Rebelo M, Burlando B, Viarengo A. Quantitative PCR analysis of two molluscan metallothionein genes unveils differential Ethics approval and consent to participate expression and regulation. Gene. 2005;345:259–70. Not applicable. English TE, Storey KB. Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine Publisher’sNote gastropod Littorina littorea. J Exp Biol. 2003;206:2517–24. Springer Nature remains neutral with regard to jurisdictional claims in Faller P. Neuronal growth-inhibitory factor (metallothionein-3): reactivity and published maps and institutional affiliations. structure of metal–thiolate clusters. FEBS J. 2010;277:2921–30. Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 17 of 18 Fang Y, Yang H, Wang T, Liu B, Zhao H, Chen M. Metallothionein and superoxide Lü D, Luo KY, Pan BP, Gao H. Expression of metallothionein and thioredoxin gene dismutase responses to sublethal cadmium exposure in the clam Mactra in Cyclina sinensis exposed to cadmium. Oceanol Limnol Sin. 2012;43:47–51. veneriformis. Comp Biochem Physiol C Toxicol Pharmacol. 2010;151:325–33. Lynes MA, Hidalgo J, Manso Y, Devisscher L, Laukens D, Lawrence DA. Fang Y, Yang H, Liu B, Zhang L. Transcriptional response of lysozyme, Metallothionein and stress combine to affect multiple organ systems. Cell metallothionein, and superoxide dismutase to combined exposure to heavy Stress Chaper. 2014;19:605–11. metals and bacteria in Mactra veneriformis. Comp Biochem Physiol C Toxicol Mao H, Wang DH, Yang WX. The involvement of metallothionein in the Pharmacol. 2013;157:54–62. development of aquatic invertebrate. Aquat Toxicol. 2012;110–111:208–13. Geffard A, Geffard O, Amiard JC, His E, Amiard-Triquet C. Bioaccumulation of Marie V, Gonzalez P, Baudrimont M, Boutet I, Moraga D, Bourdineaud JP, Boudou metals in sediment elutriates and their effects on growth, condition index, A. Metallothionein gene expression and protein levels in triploid and diploid and metallothionein contents in oyster larvae. Arch Environ Contam Toxicol. oysters Crassostrea gigas after exposure to cadmium and zinc. Environ 2007;53:57–65. Toxicol Chem. 2006;25:412–8. Guo F, Tu R, Wang W-X. Different responses of abalone Haliotis discus hannai to Maroni G, Wise J, Young JE, Otto E. Metallothionein gene duplications and metal waterborne and dietary-borne copper and zinc exposure. Ecotoxicol Environ tolerance in natural populations of Drosophila melanogaster. Genetics. 1987; Saf. 2013;91:10–7. 117:739–44. Gupta SK, Singh J. Evaluation of mollusc as sensitive indicator of heavy metal Mehra RK, Garey JR, Winge DR. Selective and tandem amplification of a member pollution in aquatic system: a review. IIOAB. 2011;2:49–57. of the metallothionein gene family in Candida glabrata. J Biol Chem. 1990; Inoue K-I, Takano H, Shimada A, Satoh M. Metallothionein as an anti- 265:6369–75. inflammatory mediator. Mediat Inflamm. 2009;101659. Munoz A, Petering DH, Shaw CF. 3rd. Structure-reactivity among metallothionein Isani G, Carpenè E. Metallothioneins, unconventional proteins from three-metal domains: role of noncysteine amino acid residues in lobster unconventional animals: a long journey from nematodes to mammals. metallothionein and human metallothionein-3. Inorg Chem. 2000;39:6114–23. Biogeosciences. 2014;4:435–57. Nielson KB, Winge DR. Order of metal binding in metallothionein. J Biol Chem. Jenny MJ, Ringwood AH, Schey K, Warr GW, Chapman RW. Diversity of 1983;258:13063–9. metallothioneins in the American oyster, Crassostrea virginica, revealed by Nielson KB, Winge DR. Preferential binding of copper to the beta domain of transcriptomic and proteomic approaches. Eur J Biochem. 2004;271:1702–12. metallothionein. J Biol Chem. 1984;259:4941–6. Jenny MJ, Warr GW, Ringwood AH, Baltzegar DA, Chapman RW. Regulation of Palacios O, Pagani A, Perez-Rafael S, Egg M, Hockner M, Brandstatter A, Capdevila metallothionein genes in the American oyster (Crassostrea virginica): ontogeny and M, Atrian S, Dallinger R. Shaping mechanisms of metal specificity in a family differential expression in response to different stressors. Gene. 2006;379:156–65. of metazoan metallothioneins: evolutionary differentiation of mollusc Jenny MJ, Payton SL, Baltzegar DA, Lozier JD. Phylogenetic analysis of molluscan metallothioneins. BMC Biol. 2011;9:4. metallothioneins: evolutionary insight from Crassostrea virginica. J Mol Evol. Paul-Pont I, Gonzalez P, Montero N, Montaudouin X, Baudrimont M. Cloning, 2016;83:110–25. characterization and gene expression of a metallothionein isoform in the Jiang LJ, Maret W, Vallee BL. The glutathione redox couple modulates zinc edible cockle Cerastoderma edule after cadmium or mercury exposure. transfer from metallothionein to zinc-depleted sorbitol dehydrogenase. Proc Ecotoxicol Environ Saf. 2012;75:119–26. Natl Acad Sci. 1998;95:3483–8. Pedersen KL, Pedersen SN, Højrup P, Andersen JS, Roepstorff P, Knudsen J, Depledge Jiang LJ, Vasak M, Vallee BL, Maret W. Zinc transfer potentials of the alpha- and MH. Purification and characterization of a cadmium-induced metallothionein from beta-clusters of metallothionein are affected by domain interactions in the the shore crab Carcinus maenas (L.). Biochem J. 1994;297:609–14. whole molecule. Proc Natl Acad Sci. 2000;97:2503–8. Perez-Rafael S, Monteiro F, Dallinger R, Atrian S, Palacios O, Capdevila M. Jiang GP, Cheng XY, Teng SS, Chai XL, Lin XG, Liu GX, Xiao GQ. Cloning and Cantareus aspersus metallothionein metal binding abilities: the unspecific expression of metallothionein gene in Meretrix lamarckii. Acta Hydrobiol Sin. CaCd/CuMT isoform provides hints about the metal preference determinants 2016;40:914–20. in metallothioneins. Biochim Biophys Acta. 1844;2014:1694–707. Khoo HW, Patel KH. Metallothionein cDNA, promoter, and genomic sequences of Perez-Rafael S, Mezger A, Lieb B, Dallinger R, Capdevila M, Palacios O, Atrian S. the tropical green mussel. Perna viridis J Exp Zool. 1999;284:445–53. The metal binding abilities of Megathura crenulata metallothionein (McMT) in Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis the frame of Gastropoda MTs. J Inorg Biochem. 2012;108:84–90. version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4. Raspor B, Dragun Z, Erk M, Ivankovic D, Pavicic J. Is the digestive gland of Mytilus Kurasaki M, Yamaguchi R, Linde Arias R, Okabe M, Kojima Y. Significance of alpha- galloprovincialis a tissue of choice for estimating cadmium exposure by fragments of metallothionein in cadmium binding. Prot Eng. 1997;10:413–6. means of metallothioneins? Sci Total Environ. 2004;333:99–108. Ladhar-Chaabouni L, Mokdad-Gargouri R, Denis F, Hamza-Chaffai A. Cloning and Sarkar A, Ray D, Shrivastava AN, Sarker S. Molecular biomarkers: their significance characterization of cDNA probes for the analysis of metallothionein gene and application in marine pollution monitoring. Ecotoxicology. 2006;15:333–40. expression in the Mediterranean bivalves: Ruditapes decussatus and Serén N, Glaberman S, Carretero MA, Chiari Y. Molecular evolution and functional Cerastoderma glaucum. Mol Biol Rep. 2009;36:1007–14. divergence of the metallothionein gene family in vertebrates. J Mol Evol. Le TTY, Zimmermann S, Sures B. How does the metallothionein induction in 2014;78:217–33. bivalves meet the criteria for biomarkers of metal exposure? Environ Pollut. Stephan W, Rodriguez VS, Zhou B, Parsch J. Molecular evolution of the 2016;212:257–68. metallothionein gene Mtn in the melanogaster species group: results from Lee SY, Nam YK. Transcriptional responses of metallothionein gene to different Drosophila ananassae. Genetics. 1994;138:135–43. stress factors in Pacific abalone (Haliotis discus hannai). Fish Shellfish Tang RS, Xia JH, Wang YM, Gong SY, Yu DH. Analysis on cloning and sequence Immunol. 2016;58:530–41. characteristics of metallothionein cDNA in Pinctada maxima. J Anhui Agricult Leignel V, Laulier M. Isolation and characterization of Mytilus edulis metallothionein Sci. 2009;37:2888–90. genes. Comp Biochem Physiol C Toxicol Pharmacol. 2006;142:12–8. Tanguy A, Moraga D. Cloning and characterization of a gene coding for a novel Leignel V, Hardivillier Y, Laulier M. Small metallothionein MT-10 genes in coastal metallothionein in the Pacific oyster Crassostrea gigas (CgMT2): a case of and hydrothermal mussels. Mar Biotechnol. 2005;7:236–44. adaptive response to metal-induced stress? Gene. 2001;273:123–30. Lemoine S, Bigot Y, Sellos D, Cosson RP, Laulier M. Metallothionein isoforms in Tanguy A, Mura C, Moraga D. Cloning of a metallothionein gene and Mytilus edulis (Mollusca, Bivalvia): complementary DNA characterization and characterization of two other cDNA sequences in the Pacific oyster quantification of expression in different organs after exposure to cadmium, Crassostrea gigas (CgMT1). Aquat Toxicol. 2001;55:35–47. zinc, and copper. Mar Biotechnol. 2000;2:195–203. Tanguy A, Boutet I, Riso R, Boudry P, Auffret M, Moraga D. Metallothionein genes Leung PTY, Park TJ, Wang Y, Che CM, Leung KMY. Isoform-specific responses of in the European flat oyster Ostrea edulis: a potential ecological tool for metallothioneins in a marine pollution biomonitor, the green-lipped mussel environmental monitoring? Mar Ecol Prog Ser. 2003;257:87–97. Perna viridis, towards different stress stimulations. Proteomics. 2014;14:1796–807. Tschuschke S, Schmitt-Wrede HP, Greven H, Wunderlich F. Cadmium resistance Lieb B. A new metallothionein gene from the giant keyhole limpet Megathura conferred to yeast by a non-metallothionein-encoding gene of the crenulata. Comp Biochem Physiol C Toxicol Pharmacol. 2003;134:131–7. earthworm Enchytraeus. J Biol Chem. 2002;277:5120–5. Liu WQ, Ni DJ, Song LS, Wu LT, Xu W, Kong XY. Cloning and characterization of a Vergani L, Grattarola M, Borghi C, Dondero F, Viarengo A. Fish and molluscan metallothionein gene in bay scallop Argopecten irradians. Oceanol Limnol metallothioneins: a structural and functional comparison. FEBS J. 2005;272: Sin. 2006;37:444–9. 6014–23. Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 18 of 18 Vergani L, Grattarola M, Grasselli E, Dondero F, Viarengo A. Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis. Arch Biochem Biophys. 2007;465:247–53. Wang H, Zhang Q, Cai B, Li H, Sze KH, Huang ZX, Wu HM, Sun H. Solution structure and dynamics of human metallothionein-3 (MT-3). FEBS Lett. 2006; 580:795–800. Wang L, Song L, Ni D, Zhang H, Liu W. Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge. Comp Biochem Physiol C Toxicol Pharmacol. 2009;149:50–7. Wang Q, Wang X, Wang X, Yang H, Liu B. Analysis of metallothionein expression and antioxidant enzyme activities in Meretrix meretrix larvae under sublethal cadmium exposure. Aquat Toxicol. 2010;100:321–8. Wang W-C, Mao H, Ma D-D, Yang W-X. Characteristics, functions and applications of metallothionein in aquatic vertebrates. Front Mar Sci. 2014;1:34. Wang C, Sheng J, Hong Y, Peng K, Wang J, Wu D, Shi J, Hu B. Molecular characterization and expression of metallothionein from freshwater pearl mussel. Hyriopsis schlegelii Biosci Biotechnol Biochem. 2016;80:1327–35. Xiong Y, Chen Y, Ru B. The expressed alpha domain of mouse metallothionein-I from Escherichia coli displays independent structure and function. Biochem Mol Biol Int. 1998;46:307–19. Yang S, Wei M, Yang X, Wang H, He L, Li C. A novel metallothionein gene from mussel, Hyriopsis cumingii: Identification and expression under lanthanum exposure. J World Aquacult Soc. 2014;45:454–60. Zhang G, Fang X, Guo X, Li L, Luo R, Xu F, Yang P, Zhang L, Wang X, Qi H, et al. The oyster genome reveals stress adaptation and complexity of shell formation. Nature. 2012;490:49–54. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries � Our selector tool helps you to find the most relevant journal � We provide round the clock customer support � Convenient online submission � Thorough peer review � Inclusion in PubMed and all major indexing services � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Fisheries and Aquatic Sciences Springer Journals

Diversification and domain evolution of molluskan metallothioneins: a mini review

Fisheries and Aquatic Sciences , Volume 20 (1) – Jun 13, 2017

Loading next page...
