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Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis... Multiple sclerosis (MS) is associated with breakdown of the myelin sheath that coats neurons in the central nervous system. The cause of MS is not known, although the pathogenesis involves destruction of myelin by the immune system. It was the aim of this study to examine the abundant myelin protein, myelin basic protein (MBP), to determine if there are sites of modification that may be characteristic for MS. MBP from the cerebellum was examined from controls and MS patients across the age range using mass spectrometry and amino acid analysis. Amino acid racemization data indicated that myelin basic protein is long-lived and proteomic analysis of MBP showed it to be highly modified. A common modification of MBP was racemization of Asp and this was significantly greater in MS patients. In long-lived proteins, L-Asp and L-Asn can racemize to three other isomers, D-isoAsp, L-isoAsp and D-Asp and this is significant because isoAsp formation in peptides renders them immunogenic. Proteomic analysis revealed widespread modifications of MBP with two surface regions that are altered in MS. In particular, isoAsp was significantly elevated at these sites in MS patients. The generation of isoAsp could be responsible for eliciting an immune response to modified MBP and therefore be implicated in the etiology of MS. Introduction age-related post-translational modifications (PTMs) were MS has long been thought to be an autoimmune disease. localized to unstructured regions [19] and racemization, Injection of brain homogenates into animals results in which involves conversion of L- to D-amino acid re- demyelination resembling that seen in MS [33] and a sidues, was found to be the most abundant type of body of data, including the presence of antibodies to PTM [39]. D-amino acids at some sites were present myelin components in MS patients, supports a role at levels that exceeded the amount of the precursor for an autoimmune response in the genesis of human L-amino acid [21]. MS [32, 42]. In humans, major proteins from cataract lenses are MBP accounts for 35 % of myelin protein and is characterized by specific racemization sites that differ intrinsically unstructured [16] Recently, MBP and other from those in comparable age-matched normal lenses proteins in myelin were shown to be long-lived [35]. [18, 20, 21]. This finding implies that pathways of Over time, long-lived proteins degrade and this time- protein degradation in the body may possibly determine dependent deterioration has been well studied in the disease outcome. In long-lived proteins, Asp and Asn lens, a tissue with no protein turnover. In lens proteins, residues are particularly sensitive to racemization, undergoing a spontaneous cyclisation reaction that leads to the formation of L-Asp, D-Asp, L-isoAsp and D- * Correspondence: rjwt@uow.edu.au isoAsp (see Fig. 2). isoAsp residues are typically present Illawarra Health and Medical Research Institute, University of Wollongong, in the highest amounts [13]. Wollongong, NSW 2522, Australia Full list of author information is available at the end of the article © 2016 The Author(s). 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. Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 2 of 12 Modifications on this scale inevitably have conse- by SDS-PAGE (Additional file 1: Figure S2). Since quences for protein structure and can lead to a func- PTMs, such as those associated with aging, can cause tional decline of the protein [34]. Recently it has been major alterations to the properties of proteins, MBP shown that the formation of isoAsp can also lead to was not separated into isoforms (charge isomers) prior altered immunogenicity of peptides/proteins, inducing to proteomic analyses [22]. both T and B cell immunity [11]. Thus, like citrulline formation from Arg residues, conversion of Asp/Asn to Amino acid analysis isoAsp, can potentially induce an immune response to D-amino acid content of MBP samples was determined self-antigens [10]. In the current study we examined the by amino acid analysis as described [18]. Three separate hypothesis that since MBP is long-lived and unstruc- runs were carried out for each sample on an Agilent tured it, like lens proteins, may also undergo significant 1100 HPLC system. covalent alterations. For the reasons outlined above, particular reference was paid to Asp/Asn racemizaton to Capillary LC mass spectrometry isoAsp. If specific PTMs such as these were found in the Capillary LC mass spectrometry was undertaken as MBP from MS patients, then they could play a role in described [21]. Briefly, MBP (~50 μg) was digested with the etiology of MS by provoking an immune response to sequence grade trypsin (1 μg) (Promega) for 16 h at 37 ° the selectively modified myelin. C. Peptides were desalted and concentrated using a Ziptip (0.6 μL, C18 resin; Millipore) and freeze dried. Materials and methods The lyophilised peptides were re-suspended in formic Tissue samples acid:heptafluorobutyric acid:water (0.1:0.05:98.85). Tan- Cerebellum samples from control (n = 21) and MS pa- dem mass spectra were acquired after LC using a Thermo tients (n = 8) were obtained from the New South Wales LTQ Orbitrap as described [21]. Tissue Resource Centre at the University of Sydney with Data were searched against the Swiss-Prot database approval from the University of Wollongong ethics com- with a range of PTMs using MASCOT (Matrix Science, mittee (Ethics #11/267). MS patients were diagnosed as UK), with enzyme specificity set to trypsin. Peptide follows: four with secondary progressive MS (ages 65, tolerance: 1 ppm; fragment tolerance: 0.6 Da with 1 68, 48, 60), two with relapsing remitting MS (ages 70 missed cleavage. The following PTMs were listed as and 72) and two with primary progressive MS (ages 36 variable modifications: deamidation (N,Q,R), oxidation and 62). All MS samples displayed microscopically small (H,W,M), methylation (R) and phosphorylation (S,T). lesions with the exception of the 68 year-old patient. Routinely greater than 80 % sequence coverage of MBP Control and MS samples were analysed separately, blind was observed. To confirm assignments, tandem mass with respect to age and severity of MS. Further details spectrometric fragmentation of each peak was per- are provided in Additional file 1: Tables S1 and S2. formed and synthetic peptides incorporating the par- ticular modified amino acid (see below for list of Homogenization of human brain tissue commercial standards) were run using the same Frozen cerebellum (Gyri of the posterior lobe) from con- method to confirm identity by comparison of retention trols (105 +/− 11 mg) and MS patients (115 +/− 13 mg) time and MS/MS. was pulverized then homogenized as detailed in Norris et. al. [30] After homogenization, each sample was Data analysis transferred to a 5 mL glass tube and centrifuged (1000 g, The doubly charged ions [FFGGDR m/z = 349.66, FFG 10 min) at 4 °C. Myelin was enriched by use of a sucrose GD(Cit)GAPK m/z = 526.76, GVDAQGTLSK m/z = gradient as described in Larocca and Norton [23]. 488.25, YLATASTMDHAR m/z = 668.82, TAHYGSLP QK m/z = 551.28 and TAHYGSLPEK m/z = 551.78] and Purification of MBP triply charged ions [TQDENPVVHFFK m/z = 487.57 and Myelin basic protein was enriched with modifications to HRDTGILDSIGR m/z = 447.24] for MBP-derived tryptic the protocol of Chevalier and Allen [7]. Briefly, the peptides were utilised for relative quantification. Their enriched myelin fraction was re-suspended in 50 mM intensities from the extracted ion chromatogram (XIC) Tris buffer (pH 7.4) and centrifuged (21,000 g, 20 min). were determined using Xcalibur software. Peak areas of The supernatant was discarded and the pellet re- each peptide were calculated using a smoothing method extracted. The pellet was re-suspended in 50 mM [Gaussian, 7 points]. The MS/MS spectrum of each pep- acetic acid (1 mL) and centrifuged (21,000 g,20min) tide was matched to the XIC, ensuring that the peak and the pellet re-extracted with 50 mM acetic acid. areas used corresponded to that of the matched peptide. The acetic acid extracts were combined and freeze The percentage of modification was calculated by using: dried. Greater than 90 % purity of MBP was confirmed [Modified/(Modified + Non- modified)] × 100. Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 3 of 12 Peptide standards residues present in MBP, on average, approximately one FFGGDR (MBP tryptic peptide 44–49), FFGGDRGAPK Asp residue in every MBP polypeptide is racemized MS and FFGGD(Cit)GAPK (MBP tryptic peptide 44–53), patients. In order to pinpoint the exact sites of modifica- GVDAQGTLSK, (MBP tryptic peptide 143–152), HRD tion of MBP, samples were treated with trypsin and the TGILDSIGR (MBP tryptic peptide 32–43), YLATAST peptides characterized by capillary liquid chromatog- MDHAR (MBP tryptic peptide 14–25), TAHYGSLPQK raphy tandem mass spectrometry (LC-MS). (MBP tryptic peptide 66–75), TAHYGSLPEK (MBP tryptic peptide 66–75) and TQDENPVVHFFK MBP MBP from MS patients differs from controls (tryptic peptide 80–91) were synthesized by GLS Bio- To determine the exact sites of modification, MBP chem (Shanghai, China). FFGGDR, FFGGDRGAPK, FFG was digested with trypsin, which cleaves only at ar- GD(Cit)GAPK, HRDTGILDSIGR, YLATASTMDAR, TQ ginine and lysine residues, and the peptide mixture DENPVVHFFK and GVDAQGTLSK were synthesized examined by LC–MS. LC enables the separation of with aspartic acid in four structural isomers, i.e., L-aspartic racemized forms of each peptide in MBP and tandem acid, L-isoaspartic acid, D-aspartic acid, or D-isoaspartic MS/MS fragments each peptide giving its sequence acid. TAHYGSLPQK was synthesized with L- and D- and sites of modification. Initial proteomic data versions of Ser. showed that the degree of PTM of all MBP samples, both in terms of the number of sites modified, as well Statistical analysis as the extent of modification, was considerable. At Statistical analysis was performed using SPSS Statistics several sites in MBP there were significant differences (version 19, IBM Corp. NY, USA) and R (version 3.1.1). in the degree of PTM between controls and MS pa- Comparison of controls and MS patients was made tients. Individual sites are discussed below with a using a Mann Whitney U test with a significance level of more comprehensive analysis of all detected PTMs p = 0.05. Changes to controls with age were analysed by provided in Additional file 1. linear regression. Prior to performing linear regression, normality of the dependent variable was assessed by Aspartic acid examining the histograms of the standardised residuals L-Asp and L-Asn residues in long-lived proteins can and non-normal data were transformed where required. undergo age-related racemization via an intramolecular condensation involving a succinimide [13]. Hydrolysis of Results the succinimide produces four structural isomers: L- Adult human MBP is extensively racemized Asp, D-Asp, L-isoAsp and D-isoAsp [13] (see Fig. 2). Racemization involves the conversion of an L-amino The formation of D-Asp, D-isoAsp and L-isoAsp from 32 43 acid to a D-amino acid and is a defining feature of long- L-Asp34 in MBP ( HRDTGILDSIGR ) as a function of lived proteins e.g. [13, 18]. It significantly affects protein age is illustrated in Fig. 3. For each of the abnormal Asp structure and can lead to the formation of epitopes that isomers there was a significantly greater amount present the body recognizes as being foreign [8]. Initial experi- in MBP from MS patients. Of particular importance, ments established the overall degree of racemization of neither D-isoAsp or nor L-isoAsp was detected in the MBP using acid hydrolysis followed by separation of the control samples. It should be noted that this peptide L- and D-forms of individual amino acids by HPLC [18]. contains an internal Arg33 residue that trypsin would All samples showed substantial racemization, even MBP normally cleave. We suspected on the basis of previous from a