 
/lp/springer-journals/diversification-and-domain-evolution-of-molluskan-metallothioneins-a-KGJBNxZdJT
Publisher
Springer Journals
Copyright
Copyright © 2017 by The Author(s)
Subject
Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
eISSN
2234-1757
DOI
10.1186/s41240-017-0054-z
Publisher site
See Article on Publisher Site

Abstract

Background: Metallothionein (MT) is a multifunctional protein playing important roles in homeostatic regulation and detoxification of metals. Mollusk species have been considered as useful sentinel platforms for MT-based biomarker approaches, and they have been reported to display an extraordinary structural diversity of MT proteins. However, potential diversity of molluskan MTs has not been fully explored and recent updates have suggested the need of revision of evolutionary hypothesis for molluskan MTs. Results: Based on bioinformatic analysis and phylogenetic evidences, novel divergence mechanisms and paths were hypothesized in both gastropod and bivalve MT groups. Our analyses are suggestive of the taxon- or lineage- specific domain multiplication/duplication from the ancestral or prototypic MT. Diversification and selection of molluskan MTs might be driven by the needs for acquiring metal selectiveness, specialized novel function, and improved capacity of metal detoxification under environmentally stressed conditions. Conclusion: The structural diversity and variations of molluskan MTs are significantly larger than previously understood. Undoubtedly, molluskan MTs have undergone dynamic divergent processes in their evolutionary histories, giving rise to the great diversity of domain structures in extant MT isoforms. Novel evolutionary paths for molluskan MTs newly proposed in this review could shed additional light onto the revision of the hypothesis for evolutionary differentiation of MTs in the molluskan lineage. Keywords: Metallothionein, Phylum Mollusca, Structural diversity, Domain evolution Background Benthic mollusks have been proposed as the useful Metallothionein (MT) is a metal-binding protein identified sentinel platforms for biomarker approaches to aquatic in almost living organisms. This housekeeping protein is a and marine environments, in relation with their high multifunctional, non-enzymatic effector playing important bioaccumulation capacity of chemical elements from both roles in various criteria of physiology of organisms under water and sediment (Amiard et al. 2006; Geffard et al. basal and stressed conditions (Carpenè et al. 2007; Mao et 2007; Le et al. 2016). Further, their sedentary nature also al. 2012). Owing to the high inducibility of its expression makes it possible that biomagnification effects of the upon metal exposure, MT has been long given attention as pollution could be effectively visualized without a signifi- one of core suits for biomarkers to address risks associated cant consideration of complex migratory factors in the with metal-related environmental problems (Sarkar et al. interpretation of bioaccumulation data (Gupta and Singh 2006). Besides its fundamental roles in the homeostatic 2011). Accordingly, the exploitation of genetic determi- regulation of essential metals and detoxification of trace nants of molluskan MTs has been a progressively growing metals, MT plays important roles in host protective path- domain in the field of MT researches. To date, a number ways under environmentally or physiologically perturbed of previous literatures have claimed that molluskan conditions (Inoue et al. 2009; Chiaverini and Ley 2010; species should represent a great structural diversity of MT Lynes et al. 2014). proteins (Jenny et al. 2004; Jenny et al. 2006; Leignel and Laulier 2006). Moreover, some mollusk species particu- larly including American oyster Crassostrea virginica have * Correspondence: yoonknam@pknu.ac.kr shown extraordinarily large-sized MT isoforms compris- Department of Marine Bio-Materials and Aquaculture, Pukyong National ing of over 100 amino acid (aa) residues (Jenny et al. 2004; University, Busan 48513, South Korea © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 2 of 18 Jenny et al. 2006; Tanguy and Moraga 2001), which have reported yet from classes such as Aplacophora, Mono- not been usually observable in vertebrate orthologs placophora, Polyplacophora and Scaphopoda. No cloned (Blindauer and Leszczyszyn 2010; Serén et al. 2014). Based MT sequence is available in the class Cephalopoda, bar- on this, it has been widely proposed that the mollusks ring couples of uncharacterized sequences predicted might have undergone evolutionary unique history in the from the octopus (Octopus bimaculoides) genome scaf- divergence of MT proteins (Serén et al. 2014; Isani and fold. Hence, it is evident that genetic determinants of Carpenè 2014; Wang et al. 2014; Jenny et al. 2016). MT have been yet narrowly explored in the phylum Undoubtedly, structural diversifications would confer Mollusca. functional variations on these molluskan MTs, leading Many gastropod and bivalve species possess multiple species-specific adaptation to environmental changes. paralogue MT isoforms. American oyster Crassostrea virgi- Even yet fully elucidated, the prevalence of diversified nica (Ostreidae; Bivalvia) is the top species showing the isoforms are also likely in relation with significant variabil- greatest number of coexisting MT sequences in a single ities and inconsistencies in the responses of molluskan MTs species (n = 17; non-redundant paralogue sequences at aa to metal or other stress exposures (in both laboratories and levels) (Jenny et al. 2004; Jenny et al. 2016); this isoform fields) (Amiard et al. 2006; Le et al. 2016). number is also the highest among all metazoans. Currently, Evolution of molluskan MTs has been proposed to be in the Bivalvia class, 34 species belonging to four subclasses fundamentally based on the duplication events of their (Pteriomorphia, Anomalodesmata, Heteroconchia, and metal-binding domains. Domain duplication(s) from a Palaeoheterodonta) have been recorded to reveal MT common ancestral MT (a singular domain MT) might isoform(s) under GenBank accession codes. However, the have given rise to multi-domain structured MTs (Jenny species distribution for GenBank-deposited MT sequences et al. 2004; Palacios et al. 2011). The prototypic MT has in bivalves is highly skewed, in which only a few popularly been supposed to go through further diversification into studied species have dominated MT sequences. More than various distinguished isoforms in different taxa. This 50% of the total MT sequences have been from only the evolutionary theory has been comprehensively addressed top five species belonging to either Mytilidae (mussels) or with several well-known molluskan models such as Ostreidae (oysters). Meanwhile, in the Gastropoda class oysters, mussels and air-breathing snails (Leignel and (the largest and primitive class in the phylum Mollusca), Laulier 2006; Jenny et al. 2016; Palacios et al. 2011; only less than 20 non-redundant full-length MT sequences Aceto et al. 2011; Leung et al. 2014). However, in contrast have been exploited from twelve different species to rich information on these popularly studied species, belonging to one of seven families. These gastropod divergent processes of MTs in other mollusk species (in species include air-breathing pulmonate snails belong- both gastropods and bivalves) have been quite limitedly ing to the Heterobranchia (species from four families, explored yet. As the research on molluskan MTs pro- Planorbidae, Physidae, Helicidae, and Bradybaenidae), aba- gresses, it is becoming increasingly evident that structural lones (Haliotidae; Vetigastropoda), a limpet (Fissurellidae; variations and deviations of MT domains in this phylum Vetigastropoda), and a periwinkle (Littorinidae; Caenogas- may be significantly larger than previously understood. tropoda). The species information and the accession code Recently, there have been considerable efforts on the iso- for each MT sequence used in this review are referred to lation of novel MTs or MT-like proteins from different in Additional file 1: Table S1. molluskan taxa. Now it is not difficult to find certain A number of previous publications have reportedly in- molluskan MT isoforms of which domain structures are dicated that most vertebrate MTs including mammalian hardly assigned into one of traditionally described categor- and teleostean MTs may exhibit well-conserved primary ies. Hence, with this viewpoint, we aimed to review the structures. They are typically characterized by common structural diversity of molluskan MT domains with an features in polypeptide length (60~65 aa), calculated mo- angle to shed additional light onto the evolutionary path lecular weight (6~7 kDa), proportion of cysteine (Cys) of MT domains in the molluskan lineage. residues (30~35%; arranged typically as Cys-X-Cys, Cys- X-X-Cys and Cys-Cys where the X is any non-Cys aa), Status of MT isoform sequence data in public and theoretical pI values (8.0~8.5) (Wang et al. 2014; database Capasso et al. 2003; Cho et al. 2005; Cho et al. 2008; Currently the number of publically released MT gene Cho et al. 2009) (Additional file 1: Figure S1A). Such a sequences (both genomic and mRNA sequences; https:// structural homology among vertebrate MTs may also attri- www.ncbi.nlm.nih.gov/genbank/) from the mollusk spe- bute to a considerable degree of functional orthology be- cies has exceeded over 100 excluding the partial se- tween vertebrate species. However, it may not apply to the quences or untrimmed ESTs. Almost all molluskan MT mollusk species. Remarkably variable or deviated scores in genes have been obtained from bivalves and gastropods, theprimarystructurehavebeenfrequentlyobservablein whereas no characterized MT sequence has been molluskan MTs (Jenny et al. 2004; Tanguy and Moraga Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 3 of 18 2001; Baršytė et al. 1999; Tanguy et al. 2001) (Additional file the numbers of taxa available for MTs from Gastropoda 1: Figure S1B). First, molluskan MTs reveal often significant class were currently limited, theoretical pI values esti- variations in polypeptide lengths among paralogue and mated from the category group Vetigastropoda (pI orthologue isoforms. Median and mean values for the num- 5.16~6.00) were relatively lower than those from Hetero- ber of aa residues estimated from all the mollusk MT poly- branchia (pI 6.10~8.28). Possibly, MT proteins with peptide sequences addressed in this study (n = 109) are 73.0 largely different pI values might exhibit differential (7.2 kDa) and 80.2 (7.8 kDa) aa, respectively. Overall, these capability to bind or interact with charged molecules in values are higher than that estimated with representative the host protective process. In addition, contents of vertebrate MT orthologs. More noticeably in molluskan some positively charged aa (e.g., Lys) may influence group, considerable numbers of MTs represent extraordin- importantly the metal-binding function of MT protein. arily short or long lengths apparently deviated from the These aa residues (particularly at positions in vicinity to mean (and median) value above. Within the Bivalvia class, Cys) are thought to be involved in the stabilization of the length of MT polypeptides ranges from 43 aa (4.2 kDa) the interaction between MTs and metal ions through the to 204 aa (20.3 kDa); noticeably both are recorded as para- electrostatic interactions to bridge the protonated basic logs from a single species C. virginica (Ostreidae) (Gen- residues and the negatively charged metal-thiolate com- Bank accession numbers, AY331700/AY331701 and plex (Pedersen et al. 1994). Thereby, such a wide range AY331706, respectively). Other oyster species also dis- of pI values may be potentially suggestive of, at least in play the significant variations in the sizes of MT iso- part, dynamic diversification and subfunctionalization forms. However, the mussel species belonging to the among molluskan MT isoforms, although the biological order Mytiloida represents relatively a uniform feature and/or functional implications of such a wide range of in the range of the MT protein lengths (66~75 aa). Be- pI values should be explored in the future. sidesthe twomainbivalve taxa,itisworthytonoteun- Third, molluskan taxa are found to have a tendency of usually long MT isoforms reported from Argopecten relatively lower Cys content (mean ± s.d. = 28.1 ± 1.8%) in irradians (Pectinidae; EF093795, and EU734181) and their MT proteins than are mammalian (32.4 ± 1.3%) or Pisidium coreanum (Sphaeriidae; GQ268325). teleostean (33.3 ± 0.3%) groups. Also, the intra- and inter- On the other hand, mean and median values for MT species variations in Cys contents of MTs are larger in polypeptide lengths available for gastropod MTs were 67.5 mollusks (especially in bivalves; ranging 21.2 ~ 31.9%) than and 65.0 aa, respectively. Currently, the shortest gastropod in mammals (26.2~34.4%; 31.1~34.4% if MT-IIIs excluded) MT sequence is found to be Physa acuta (Physidae; and fish (32.8~35.0%) (Additional file 1: Figure S1). Metal GU259686) MT consisting of 59 aa, whereas the longest binding property and capacity of molluskan MTs have been one is the MT isoform with 100 aa reported in a periwinkle studied in only a few species (Palacios et al. 2011). How- species Littorina littorea (Littorinidae; AY034179). Except ever, Cys residues are known to be essential for the affinity these shortest and longest sequences, the polypeptide to bind metal ions where the number of metal ions bound lengths of other gastropod MTs are found to fall in the by the MT may be fundamentally determined by the num- range from 64 to 70 aa. Similarly with bivalves, multiple ber of Cys residues (Amiard et al. 2006; Jenny et al. 2016; isoform MTs (n = 2~4) have been reported in pulmonate Vergani et al. 2005). Hence, it is able to suggest that snails belonging to Helicidae or Planorbidae, while marine molluskan MTs may display relatively larger variations in gastropods including abalones have shown only a single the metal binding capacity among isoforms than vertebrate MT isoform; yet still unclear if they have additional MT orthologue groups. isoform gene(s) or not (Additional file 1: Table S1; Taken together, all the three parameters above mentioned Additional file 1: Figure S1). are undoubtedly indicative of large structural variations and Second, molluskan group shows a wider range of divergence of MT proteins among molluskan species. Based distribution for theoretical isoelectric point (pI) values on this overview, taxa (or lineage)-specific patterns for across MT isoforms as compared to mammalian and structural diversification of MTs are described more details teleostean MT groups (Additional file 1: Figure S1). In in following sections, with a particular attention on the mammals, MT-IIIs are only the isoform family (but arrangement of Cys motifs. excluding murine MT-IIIs) to show a significantly acidic pI values (4.79~4.82), while most other isoforms belong- Structural diversity of MT isoforms in major ing to MT-I, −II or -IV class typically represent pI values molluskan taxa higher than at least 7.5. Most teleostean MTs show pI Nomenclature and classification of MTs used in this values ranging from 7.8 to 8.4. On the other hand, bi- paper were referred to GenBank (NCBI) based on the valve MTs display pI values ranging 4.31 to 8.56 depend- definition of each sequence. If the MT sequence was ing on isoforms. In the Gastropoda group, the pI values published in scientific paper(s), its classification was of the MT proteins range from 5.16 to 8.28. Although checked again. Within a given species, the redundant Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 4 of 18 MT sequences at aa level were not included in analyses MT is proven to show a conserved pattern for Cys (Additional file 1: Table S1). Classification of putative arrangements in N- and C-terminal parts (i.e., nine Cys domain structure in each MT sequence was based on respectively in putative N- and C-terminal domain re- the number and arrangement pattern of Cys motifs as gions). Considering the Cys motif patterns, the N- and C- described previously (Jenny et al. 2016), since there have terminal parts of the L. littorea MT could be designated been no empirical studies on three-dimensional struc- β -and β -domains, respectively. Further, a closer examin- 2 1 tures of molluskan MT proteins. In general, domain ation on the intervening region (comprised by 32 aa) be- structure in MT is designated α and β (Braun et al. tween the β - (N-terminal) and β - (C-terminal) domains 2 1 1986; Binz and Kagi 1999). Usually, the α-domain con- has indicated that the 32-aa internal segment has been po- tains eleven to twelve Cys residues, binds four divalent tentially a duplicated copy of the N-terminal β -domain. It metal ions and confers structural stability on the MT conserves clearly 9 Cys residues and shows a considerably polypeptides. On the other hand, the β-domain, binds high sequence identity (75%) to the N-terminal (β -do- three divalent metal cations through the nine Cys resi- main) domain. Based on our peer review, the structure of dues and participates in metal exchange reactions via L. littorea MT could be considered as a novel shape of glutathione-shuttling with metal-requiring apoproteins gastropod MT characterized by β β β -domain form. 2 2 1 (Jiang et al. 1998; Jiang et al. 2000). Hence, this newly proposed structure suggests that domain duplication event might have served as a driving Gastropoda MTs force to figure the large MT in certain gastropod taxa. In a total, nineteen non-redundant MTs (from twelve Another example for the domain duplication in gastropod species belonging to seven families) including a putative MT is the 124-aa B. glabrata MT (XP_013080485). In that MT predicted in the unplaced genomic scaffold se- MT polypeptide sequence, three putative domain regions quence (Biomphalaria glabrata; Planorbidae) were sharing a considerable sequence similarity in one another analyzed with sequence alignments. Among the nineteen could be identified, and each of the three putative domains sequences, 17 sequences with 59 to 70 aa residues are maybedesignated β -structure based on their Cys arrange- found to be fairly aligned in the multiple sequence align- ment patterns (Additional file 1: Figure S2). As numbered ment trials. In spite of substantial differences in non-Cys from theN-terminal, thefirst β -domain and the second β - 2 2 residues among taxa, they share a conserved pattern of domain share the conserved Cys motif frame (except one Cys motifs. Eighteen Cys in these gastropoda MTs are additional Cys in the second β-domain). They also show the arranged as [Cys-X-X-X-Cys→ (Cys-X-Cys) → Cys]→ [( high sequence homology (76.5%) each other. The putative Cys-X-Cys) → Cys→ (Cys-X-Cys) ] (Additional file 1: third β -domain linked to the second β -domain is found to 2 2 2 2 Figure S2). It indicates that gastropoda MTs represent display 66.7% of sequence homology to the first and second the protein structure comprising of two distinguished β- β -domains. Within this context, the B. glabrata MT could domain forms (i.e., β β -form) designated by the recent be proposed to possess at least three duplicated β -domains 2 1 2 suggestion to propose the presence of two hypothetical tandemly arrayed in a tail-to-head fashion. On the other ancestral β-domains (Jenny et al. 2016). The β -domain hand, the remaining C-terminal part (23-aa) following the structure at the N-terminal of gastropod MT protein is β β β -domain region is found to contain five Cys (three 2 2 2 similarly observed in C. virginica MT-IIIs as well as in singlet Cys and a Cys-Cys doublet motif). Unlike the L. the β-domains of vertebrate (mammals and fish) MTs. littorea MT above, the C-terminal region of this B. glabrata On the other hand, the C-terminal β -domain of the MT display no typical shape to be categorized into one of gastropod MT is commonly found in various molluskan known domain structures (Additional file 1: Figure S2). MTs. According to the β β -structural scheme, the Currently, the origin of this C-terminal region has been 2 1 shortest P. acuta MT (comprised by 59 aa) is thought to unknown. Further validation of scaffold genomic sequences have lost two Cys in its β -domain. In that alignment, along with mining of similarly organized MTs from MTs from Vetigastropoda species possess more aa resi- other Heterobranchia genomes would be needed to dues (5 or 8 aa) in the intervening region between the get a deeper insight into the mechanism responsible β - and β -domains than those from Heterobranchia for the formation of the array of three β -domain 2 1 2 species (2 aa). region in this gastropod MT. This pulmonate snail Besides the common β β -shape, gastropod group species has already been knowntoexpress functionally 2 1 represents two significantly lengthy MT polypeptides. diversified (i.e., different metal selectiveness) MT iso- One is 100-aa MT from Littorina littorea (Caenogastro- forms [(i.e., Cd-MT (GQ205374), Cu-MT (GQ205373) poda; AY034179) (English and Storey 2003) and the and intermediate Cd/Cu-MT (GQ205375)] (Berger et other is 124-aa MT (XP_013080485; deduced from the al. 1997). Hence, it would also be valuable to examine unplaced genomic scaffold) from B. glabrata (Additional the expression patterns of this newly identified MT file 1: Figure S2). Based on manual alignment, L. littorea regarding its potential differentiation in physiological Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 5 of 18 function and/or metal responsiveness in comparison with IID/-IIE/-IIF and MT-IIG/-IIH display tandem arrays previously characterized paralogs from this species. comprised of three and four α-domain copies, respect- ively (Jenny et al. 2004). Between and among duplicated Bivalve-ostreidae MTs (repetitive) copies, a few aa substitutions have been Structural diversity of MT families in Ostreidae has been found. Collectively, the divergent process of C. virginica comprehensively described with C. virginica model MT-II family has occurred through the loss of β -do- (Jenny et al. 2004; Jenny et al. 2006). Currently, seven- main (e.g., MT-IIA/-IIB; α-domain-structure) followed teen C. virginica MT sequences available in GenBank by further divergence into various subisoforms having could be classified into one of four MT isoform families differential numbers of duplicated α-domains (e.g., MT- (MT-I, −II, −III, and IV). The MT families consist of 2 IIC to MT-IIH; α -domain structure) (Jenny et (n = 2 ~ 4) (MT-IA and MT-IB), 8 (MT-IIA, MT-IIB, MT-IIC, MT- al. 2016). IID, MT-IIE, MT-IIF, MT-IIG, and MT-IIH), 3 (MT- However, this divergence pattern has not been always IIIA, MT-IIIB, and MT-IIIC), and 3 (MT-IVA, MT-IVB, a common finding in the Ostreidae lineage (Additional and MT-IVC) subisoforms, respectively. In addition to file 1: Figure S3A). Rather than the loss of β-domain, C. these 16 sequences, one MT sequence named MTA gigas MT-II family has represented the tandem duplica- (GenBank accession no. AF506977) is independently tion of 32-aa β -domain with retaining the α-domain, recorded in this oyster species (see Additional file 1: giving rise to the αβ β -domain structure (Tanguy and 1 1 Table S1). Of the 16 isoforms, the prototypical MT Moraga 2001). Further, unlike in C. virginica, there has structure (corresponding to MT-I form; MT-IA and been no variation in repeat numbers among C. gigas MT-IB) is 75-aa MT possessing 21 Cys residues (28% of MT-II subisoforms. MT (AF349907) from another Cys content). The MTA isoform can also be classified to Crassostrea species, Portuguese oyster (C. angulata)has the same prototype (i.e., MT-I) with an addition of Asp exhibited the duplication of only a short, partial β -do- in the region between 16th and 17th Cys residues. They main fragment (7-aa region), giving rise to a non- share a high sequence homology one another in a proto- canonical αβ β -structure with truncated C-terminus. 1 1P typic αβ -domain-structure (Jenny et al. 2004). This αβ - Because there has been no other publically released MT 1 1 structure is well conserved also in other oyster species, paralog from C. angulata, it has been yet unclear if this including C. gigas (Pacific oyster; AJ242657), C. ariaken- oyster species may possess any paralog copies represent- sis (the Suminoe or Asian oyster; DQ342281) and C. ing the complete αβ β -domain structure or not. Beyond 1 1 rivularis (the Jinjiang oyster; JN225502). The same the Crassostrea genus, our survey against GenBank has domain structure is also relevant with isoforms from identified that Alectryonella plicatula MT (KP875559; non-Crassostrea species Ostrea edulis (Tanguy et al. 107-aa) should also display the typical αβ β -domain 1 1 2003). However, relative large substitutions of non-Cys structure. Moreover, the A. plicatula MT shows very aa (also the replacement of three Cys with other aa in high sequence homology to its Crassostrea orthologs the O. edulis MTb isoform) have been found in the O. (MT-IIs), indicating the common origin of this multi-do- edulis MTs compared to MT-I orthologs from Crassos- main structure. Currently, the known αβ β -structured 1 1 trea species (Additional file 1: Figure S3A). MT subisoforms in Ostreidae (except for the C. angulata C. virginica MT-II family includes eight subisoforms MT with a truncated C-terminus) share the same N- (MT-IIA to MT-IIH), and this MT-II family has been terminal (Met-Ser-Asp-Pro) and C-terminal (Cys-Lys-Lys) known to be classified into two subgroups. First, the motif residues (Additional file 1: Figure S3A). MT-IIA/-IIB group possesses a sole α-domain of which On the other hand, the C. virginica MT-III group consists sequence is highly conserved with that of the prototypic of three homogenous subisoforms MT-IIIA/-IIIB/-IIIC. αβ -MT-I. The loss of functional β -domain has been They share each other high sequence identity including 18 1 1 proposed to occur due to the point mutation in the Cys residues, and the distribution pattern of the 18 Cys linker region (i.e., non-sense mutation resulting in a stop have been proposed as the array of two β-domains as codon). Consequently, MTs belonging to this group re- [(Cys-X-Cys) → Cys] × 2 (i.e., β β -MT) (Additional 4 2 2 veal noticeably short polypeptide length (43 aa) (Jenny file 1: Figure S3B). The arrangement pattern of nine et al. 2004). Second, on the contrary, the remaining six Cys is obviously similar with the β -domain of the C. virginica MT-II isoforms are lengthy polypeptides gastropod β β -MTs (Jenny et al. 2006; Jenny et al. 1 2 (94-aa MT-IIC, 149-aa MT-IID/-IIE, 145-aa MT-IIF, 2016). The same β β -domain structure has also been 2 2 204-aa MT-IIG, and 200-aa MT-IIH). They have tan- found in C. gigas MT-III (JF781299); however unlikely demly duplicated copies of α-domain where the numbers in C. virginica, multiple MT-III subisoforms have not of repetitive duplications are variable among isoforms. been characterized in C. gigas (Cong et al. 2012). The C. In MT-IIC, only one duplication event is predicted (i.e., gigas MT-III shows a series of substitutions of non-Cys aa two tandem duplicate copies), while subisoforms MT- from the C. virginica MT-III isoforms. The overall Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 6 of 18 sequence identity of MT-IIIs between the two Crassostrea diverged from the prototypic MT-I through the non- species is about 70% (Additional file 1: Figure S3B). Mean- Cys aa changes. This isoform is likely to show, at least while, Crassostrea MT-III isoforms reveal considerably in part, certain functional orthology to the C. virginica low pI values (4.38~4.77 for C. virginica and 4.31 for C. MT-III, although they have different domain structures. gigas) as compared to other MT isoforms showing pI Finally, the MT-IV isoforms (MT-IVA, MT-IVB and values > 7.5. With the viewpoint of the low pI value, C. vir- MT-IVC; 83 aa in length) from C. virginica have been ginica MT-III may resemble mammalian MT-III family. proposed as variant forms of αβ -MT. This isoform Most known mammalian MT-III isoforms except murine group has been supposed to have experienced a series MT-IIIs reveal acidic pI ranges (4.79~4.82) with acidic 6- of aa substitutions including Cys residues, giving rise amino-acid insert in the C-terminal region. Synthesis of to 25 Cys with the formation of a Cys-Cys doublet (in the mammalian MT-III is not inducible by heavy metals and α-domain) and three Cys-Cys-Cys triplet motifs (in the localized predominantly in the central nervous system β -domain). The C-terminal residue of the C. virginica (Faller 2010). Unique roles of mammalian MT-III differing MT-IV isoform (glutamine-alanine-threonine) is also not- from other MT isoforms have been characterized as the ably different from those of other paralog isoforms. neuronal growth-inhibitory factor to inhibit neuronal out- Thereby, the proposed designation of domain structure growth (Wang et al. 2006). Specific roles of bivalve MT-III for MT-IVs could be α′β ′-form. In addition to C. virgi- differing other MT family groups have not been yet exten- nica,two Crassostrea species (C. gigas and C. ariakensis) sively addressed. However, the expression study with C. possess MT-IVs of which primary domain structures are virginica MT-III has indicated that the C. virginica MT-III fairly conserved with that of C. virginica MT-IV. However, showed quite a low basal level of expression in adult tis- C. gigas MT-IV (87 aa; AM265551) and C. ariakensis MT- sues (i.e., only actively expressed in early larvae). Further, IV (86 aa; JF919323) represents one additional Cys residue C. virginica MT-III represented only a moderate respon- at C-terminal region (Additional file 1: Figure S3B). siveness to heavy metal exposures in both larvae and Besides the above C. gigas MT-IV clone (AM265551), gen- adults (Jenny et al. 2006). However, on the contrary, the ome sequencing of C. gigas (Zhang et al. 2012) represents C. gigas MT-III has been reported to be significantly in- two unplaced genomic scaffolds [scaffold852 (JH816574) duced by zinc (as a main regulator for zinc homeostasis), and scaffold1297 (JH818394)] each containing the puta- and it may be a participating member for cadmium de- tive gene encoding MT (EKC32371 and EKC28510, re- toxification in the adult tissues (Cong et al. 2012). spectively, in the two scaffolds). The deduced sequences Thereby, it suggests the functional differentiation/diver- of these MTs are 128 and 137 aa in length, respectively. gence of MT-III isoforms during speciation events in the Both MTs are predicted to possess unusual N-terminal Crassostrea lineage. Within the context of this hypothesis, regions (41-aa for EKC32371 and 50-aa for EKC28510). it is worthy to have an attention on another C. gigas MT However, immediately following the N-terminal region, isoform named mt3 under the accession number the two putative MTs represent the 87-aa-structure that is AJ295157. This C. gigas mt3 isoform is not a true MT-III apparently homologous to the C. gigas MT-IV isoform family member with the β β -domain structure. Rather (AM265551). When the unusual N-terminal regions are 2 2 than, mt3 should be considered as a MT-I member be- excluded from these two MT-IV-like sequences, the cause it represents the αβ -domain structure (see three MT sequences (one characterized MT-IV and Additional file 1: Figure S3A). However, due to the two in-scaffold sequences) reveal only one aa substitu- significant non-Cys aa substitutions, C. gigas mt3 tion, although the scaffold sequences should be further displays only 67% sequence identity to its paralogue validated in future. MT-I isoform. Moreover, the substitutions include the change of three Lys residues to uncharged aa as well Bivalve-mytilidae MTs as the replacement of uncharged aa with negatively In Mytilidae, 33 full-length, non-redundant MT aa se- charged aa. Such aa substitutions might give rise to quences were retrieved from five taxa (12 sequences lower pI value (5.98) of mt3 than those of its paralo- form Mytilus edulis, 6 from M. galloprovincialis, 2 from gue MT-I members. A previous study has reported Mytilus sp., 11 from Perna viridis and 2 from Bathymo- that mt3 should have the extremely low basal expres- diolus azoricus). Mytilid MTs are a structurally more sion level with only moderate or minute responsive- homogeneous group as compared to Ostrea MT group. ness to metal exposure (Marie et al. 2006). The mt3 They represent 66-aa to 75-aa polypeptides containing has been suggested to have probably no significant 19 to 23 Cys residues, and display typically the αβ -do- physiological functions under metal exposure and to main structure (Additional file 1: Figure S4). be expressed only in particular developmental stages In the mytilid mussels, two types of MT isoforms, (Marie et al. 2006). Hence, taken together, it could be MT10 and MT20 have been described previously (Aceto hypothesized that C. gigas mt3 might have been et al. 2011; Leung et al. 2014). These two MT types Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 7 of 18 differ in mass and Cys arrangement. The monomeric (alignment position; Ser in P. viridis MTs and MT20s vs. form MT10 (~10 kDa) represents generally 73-aa poly- Gly in other mytilid MT10s), 39th (Gly vs. Lys), and 73rd peptides including 21 Cys residues mainly arranged as (Ser vs. Pro). Finally, the third group is comprised of six nine Cys-X-Cys motifs. On the other hand, the dimeric MT sequences from three Mytilus species. They are 72-aa form MT20 (~20 kDa) is typically 72-aa polypeptides polypeptide containing 23 Cys residues (i.e., MT20 type). containing 23 Cys residues. Unlike MT10, the MT20 iso- Only one exception is the substitution of Cys to Arg in the forms show a Cys-Cys doublet in the α-domain. MT10 M. edulis MT-20 clone. From the alignment, two MT20- and MT20 have been reported to be functionally differ- specific residues could be found at positions 24th (Lys entiated. MT10 has been found to be more abundant in MT20s vs. Glu in all MT10s) and 68th (Asn vs. Thr) than MT20, and hence it could be considered as a main (Additional file 1: Figure S4). In particular, the change player for the regulation of homeostasis under basal from negatively charged aa (e.g., Glu) to Lys at position conditions (Leung et al. 2014; Lemoine et al. 2000). 24th is likely related with the MT20-specific formation Under metal-exposed conditions, MT10 and MT20 have of Cys-Cys doublet motif, since the positively charged aa been known to display differential responses and binding (e.g., Lys) at vicinity to Cys motif is considered to play ability to essential and non-essential metals. MT10 could important roles in the stabilization of the metal binding be actively inducible by various metals while MT20 be reaction in most MT proteins (Pedersen et al. 1994). more preferentially associated with non-essential metals such as Cd and/or Hg (Raspor et al. 2004; Dondero et al. Bivalve-other taxa 2005; Vergani et al. 2007). Besides the two main bivalve taxa (Ostreidae and Mytilidae), From multiple sequence alignment, mytilid MTs could 27 non-redundant, full-length MT isoforms have been be categorized into three main groups (Additional file 1: exploited from 21 bivalve species belonging to one of six Figure S4). The first group consists of three MT10B orders Pterioida, Arcoida and Pectinoida (subclass sequences (two from M. edulis and one from M. gallopro- Pteriomorphia), Pholadomyoida (subclass Anomalodes- vincialis; AJ577126, AJ577127 and DQ848984, respect- mata), Veneroida (subclass Heteroconchia), and Union- ively). They represent 66-aa polypeptides containing 19 oida (belonging to Palaeoheterodonta). From the multiple Cys residues with the deletion of one Cys-X-Cys motif in sequence alignment, most of them are found to represent the α-domain. All the three MT sequences are originated αβ -domain-structure with a conserved 21-Cys-frame from intronless MT genes (Leignel et al. 2005; Yang et al. (Additional file 1: Figure S5A). However, several variant 2014). The second group contains twenty-four MT10 isoforms are also found to show modification(s) of Cys isoform sequences. They reveal 72 to 75 aa in lengths and motifs in one or two positions, giving rise to the gener- possess 21 Cys residues in conserved positions (Lemoine et ation of Cys-Cys doublet, substitution, insertion or dele- al. 2000; Khoo and Patel 1999). There are only two tion. Several variant isoforms are found to retain the total exceptions; one is the replacement of a Cys with Arg in P. number of Cys residues (i.e., 21 Cys) while others show viridis MT-IA (JN596471) and the other is an insertion of changes of the total number of Cys residues. MT isoforms an additional Cys in P. viridis MT (AF036904). Within the from Pinctada maxima (pearl oyster; FJ389580) (Tang et second group, theMTs are likelytobesub-grouped al. 2009) and Laternula elliptica (Atlantic clam; according to known taxonomic appraisal at genus level DQ832722/DQ832723) display the insertion of an add- (i.e., Bathymodiolus, Mytilus and Perna). All the P. viridis itional Cys in the third Cys-X-Cys motif, resulting in the MT-II isoforms (named MT-IIA, −IIB, −IIC and -IID) are Cys-Cys-Cys motif at that position. On the contrary, an found to possess 72 aa, as similarly with the mytilid MT20 MT isoform from Hyriopsis schlegelii (freshwater pearl isoforms. Nevertheless, based on their Cys motifs frame, mussel; MT2; KJ019821) has lost one Cys residue at the these P. viridis MT-IIs (JN596477 to JN596480) have been second Cys-X-Cys motif along with considerable alter- proposed as MT10 members. Previous phylogenetic ana- ations in non-Cys aa residues. Unlike its paralog (H. schle- lysis has claimed an early divergence of P. viridis MTs from gelii MT1; KJ019820), the H. schlegelii MT2 has been the main mytilid MT10/MT20 groups (Leung et al. 2014). proposed as a genetically separated isoform (Wang et al. When aligned with other mytilid orthologs, P. viridis 2016). A recent study has indicated that these two H. MT10-I and/or MT10-II represent several residues distinct schlegelii MT paralogs might have been subfunctionalized from other mytilid MT isoforms. They include positions as evidenced by clearly distinct tissue expression pat- 11th (alignment position; Gln/Lys in P. viridis MTs vs. Asn terns (i.e., constitutive expression of H. schlegelii MT1 in all other mytilid MT10 and MT20 isoforms), 62nd (Gln vs. gonad-specific or predominant expression of H. vs. Gly/Asp) and 74th (Ser vs. Gly). Further, P. viridis schlegelii MT2) (Wang et al. 2016). Another example MT10 isoforms (both MT10-I and MT10-II) are found to for the large difference between paralog MT isoforms is share the same aa in several positions with mytilid MT20 P. martensi MT isoforms. Even though the P. martensi isoforms. These could be exemplified by positions 34th MT1 (KC197172.1) represents the common αβ -shape, 1 Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 8 of 18 its paralog MT2 (KC832833.1) exhibits an apparently domains indicate that they share little sequence non-canonical pattern of Cys arrangement (20 Cys). similarity one another except conserved Cys residues On the other hand, two MT isoforms from Veneroida (Additional file 1: Figure S5B). Within this scheme, the A. are found to have noticeably less number of Cys residues irradians MT2 could be treated as a paralog having the than others: one is the duck clam Mactra veneriformis less number of β -domains (i.e., putatively designated (Mactridae) MT with 18 Cys (59-aa; Cys content = β β β -like structure). The A. irradians MT2 also reveals 2 2 1 30.5%; FJ611963) (Fang et al. 2010; Fang et al. 2013) and some non-canonical attributes including the lack of one the other is Venus clam Cyclina sinensis (Veneridae) Cys residue and the formation of Cys-Cys doublet in the MT with 16 Cys (74-aa; Cys content = 21.6%; N-terminal β -like domain. These two A. irradians MT HM246244) (Lü et al. 2012). The M. veneriformis MT isoforms are found to share only a little sequence hom- seems to have lost an internal fragment near N-terminal ology, indicating that they may be quite distantly related region (possibly corresponding to the α-domain) con- paralogs (Wang et al. 2009). taining three Cys residues (likely a Cys-X-Cys motif and On the other hand, the 105-aa P. coreanum (Sphaeriidae) one conserved Cys). The C. sinensis MT lacks a Cys-X- MT (GQ268325; 31 Cys) (Baek et al. 2009) is found to Cys motif in α-domain and additionally three Cys resi- show the domain multiplication to resemble the αβ β - 1 1 dues (Cys-X-Cys motif and one Cys residue) probably in structure, of which Cys arrangement is similar with that of the β-domain. C. gigas MT-II. However, unlike C. gigas MT-II to show Importantly, three MT isoforms display large polypep- thetandemarrayoftwo homogenous β -domains, P. tide sizes comprising of more than two putative domains coreanum MT contains the two heterogeneous β -domains (Additional file 1: Figure S5B). Of the three MTs, two with no apparent sequence homology between the two MT sequences (MT1 and MT2) are from the bay scallop domains. The P. coreanum MT lacks a common triplet Argopecten irradians (Pectinidae) and remaining one linker sequence (Lys-Val-Lys/Val) between α- and first β - isoform is from the fingernail clam Pisidium coreanum domain. The array of two heterogeneous β -domains (Sphaeriidae). These sequences have been reported linked to N-terminal α-domain observed in P. coreanum earlier but their domain structures have never been ad- MT could be the novel structure of bivalve MT proteins dressed clearly. Even though A. irradians MT1 (145-aa; (Additional file 1: Figure S5B). 40 Cys; EF093795; (Liu et al. 2006)) and MT2 (110-aa; 28 Cys; EU734181; (Wang et al. 2009)) exhibit essential features of mollusk MTs (i.e., the presence of character- Domain evolution in molluskan MTs istic Cys-X-Cys motifs), their overall structures are more Current theory for domain evolution of molluskan MTs or less complicated and difficult to be simply categorized Currently, the proposed hypothesis for the evolution of into one of currently known shapes of bivalve MTs. molluskan MT has been based on the domain duplica- However, in a broad sense, these isoforms may bear a tion event(s) from an ancestral single domain-structured resemblance to the MT isoforms with multi-β-domain- MT, in which the β-domain has been considered as the structure. For both A. irradians MT isoforms, the C- ancestral shape (Cols et al. 1999). After an early duplica- terminal region may be considered the β -domain posses- tion event of the ancestral β-domain, the resultant ββ- sing nine Cys as (Cys-X-Cys) → Cys→ (Cys-X-Cys) .In domain MT has undergone divergent processes, given 2 2 addition to the C-terminal β -domain, A. irradians MT1 rise to the αβ-structure in certain taxa (Jenny et al. 2016; potentially exhibits three tandemly arrayed β -like do- Braun et al. 1986; Cols et al. 1999). Difference in the mains. However, each β -like domain in the A. irradians metal-binding properties between the α-domain and the MT1 displays some non-canonical arrangement of Cys. β-domain makes the two domains to represent differen- First, two Cys of the first Cys-X-Cys motif in each domain tiated roles in the cellular physiology. Generally, α- is separated further by intervening 2~4 aa residues (i.e., domain plays a more prevalent role in Zn homeostasis similar with the pattern found in N-terminal regions of and detoxifying sequestration of toxic metals (e.g., Cd) gastropod β -domains). Second, Cys-Cys doublet motifs whereas the β-domain is primarily responsible for the rather than a canonical Cys-X-Cys motif are present in the homeostatic regulation of essential metals (e.g., Cu) first and third β -like domains. Third, an additional Cys- (Jenny et al. 2004; Cols et al. 1999; Nielson and Winge X-Cys motif exists in the flanking regions between the first 1984; Xiong et al. 1998). Consequently, the multi- and second β -like domains as well as between the second domain MT in specific taxa acquiring both α- and β- and third β -like domains. Nevertheless, the overall shape domains was able to perform the dual functions; the of A. irradians MT1 may be designated β β β β -like detoxification of toxic metals by the α-domain and the 2 2 2 1 structure, although this novel proposal should be further homeostasis of physiologically relevant metals by the β- challenged with empirical structural analyses. Sequence domain (Jenny et al. 2016; Cols et al. 1999; Nielson and comparisons among/between these successive β -like Winge 1984; Nielson and Winge 1983). 2 Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 9 of 18 The latest phylogenetic work has proposed that two domains. Based on this assumption, the unusual C- distinct ancestral β-domains (designated β and β terminal part of B. glabrata MT might be a reminiscent, 1 2 domains) might have existed and given rise to the struc- partial segment (designated β here) originated from 1P tural diversity of all molluskan MTs (Jenny et al. 2016). the early β -domain. Hence, the tandem duplications of In that literature, they have hypothesized separate paths β -domain accompanied with partial loss of the C- of the evolution of the two ancestral MTs in the major terminal β -domain may be a plausible mechanism to taxa within the mollusk phylum. The two β-domains ap- produce the current β β β β structure in this pulmon- 2 2 2 1P pear to have diverged into two structurally different MT ate species. We performed additional analyses on the isoform types (i.e., αβ -MT and the β β -MT) in bivalves duplicated β -domains of this large MT (first and second 1 2 2 2 whereas in gastropods, the two ancestral β-domains β - domains used for analysis) in order to hypothesize a form a single structural β β -MT isoform. With C. virgi- plausible reason responsible for the happening of this 2 1 nica model, the structural diversity of bivalve MT iso- evolutionary episode. For this, β -domains of previously forms has been highlighted to demonstrate evolutionary known MTs from pulmonate species (i.e., Cd-MT, Cu- paths from not only αβ -domain but also β β -domain MT and intermediate Cd/Cu-MT) were included in 1 2 2 (Jenny et al. 2016). On the contrary, in the gastropoda analyses together with the β -domains of this large B. lineage, the β β -domain structure has been proposed as glabrata MT. From the sequence alignment, the dupli- 2 1 a typical appearance common to most extant gastropod cated β -domains of the B. glabrata β β β β -MT 2 2 2 2 1P MTs. Instead of a series of domain duplications seen in revealed a more sequence similarity to Cd-MT than to bivalve MTs, gastropod MTs appears to have diverged to Cu-MT and Cd/Cu-MT. Phylogenetic analysis of β -do- functionally differentiated isoforms (i.e., Cd-MT, Cu-MT mains from B. glabrata paralogs also showed a close or intermediate Cd/Cu-MT) through the composition relationship between the β β β β -MT and Cd-MT 2 2 2 1P changes of non-Cys aa residues (Jenny et al. 2016; (Additional file 1: Figure S6). Although the tree topology Palacios et al. 2011; Cols et al. 1999). However, our bio- on the affiliation was not statistically supported, it could be informatic analyses in this study suggest that the current enough to hypothesize that the emergence of β β β β - 2 2 2 1P theory on MT domain evolution in the phylum Mollusca MT in B. glabrata might have been an evolutionary could be revised based on newly recognized evidences. process toward the need of more specificity for detoxifica- Novel hypothetical paths and additional insights into the tion of non-essential metals (i.e., primarily Cd). This domain evolution of molluskan MTs are proposed in hypothesis is congruent with the evolutionary theory of following sections. multi-domain MTs in bivalves. In bivalves, development of Cd-preferring MT has been proposed to be based on the Novel evidences for domain duplication in gastropod MTs conversion of a Cu-preferring β-domain to the Cd- The non-canonical domain structure of large MTs from preferring α-domain by the acquisition of additional Cys, two gastropod species (L. littorea and B. glabrata) may followed by subsequent domain duplications (Jenny et al. be considered as novel shapes. Phylogenetic analysis of 2004; Jenny et al. 2006). On the other hand, in pulmonate gastropoda MT domains (β or β ) has generated the gastropods, it has been widely proposed insofar that fur- 1 2 two major clades separated depending on the types of β- ther domain duplication from the prototypic β β -MT 2 1 domains (β or β ) (Fig. 1). The gastropod β -clade has form has unlikely happened. Instead, specific MT isoforms 1 2 2 been proven to contain all previously proposed gastro- with different metal selectiveness in pulmonate gastropods poda β -domains together with the putative β -domains have been achieved mainly through the composition 2 2 of the large MTs proposed in this study. For the large B. changes of non-Cys aa (Jenny et al. 2016; Palacios et al. glabrata MT, three β-domains (the first to third domains 2011). However, from the new evidence in this study, numbered from the N-terminal) are closely clustered domain duplication giving rise to large MTs should be con- together and placed in the major clade comprising the sidered as one of the important mechanisms permitting gastropod β -domains. This result suggests obviously pulmonate MTs to achieve more specificity for their that they are duplicated copies of β -domains that might cognate heavy metals. Taking into account that β β β β - 2 2 2 2 1P have evolved through the tandem duplication events. On MT-originated β -domains display much closer relation- the other hand, the C-terminal region containing only ship among themselves than with the Cd-MT-originated five Cys is not clustered with any typically known β or β -domain in the phylogenetic analysis, it is likely that the 1 2 β -domain sequences. Although we did not provide clear divergence to the β β β β -MT in the B. glabrata genome 2 2 2 2 1P evidence for the origin of this C-terminal region, the might have occurred through a separate path independent most likely scenario is that the originally existed β -do- of the pathway for generating the Cd-MT (Fig. 2). Hence, main at C-terminal in the ancestral β β -MT might have exposure experiments to examine metal selectiveness or 2 1 undergone certain recombination(s) including the loss of binding property of β β β β -MT domains would be 2 2 2 1P some parts during duplication events of neighboring β helpful to test this hypothesis. 2 Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 10 of 18 Fig. 1 Neighbor-joining tree showing the phylogenetic relationship among gastropoda MT β-domains (DM) analyzed with MEGA7software(ver. 7.0.21) (Kumar et al. 2016). Based on the bootstrap tests (1000 replicates), only the clades supported by higher than 50% (condensed tree cutoff value = 50%) are visualized. For MT abbreviations and species, refer to Additional file 1: Table S1. In the NJ tree, two distinct clades, respectively, comprising of β -and β - 1 2 domains indicate the presence of two ancestral β-domains in the gastropod lineage (Jenny et al. 2016). Typical arrangement patterns of Cys motifs for gastropod β -and β -domains are noted at the right side of the tree. Most gastropod MTs are structured as N-terminal β -domain linked to C-terminal 1 2 2 β -domain. However, noticeably, some gastropod MTs are proposed to possess more than two β-domains (likely caused by tandem duplication) based on newly recognized evidences from B. glabrata MT (β β β β -like shape) and L. littorea MT (β β β -structure). Evidence for the β -domain duplication 2 2 2 1P 2 2 1 2 from the sequence alignment analysis can also be referred to in Additional file 1: Figure S2 The other evidence for the β -domain duplication in successive β -domains from the N-terminal that is linked 2 2 the gastropod MTs is observable in the periwinkle (L. lit- to the C-terminal β -domain (i.e., β β β -MT). Like the 1 2 2 1 torea; Caenogastropoda; Hypsogastropoda) MT. In the B. glabrata β β β β -MT above, the first and second β 2 2 2 1P 2 molecular phylogenic tree, a subclade consisting of two domains in the L. littorea MT share high sequence simi- closely affiliated L. littorea β-domains (first and second larity including the conserved Cys motifs, suggesting domains numbered from the N-terminal) was placed that they might have evolved from a tail-to-head tandem within the gastropod β -clade, whereas the third β- duplication event (Additional file 1: Figure S2). Further domain of the L. littorea MT was positioned in the efforts to exploit potential paralog isoforms from this gastropod β -clade (Fig. 1). Based on the phylogenetic species or closely related species are needed to separation between first, second, and third β-domains hypothesize potential factor(s) to drive the domain indicates that the L. littorea MT is comprised of two duplication in L. littorea MT. Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 11 of 18 Fig. 2 (See legend on next page.) Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 12 of 18 (See figure on previous page.) Fig. 2 A schematic representation to suggest the revised hypothesis including the newly proposed evolutionary paths (thickened arrows) for the divergence of MTs in the molluskan lineage. For MT abbreviations and species, refer to Additional file 1: Table S1. Ancestral β-domain-MT has been duplicated to early β β - (eventually to αβ ), β β - and β β -structured MTs and then further diverged into various shapes of extant MTs in 1 1 1 2 2 2 1 the bivalve and gastropoda classes through domain duplication and/or aa substitutions in species/lineage-specific fashion. For MT isoforms with more than two metal-binding domains, the presumed duplicated domains are underlined with either a solid line (for homologous duplication giving rise to tandem array of domains with high sequence similarity) or dashed line (for heterologous duplication resulting in domains sharing little sequence similarity). Representative species examples for each MT type are noted below the schematic representation Postmortem studies have claimed that most gastropod MT would be responsive to not only various heavy MTs would be very conservative in the Cys arrangements metals including Cu, Cd, and Zn but also non-metal as a β β -domain structure (Jenny et al. 2016; Berger et al. stimulating stress treatments such as induced hypoxia, 2 1 1997; Dallinger et al. 1993). On the contrary, substitu- immune challenge and heat shock (Lee and Nam 2016; tions/replacements of non-Cys aa residues while retaining Guo et al. 2013). Based on this observation, the MT, at the β β -frame have been thought as the major process least in this vetigastropod species, is thought to have 2 1 for the evolutionary divergences of MTs in this primitive evolved to play readily multifunctional roles in diverse class Gastropoda. The Cu homeostatic requirements pathways involved in stress physiology. (thought to be mainly operated by β-domains) from the use of hemocyanin as a respiratory pigment in these Updates in ostreidae and mytilidae MTs gastropods, which is not present in oysters, has also been The evolutionary theory of MT based on domain dupli- proposed as one of plausible factors responsible for the cation has been the most comprehensively highlighted in lack of divergently duplicated domains in gastropod MTs the Crassostrea species, particularly in C. virginica.Two (Jenny et al. 2016; Berger et al. 1997; Perez-Rafael et al. ancestral β-domains (i.e., β and β ) appear to have di- 1 2 2012; Perez-Rafael et al. 1844). However, the present study verged to produce two different structural MT isoforms, claims that the formation of large MT with more than two i.e., αβ -MT and β β -MT in the oysters belonging to 1 2 2 domains should not be a bivalve-exclusive episode. It Ostreidae (Jenny et al. 2004; Jenny et al. 2006). The might have been an important path allowing some ancestral β -domain appears to have duplicated to pro- gastropod MTs to better modulate metal specificity in duce a two-domain-structured MT that ultimately led to response to variations occurred in their habitat environ- the evolution of the αβ -structured MTs, which is ments (Fig. 2). For both examples (B. glabrata MT and L. observable in the Crassostrea species MT-Is and MT- littorea MT), the target domain that has undergone dupli- IVs. On the other hand, the Crassostrea species MT-IIIs cation is the β -domain. Hence, novel or specified func- reveal the typical β β -domain structure, which might 2 2 tions (e.g., detoxification of non-essential metals) could be have been a descendant shape resulted from the duplica- assigned to duplicated β -domains while original and tion event of a single ancestral β -domain (Fig. 2). In the fundamental roles (e.g., Cu homeostatic regulation) are reconstructed phylogenetic tree, Ostreidae β-domain retained in the β -domain. Extra metal-binding residues sequences are placed on one of two main clades (i.e., offered by duplicated β -domains may also be potentially either β -or β -domain clade), although several sub- 1 2 advantageous to strengthen further capacity of both metal clades within the β -clade are not supported by high reservation and resistance to excessive metal ions. Taken confidence values (Additional file 1: Figure S7). Crassos- together, hypothesis for evolutionary mechanism to mold trea MT-IVs are closely related each other and distin- gastropod MTs should be revised taking into account the guished from MT-I/-II isoforms within the β -clade, inclusion of this novel path featured by the duplication of suggesting the early divergence between MT-IV and β -domain. MT-I/-II families from the prototypic αβ -MT form. On the other hand, unlike in Heterobranchia and Also within the β -clade, it is notable that the C. virgi- Caenogastropoda, the clear sign for taxa-specific domain nica MT isoforms have a tendency to be separately multiplication has not been yet identified in Vetigastro- clustered from MTs from other Crassostrea species poda species (abalone and limpet) (Lee and Nam 2016; (such as C. gigas, C. ariakensis, C. rivularis, and C. angu- Lieb 2003). Although the characterization of MT in this lata) as seen in both MT-I and MT-IV groups. It may taxonomic group has been very limited, vetigastropod suggest the divergence of these MT families during spe- species have reportedly shown only a single MT isoform ciation in the genus Crassostrea to make C. virginica to (i.e., β β -MT) within a given species (Fig. 2). Currently, be distinguished from other Crassostrea species (Jenny 2 1 it is unclear if vetigastropods possess functionally or et al. 2016) (Additional file 1: Figure S7 and Additional structurally diverged paralogs. However, a recent study file 1: Figure S8). The most apparent difference between has reported that the abalone (Haliotis discus hannai) C. virginica and other Crassostrea species is found in the Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 13 of 18 MT-II family. C. virginica MT-IIs have been reported to essential metals than MT10 (Leung et al. 2014; Lemoine et the most recently evolved isoforms, which have formed al. 2000; Vergani et al. 2007). Conversely, in some environ- through the loss of β-domain, giving rise to the sole α- mental situations, the early αβ -MT might have diverged domain structured MTs (Additional file 1: Figure S8). into functionally differentiated isoforms in the Mytilidae: However, this divergence pattern in C. virginica is clearly MT10 to execute primarily the homeostatic regulation of contrasted by the duplication of β -domain in C. gigas physiologically relevant metals and MT20 to function in MT-IIs (i.e., αβ β -structured MT), suggesting the differ- the detoxification of trace metals (Fig. 2). 1 1 ent evolutionary paths of the MT-II between the two closely related species belonging to the same genus Novel paths for domain duplication in bivalve MTs (Jenny et al. 2004; Tanguy and Moraga 2001). Such a Exploitation of genetic determinants for MTs from other solely α-domain-based structure has been found only in bivalve taxa has often showed the species (or lineage)- C. virginica insofar, whereas αβ β -domain structure has specific variations in MT structure. However, currently 1 1 also been observed similarly in other Crassostrea species limited volume of knowledge on these MTs still hurdles and non-Crassostrea species (Additional file 1: Figure S7). to hypothesize the evolutionary mechanism of non- Meanwhile, the β -domain clade comprising of C. virgi- canonical MT forms in detail. Reconstruction of molecu- nica and C. gigas MT-III isoforms shows a monophyletic lar phylogenetic trees in this study displays two main topology, indicating the divergence from a common an- clades: one is a large clade comprising of β -domains cestral β β -MT origin. However, within the β -clade, two from various taxa and the other is a small clade consist- 2 2 2 subclades are characterized by the first and second β -do- ing of five presumed β -domain sequences deciphered 2 2 mains rather than by species. All the N-terminally present from two A. irradians (Pectinidae) paralogue MTs β -domains are clustered together in a subclade while all (Fig. 3). Within a former β -clade, paralogue isoforms 2 1 the C-terminally present β -domains in other subclade from a given single species (e.g., R. philippinarum MT1/ (see also (Jenny et al. 2016)). This finding may be indica- MT2 and L. elliptica MT10a/10b) formed subclades tive of that the first and second β -domains of the Crassos- supported by high bootstrap values. Similarly, several trea MT-IIIs might appear to have originated before the subclades consisted of orthologs from closely related separation of the two oyster species (Fig. 2). species belonging to the same genus ([e.g., MTs from Mytilidae species represent uniformly the αβ -domain genus Meretrix (Chang et al. 2007; Wang et al. 2010; structure (known as the prototypic shape of bivalve MT). Jiang et al. 2016) and genus Cerastoderma (Desclaux- Some mytilid species have been reported to possess the Marchand et al. 2007; Ladhar-Chaabouni et al. 2009; intronless, relatively short MTs in their genomes. The pres- Paul-Pont et al. 2012)). Collectively, it suggests that they ence of intronless MT genes has been proposed as the might have evolved from recent divergence at species or organisms’ strategic means for the efficient response to genus levels. In contrast, some paralogue MT isoforms changes of cellular metal circumstances through the rapid are distantly placed in the phylogenetic tree, although transcription of MT genes (Leignel et al. 2005). Molecular they are placed in the same β -clade. Such a distant rela- phylogenetic analysis of Mytilidae MT domains generated tionship is found in the genus Hyriopsis where MT1 and trees consistently comprising of three main clades; clades MT2 paralogs are not affiliated depending upon species. for mytilid MT10s, P. viridis MTs and mytilid MT20s Although nomenclatures MT1 and MT2 are not estab- (Additional file 1: Figure S9). In congruent with the previ- lished clearly in this two species, an isoform of H. ous phylogenetic results using the entire MT polypeptide cumingii MT (GQ184290) is closely related with H. region, present phylogenetic analyses using separate do- schlegelii MT1 ortholog (KJ019820), rather than its para- mains also suggest the early divergence of P. viridis MTs logue isoform (FJ861993). This finding may indicate that from the other mytilid orthologs (Leung et al. 2014). In the the divergence between MT1 and MT2 might have Mytilidae lineage, a critical event before speciation is the occurred earlier than the speciation of the two Hyriopsis divergence of MT10 and MT20 forms (Aceto et al. 2011) species (Yang et al. 2014; Wang et al. 2016). (Fig. 2). As compared to MT10, MT20s are characterized From the present molecular phylogenetic analysis, novel by the acquisition of additional Cys residues (i.e., a Cys-Cys paths of MT evolution through domain duplication giving doublet) in their α-domains (Leignel and Laulier 2006). It rise to large-sized MTs with more than two metal-binding could be thought as a process to prepare the paralogue domains could be proposed (Fig. 3). Evidences come from MT varieties with better execution in the detoxification of two bivalve species: one is P. coreanum (Sphaeriidae) non-essential metals, since more Cys residues are generally (Baek et al. 2009) and the other is A. irradians (Pectinidae) taken into account for enhanced capability for sequestrat- (Liu et al. 2006; Wang et al. 2009). In the P. coreanum,the ing the toxic metals (i.e., metal tolerance). This hypothesis two putative β-domains are placed in the β -clade. Within could be supported by the fact that MT20 would be more the β -clade, the second (numbered from the N-terminal) preferentially associated or exclusive reacted with non- β -domain of P. coreanum MT is found to form a 1 Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 14 of 18 Fig. 3 Molecular phylogenetic tree (neighbor-joining tree drawn with MEGA7 (Kumar et al. 2016)) to propose the duplication of β - (for P. coreanum MT) and β -domain (A. irradians MT1 and MT2), giving rise to MTs with more than two metal-binding domains. Clades are visualized with the condensed tree cutoff value of 50% based on 1000 replicate bootstrap tests. For MT abbreviations and species, refer to Additional file 1: Table S1. Detailed domain structures of these multi-domain MTs are provided in Additional file 1: Figure S5B subclade with two β -domains from Arcidae species Ostreidae and Sphaeriidae (Fig. 2). In Ostreidae, the β - 1 1 [Scapharca broughtonii (FJ154101) and Tegillarca granosa domain seems to have evolved from a relatively recent (AY568678)]. Considering the N-terminally present puta- gene duplication, resulting in a tandem array of the two tive α-domain, this P. coreanum MT could be designated homologous β -domains. On the contrary, the newly αβ β -structure. In the phylum Mollusca, the αβ β -do- proposed P. coreanum αβ β -MT shows no apparent se- 1 1 1 1 1 1 main structure (i.e., duplication of β -domain from the quence homology between the two β -domains. Possibly, 1 1 anticipated prototypic αβ -MT) has been previously the multiplication of β -domain in Sphaeriidae might have 1 1 reported to be the Crassostrea-specific event (Tanguy and been an earlier divergent event. Currently, the evolution- Moraga 2001). However, we have already proposed above ary route for the acquisition of additional β -domain in P. that this event has also been true for non-Crassostrea oys- coreanum MT is open to hypothesize. One possible sce- ter (i.e., A. plicatula; Ostreidae). Further, the present P. nario is the duplication of β -domain from the prototypic coreanum (Sphaeriidae) MT could indicate that this dupli- αβ -MT, followed by further divergence in non-Cys resi- cation process would not be limited to the Ostreidae. dues. Alternatively, the other possibility is that the more However, the evolutionary scheme for the domain dupli- ancestral β β -MT (not yet reported in extant bivalve 1 1 cation may be different between the two families MTs) might have acquired additional α-domain through Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 15 of 18 the duplication to β β β -MT followed by the conversion event. Although need to be further challenged, the ances- 1 1 1 of one of β -domains to α-domain (Fig. 2). Molecular tral β β -MT in the phylum Mollusca perhaps might have 1 2 1 phylogeny of bivalve α-domains also shows that P. corea- gone through two separate paths (Fig. 2). Namely, one num α-domain is independently placed without being might have been a preservative path from the ancestral affiliated with any other bivalve α-domain ortholog in a β β -MT to produce the conserved β β -structured MTs 2 1 2 1 subclade (Additional file 1: Figure S8). Hence, further which seen in most extant gastropod MTs, although some efforts to exploit paralogue isoforms from this species (P. exceptional gastropod species represent additional coreanum) and/or similarly structured orthologs from its lineage-specific, recent duplication(s) of homologous β - closely related species should be needed to get a deeper domain. On the other hand, the other path might involve insight into the mechanism responsible for the emergence earlier duplication of β -domain from the ancestral β β - 2 2 1 of αβ β -MT in Sphaeriidae. MT, giving rise to multiple β -like domains linked to the 1 1 2 From the same molecular phylogenic tree, putative C-terminal β -domain in certain bivalve taxa. Based on domains from the two A. irradians MT isoforms (Liu et this, the extant shapes of A. irradians MT paralogs may al. 2006; Wang et al. 2009) are positioned in either the reflect the consequences of differential rounds of β -do- main β -clade or a small clade consisting of only A. irra- main duplication from an ancestral β β -MT. However, 1 2 1 dians MT domains (Fig. 3). Two C-terminal domains re- further validation is needed to test whether this divergent spectively from A. irradians MT1 and MT2 are placed process might have occurred before or after speciation in the main β -clade. On the other hand, the small clade events in the Pectinoida lineage. are exclusively comprised of three putative β -like do- What are the functional or physiological implications mains from the MT1 (first to third domains predicted of domain duplications (or multiplication) in molluskan from the N-terminal side) and two from MT2 (first and MTs? The evolution of larger MT proteins has been second domains). A. irradians MTs represent non- proposed as a strategic means that might likely be canonical shape that does not perfectly match the advantageous for benthic organisms that are believed to known typical β-domain structure. Nevertheless, if the experience a greater exposure to metals due to their eco- Cys distribution pattern is fundamentally considered, the logical niche (Jenny et al. 2004; Jenny et al. 2006; Tanguy multiplied domains in A. irradians MTs could be classified and Moraga 2001; Tschuschke et al. 2002). Although the as β -like structure (possibly designated β ′-domain). paucity or limitation of the functional studies on such 2 2 Hence, the overall domain structures of A. irradians MT1 large MTs hurdles to hypothesize comprehensively the and MT2 could be designated β ′β ′β ′β -MT and relevant mechanism(s) in detail, there have been some 2 2 2 1 β ′β ′β -MT, respectively. In bivalve class, the MT pro- hypothetical evidences or suggestive assumptions. First, 2 2 1 teins possessing multiple β-domains without α-domain based on the heterologous expression assay by using the have been reported only in the Crassostrea β β -MT-IIIs, recombinant microbial systems, a couple of noteworthy 2 2 and this structure has been considered to have developed experiments have shown that large-sized multi-domain- from the early duplication of the ancestral β -MT. How- structured MT proteins would be able to confer greater ever, A. irradians MTs represent a C-terminal β -domain Cd resistance of the hosts (Tanguy et al. 2001; as well as the multiple β ′-domains (Fig. 2). Two plausible, Tschuschke et al. 2002). Second, several independent but untested, hypotheses may be possible regarding the previous studies have claimed that amplification and/or evolution of such unusual domain structure in A. irra- tandem duplications of MT genes might have been an dians MTs. One is the acquisition of C-terminal β -do- advantageous process to attain the strengthened ability main in the ancestrally duplicated β β -MT (i.e., giving of metal tolerance (Cho et al. 2009; Beach and Palmiter 2 2 rise to β β β -MT) followed by further divergent process 1981; Maroni et al. 1987; Mehra et al. 1990; Stephan et 2 2 1 resulting in current shapes of A. irradians MT isoforms. al. 1994). Because the number of Cys residues in MT The other, more plausible, hypothetical path is the multi- proteins has been taken into account as a fundamental plication of β -domain from the prototypic β β -MT factor to determine the number of metal ions bound or 2 2 1 structure that is seen in most gastropod MTs (Jenny et al. reserved by the MTs (Amiard et al. 2006), more Cys 2016; Palacios et al. 2011). As described above, the present attained by domain duplications might be beneficial in study has already noted that the multiplication of β -do- the conference of metal resistance in a broad sense. main (i.e., β β -structure) might have been one of diver- Third, domain multiplication(s) accompanied with signifi- 2n 1 gence mechanisms in certain gastropod taxa (Fig. 2). cant substitutions/replacements of non-Cys residues could However, unlike the tandem duplication of homologous offer a chance to confer some novel functions on large β -domain in gastropod MTs, A. irradians MTs represent MTs. Because aa replacements on non-Cys residues in little sequence similarity among β ′-domains, suggesting MT have been known to represent significant effects on that the multiplication of these β ′-domains in each A. metal-binding specificity and kinetic reactivity (Palacios irradians MT isoform may not be the recent duplication et al. 2011; Pedersen et al. 1994; Kurasaki et al. 1997; Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 16 of 18 Munoz et al. 2000), such a divergent pattern (domain Received: 23 January 2017 Accepted: 25 May 2017 duplication together with significant aa substitutions) might also increase the kinds of metals reacted by these References MT proteins. Taken together, the ancestral or prototypic Aceto S, Formisano G, Carella F, Vico GD, Gaudio L. The metallothionein genes of MT protein has diverged into various isoforms with a Mytilus galloprovincialis: genomic organization, tissue expression and great structural diversity in the phylum Mollusca. Struc- evolution. Mar Genom. 2011;4:61–8. Amiard JC, Amiard-Triquet C, Barka S, Pellerin J, Rainbowd PS. Metallothioneins in tural diversifications driven by both domain duplication aquatic invertebrates: their role in metal detoxification and their use as and aa replacements might have led certain subfunctiona- biomarkers. Aquat Toxicol. 2006;76:160–202. lization and/or neofunctionalization of MT proteins in an Baek MK, Lee JS, Kang SW, Lee JB, Kang HJ, et al. Phylogenetic analysis based on metallothionein gene sequence of an indigenous species Pisidium isoform-dependent fashion (Tanguy and Moraga 2001). (Neopisidium) coreanum in Korea. Kor J Malacol. 2009;25:153–60. Baršytė D, White KN, Lovejoy DA. Cloning and characterization of metallothionein cDNAs in the mussel Mytilus edulis L. digestive gland. Comp Biochem Physiol Conclusions C Pharmacol Toxicol Endocrinol. 1999;122:287–96. Phylum Mollusca represents a great structural diversity Beach LR, Palmiter RD. Amplification of the metallothionein-I gene in cadmium- of MT, a core suite playing key roles in both homeostatic resistant mouse cells. Proc Natl Acad Sci. 1981;78:2110–4. Berger B, Dallinger R, Gehrig P, Hunziker PE. Primary structure of a copper- regulation of essential metals and detoxification of trace binding metallothionein from mantle tissue of the terrestrial gastropod Helix metals in living organisms. The structural diversity of pomatia L. Biochem J. 1997;328:219–24. molluskan MTs have been achieved essentially through Binz PA, Kagi JHR. Metallothionein: molecular evolution and classification. In: Klaassen C, editor. Metallothionein. Basel: Birkhäuser; 1999. p. 7–13. the domain duplication events from an ancestral, singu- Blindauer CA, Leszczyszyn OI. Metallothioneins: unparalleled diversity in structures lar domain-MT. Domain duplication have been followed and functions for metal ion homeostasis and more. Nat Prod Rep. 2010;27: by further diversification and selection toward needs for 720–41. Braun W, Wagner G, Worgotter E, Vasak M, Kagi JH, Wuthrich K. Polypeptide fold acquiring metal selectiveness, specialized novel function, in the two metal clusters of metallothionein- 2 by nuclear magnetic and improved capacity of metal homeostasis/detoxifica- resonance in solution. J Mol Biol. 1986;187:125–9. tion. With this viewpoint, novel paths for domain diver- Capasso C, Carginale V, Scudiero R, Crescenzi O, Spadaccini R, Temussi PA, Parisi E. Phylogenetic divergence of fish and mammalian metallothionein: gences of some gastropod and bivalve MT families relationships with structural diversification and organismal temperature. J proposed in this review could shed new light onto the Mol Evol. 2003;57:S250–7. revision and update of the hypothesis for evolutionary Carpenè E, Andreani G, Isani G. Metallothionein functions and structural characteristics. J Trace Elem Med Biol. 2007;21:35–9. differentiation of MTs in the molluskan lineage. Chang YT, Jong KJ, Liao BK, Wu SM. Cloning and expression of metallothionein cDNA in the hard clam (Meretrix lusoria) upon cadmium exposure. Additional file Aquaculture. 2007;262:504–13. Chiaverini N, Ley MD. Protective effect of metallothionein on oxidative stress- induced DNA damage. Free Radic Res. 2010;44:605–13. Additional file 1: Supplementary figures and tables. (PDF 433 kb) Cho YS, Choi BN, Ha EM, Kim KH, Kim SK, Kim DS, Nam YK. Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and Acknowledgements expression analysis. Mar Biotechnol. 2005;7:350–62. Not applicable. Cho YS, Lee SY, Kim K-Y, Bang IC, Kim DS, Nam YK. Gene structure and expression of metallothionein during metal exposures in Hemibarbus Funding mylodon. Ecotoxicol Environ Saf. 2008;71:125–37. This study was supported by the grant from the Golden Seed Project (GSP), Cho YS, Lee SY, Kim KY, Nam YK. Two metallothionein genes from mud loach Ministry of Oceans and Fisheries, Republic of Korea. Misgurnus mizolepis (Teleostei: Cypriniformes): gene structure, genomic organization, and mRNA expression analysis. Comp Biochem Physiol B Availability of data and materials Biochem Mol Biol. 2009;153:317–26. Not applicable. Cols N, Romero-Isart N, Bofill R, Capdevila M, Gonzalez-Duarte P, Gonzalez-Duarte R, Atrian S. In vivo copper- and cadmium-binding ability of mammalian metallothionein beta domain. Protein Eng. 1999;12:265–9. Authors’ contributions Cong M, Wu H, Liu X, Zhao J, Wang X, Lv J, Hou L. Effects of heavy metals on the YKN designed this study, carried out bioinformatic analysis and drafted the expression of a zinc-inducible metallothionein-III gene and antioxidant manuscript. EJK carried out the management of sequence data, reference enzyme activities in Crassostrea gigas. Ecotoxicology. 2012;21:1928–36. analyses, and polishing of the manuscript. Both authors read and approved Dallinger R, Berger B, Hunziker PE, Birchler N, Hauer CR, Kägi JHR. Purification and the final manuscript. primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A. Eur J Biochem. 1993;216:739–46. Competing interests Desclaux-Marchand C, Paul-Pont I, Gonzaleza P, Baudrimont M, Montaudouin X. The authors declare that they have no competing interests. Metallothionein gene identification and expression in the cockle (Cerastoderma edule) under parasitism (trematodes) and cadmium Consent for publication contaminations. Aquat Living Resour. 2007;20:43–9. Not applicable. Dondero F, Piacentini L, Banni M, Rebelo M, Burlando B, Viarengo A. Quantitative PCR analysis of two molluscan metallothionein genes unveils differential Ethics approval and consent to participate expression and regulation. Gene. 2005;345:259–70. Not applicable. English TE, Storey KB. Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine Publisher’sNote gastropod Littorina littorea. J Exp Biol. 2003;206:2517–24. Springer Nature remains neutral with regard to jurisdictional claims in Faller P. Neuronal growth-inhibitory factor (metallothionein-3): reactivity and published maps and institutional affiliations. structure of metal–thiolate clusters. FEBS J. 2010;277:2921–30. Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 17 of 18 Fang Y, Yang H, Wang T, Liu B, Zhao H, Chen M. Metallothionein and superoxide Lü D, Luo KY, Pan BP, Gao H. Expression of metallothionein and thioredoxin gene dismutase responses to sublethal cadmium exposure in the clam Mactra in Cyclina sinensis exposed to cadmium. Oceanol Limnol Sin. 2012;43:47–51. veneriformis. Comp Biochem Physiol C Toxicol Pharmacol. 2010;151:325–33. Lynes MA, Hidalgo J, Manso Y, Devisscher L, Laukens D, Lawrence DA. Fang Y, Yang H, Liu B, Zhang L. Transcriptional response of lysozyme, Metallothionein and stress combine to affect multiple organ systems. Cell metallothionein, and superoxide dismutase to combined exposure to heavy Stress Chaper. 2014;19:605–11. metals and bacteria in Mactra veneriformis. Comp Biochem Physiol C Toxicol Mao H, Wang DH, Yang WX. The involvement of metallothionein in the Pharmacol. 2013;157:54–62. development of aquatic invertebrate. Aquat Toxicol. 2012;110–111:208–13. Geffard A, Geffard O, Amiard JC, His E, Amiard-Triquet C. Bioaccumulation of Marie V, Gonzalez P, Baudrimont M, Boutet I, Moraga D, Bourdineaud JP, Boudou metals in sediment elutriates and their effects on growth, condition index, A. Metallothionein gene expression and protein levels in triploid and diploid and metallothionein contents in oyster larvae. Arch Environ Contam Toxicol. oysters Crassostrea gigas after exposure to cadmium and zinc. Environ 2007;53:57–65. Toxicol Chem. 2006;25:412–8. Guo F, Tu R, Wang W-X. Different responses of abalone Haliotis discus hannai to Maroni G, Wise J, Young JE, Otto E. Metallothionein gene duplications and metal waterborne and dietary-borne copper and zinc exposure. Ecotoxicol Environ tolerance in natural populations of Drosophila melanogaster. Genetics. 1987; Saf. 2013;91:10–7. 117:739–44. Gupta SK, Singh J. Evaluation of mollusc as sensitive indicator of heavy metal Mehra RK, Garey JR, Winge DR. Selective and tandem amplification of a member pollution in aquatic system: a review. IIOAB. 2011;2:49–57. of the metallothionein gene family in Candida glabrata. J Biol Chem. 1990; Inoue K-I, Takano H, Shimada A, Satoh M. Metallothionein as an anti- 265:6369–75. inflammatory mediator. Mediat Inflamm. 2009;101659. Munoz A, Petering DH, Shaw CF. 3rd. Structure-reactivity among metallothionein Isani G, Carpenè E. Metallothioneins, unconventional proteins from three-metal domains: role of noncysteine amino acid residues in lobster unconventional animals: a long journey from nematodes to mammals. metallothionein and human metallothionein-3. Inorg Chem. 2000;39:6114–23. Biogeosciences. 2014;4:435–57. Nielson KB, Winge DR. Order of metal binding in metallothionein. J Biol Chem. Jenny MJ, Ringwood AH, Schey K, Warr GW, Chapman RW. Diversity of 1983;258:13063–9. metallothioneins in the American oyster, Crassostrea virginica, revealed by Nielson KB, Winge DR. Preferential binding of copper to the beta domain of transcriptomic and proteomic approaches. Eur J Biochem. 2004;271:1702–12. metallothionein. J Biol Chem. 1984;259:4941–6. Jenny MJ, Warr GW, Ringwood AH, Baltzegar DA, Chapman RW. Regulation of Palacios O, Pagani A, Perez-Rafael S, Egg M, Hockner M, Brandstatter A, Capdevila metallothionein genes in the American oyster (Crassostrea virginica): ontogeny and M, Atrian S, Dallinger R. Shaping mechanisms of metal specificity in a family differential expression in response to different stressors. Gene. 2006;379:156–65. of metazoan metallothioneins: evolutionary differentiation of mollusc Jenny MJ, Payton SL, Baltzegar DA, Lozier JD. Phylogenetic analysis of molluscan metallothioneins. BMC Biol. 2011;9:4. metallothioneins: evolutionary insight from Crassostrea virginica. J Mol Evol. Paul-Pont I, Gonzalez P, Montero N, Montaudouin X, Baudrimont M. Cloning, 2016;83:110–25. characterization and gene expression of a metallothionein isoform in the Jiang LJ, Maret W, Vallee BL. The glutathione redox couple modulates zinc edible cockle Cerastoderma edule after cadmium or mercury exposure. transfer from metallothionein to zinc-depleted sorbitol dehydrogenase. Proc Ecotoxicol Environ Saf. 2012;75:119–26. Natl Acad Sci. 1998;95:3483–8. Pedersen KL, Pedersen SN, Højrup P, Andersen JS, Roepstorff P, Knudsen J, Depledge Jiang LJ, Vasak M, Vallee BL, Maret W. Zinc transfer potentials of the alpha- and MH. Purification and characterization of a cadmium-induced metallothionein from beta-clusters of metallothionein are affected by domain interactions in the the shore crab Carcinus maenas (L.). Biochem J. 1994;297:609–14. whole molecule. Proc Natl Acad Sci. 2000;97:2503–8. Perez-Rafael S, Monteiro F, Dallinger R, Atrian S, Palacios O, Capdevila M. Jiang GP, Cheng XY, Teng SS, Chai XL, Lin XG, Liu GX, Xiao GQ. Cloning and Cantareus aspersus metallothionein metal binding abilities: the unspecific expression of metallothionein gene in Meretrix lamarckii. Acta Hydrobiol Sin. CaCd/CuMT isoform provides hints about the metal preference determinants 2016;40:914–20. in metallothioneins. Biochim Biophys Acta. 1844;2014:1694–707. Khoo HW, Patel KH. Metallothionein cDNA, promoter, and genomic sequences of Perez-Rafael S, Mezger A, Lieb B, Dallinger R, Capdevila M, Palacios O, Atrian S. the tropical green mussel. Perna viridis J Exp Zool. 1999;284:445–53. The metal binding abilities of Megathura crenulata metallothionein (McMT) in Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis the frame of Gastropoda MTs. J Inorg Biochem. 2012;108:84–90. version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4. Raspor B, Dragun Z, Erk M, Ivankovic D, Pavicic J. Is the digestive gland of Mytilus Kurasaki M, Yamaguchi R, Linde Arias R, Okabe M, Kojima Y. Significance of alpha- galloprovincialis a tissue of choice for estimating cadmium exposure by fragments of metallothionein in cadmium binding. Prot Eng. 1997;10:413–6. means of metallothioneins? Sci Total Environ. 2004;333:99–108. Ladhar-Chaabouni L, Mokdad-Gargouri R, Denis F, Hamza-Chaffai A. Cloning and Sarkar A, Ray D, Shrivastava AN, Sarker S. Molecular biomarkers: their significance characterization of cDNA probes for the analysis of metallothionein gene and application in marine pollution monitoring. Ecotoxicology. 2006;15:333–40. expression in the Mediterranean bivalves: Ruditapes decussatus and Serén N, Glaberman S, Carretero MA, Chiari Y. Molecular evolution and functional Cerastoderma glaucum. Mol Biol Rep. 2009;36:1007–14. divergence of the metallothionein gene family in vertebrates. J Mol Evol. Le TTY, Zimmermann S, Sures B. How does the metallothionein induction in 2014;78:217–33. bivalves meet the criteria for biomarkers of metal exposure? Environ Pollut. Stephan W, Rodriguez VS, Zhou B, Parsch J. Molecular evolution of the 2016;212:257–68. metallothionein gene Mtn in the melanogaster species group: results from Lee SY, Nam YK. Transcriptional responses of metallothionein gene to different Drosophila ananassae. Genetics. 1994;138:135–43. stress factors in Pacific abalone (Haliotis discus hannai). Fish Shellfish Tang RS, Xia JH, Wang YM, Gong SY, Yu DH. Analysis on cloning and sequence Immunol. 2016;58:530–41. characteristics of metallothionein cDNA in Pinctada maxima. J Anhui Agricult Leignel V, Laulier M. Isolation and characterization of Mytilus edulis metallothionein Sci. 2009;37:2888–90. genes. Comp Biochem Physiol C Toxicol Pharmacol. 2006;142:12–8. Tanguy A, Moraga D. Cloning and characterization of a gene coding for a novel Leignel V, Hardivillier Y, Laulier M. Small metallothionein MT-10 genes in coastal metallothionein in the Pacific oyster Crassostrea gigas (CgMT2): a case of and hydrothermal mussels. Mar Biotechnol. 2005;7:236–44. adaptive response to metal-induced stress? Gene. 2001;273:123–30. Lemoine S, Bigot Y, Sellos D, Cosson RP, Laulier M. Metallothionein isoforms in Tanguy A, Mura C, Moraga D. Cloning of a metallothionein gene and Mytilus edulis (Mollusca, Bivalvia): complementary DNA characterization and characterization of two other cDNA sequences in the Pacific oyster quantification of expression in different organs after exposure to cadmium, Crassostrea gigas (CgMT1). Aquat Toxicol. 2001;55:35–47. zinc, and copper. Mar Biotechnol. 2000;2:195–203. Tanguy A, Boutet I, Riso R, Boudry P, Auffret M, Moraga D. Metallothionein genes Leung PTY, Park TJ, Wang Y, Che CM, Leung KMY. Isoform-specific responses of in the European flat oyster Ostrea edulis: a potential ecological tool for metallothioneins in a marine pollution biomonitor, the green-lipped mussel environmental monitoring? Mar Ecol Prog Ser. 2003;257:87–97. Perna viridis, towards different stress stimulations. Proteomics. 2014;14:1796–807. Tschuschke S, Schmitt-Wrede HP, Greven H, Wunderlich F. Cadmium resistance Lieb B. A new metallothionein gene from the giant keyhole limpet Megathura conferred to yeast by a non-metallothionein-encoding gene of the crenulata. Comp Biochem Physiol C Toxicol Pharmacol. 2003;134:131–7. earthworm Enchytraeus. J Biol Chem. 2002;277:5120–5. Liu WQ, Ni DJ, Song LS, Wu LT, Xu W, Kong XY. Cloning and characterization of a Vergani L, Grattarola M, Borghi C, Dondero F, Viarengo A. Fish and molluscan metallothionein gene in bay scallop Argopecten irradians. Oceanol Limnol metallothioneins: a structural and functional comparison. FEBS J. 2005;272: Sin. 2006;37:444–9. 6014–23. Nam and Kim Fisheries and Aquatic Sciences (2017) 20:8 Page 18 of 18 Vergani L, Grattarola M, Grasselli E, Dondero F, Viarengo A. Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis. Arch Biochem Biophys. 2007;465:247–53. Wang H, Zhang Q, Cai B, Li H, Sze KH, Huang ZX, Wu HM, Sun H. Solution structure and dynamics of human metallothionein-3 (MT-3). FEBS Lett. 2006; 580:795–800. Wang L, Song L, Ni D, Zhang H, Liu W. Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge. Comp Biochem Physiol C Toxicol Pharmacol. 2009;149:50–7. Wang Q, Wang X, Wang X, Yang H, Liu B. Analysis of metallothionein expression and antioxidant enzyme activities in Meretrix meretrix larvae under sublethal cadmium exposure. Aquat Toxicol. 2010;100:321–8. Wang W-C, Mao H, Ma D-D, Yang W-X. Characteristics, functions and applications of metallothionein in aquatic vertebrates. Front Mar Sci. 2014;1:34. Wang C, Sheng J, Hong Y, Peng K, Wang J, Wu D, Shi J, Hu B. Molecular characterization and expression of metallothionein from freshwater pearl mussel. Hyriopsis schlegelii Biosci Biotechnol Biochem. 2016;80:1327–35. Xiong Y, Chen Y, Ru B. The expressed alpha domain of mouse metallothionein-I from Escherichia coli displays independent structure and function. Biochem Mol Biol Int. 1998;46:307–19. Yang S, Wei M, Yang X, Wang H, He L, Li C. A novel metallothionein gene from mussel, Hyriopsis cumingii: Identification and expression under lanthanum exposure. J World Aquacult Soc. 2014;45:454–60. Zhang G, Fang X, Guo X, Li L, Luo R, Xu F, Yang P, Zhang L, Wang X, Qi H, et al. The oyster genome reveals stress adaptation and complexity of shell formation. Nature. 2012;490:49–54. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries � Our selector tool helps you to find the most relevant journal � We provide round the clock customer support � Convenient online submission � Thorough peer review � Inclusion in PubMed and all major indexing services � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit

Journal

Fisheries and Aquatic SciencesSpringer Journals

Published: Jun 13, 2017

References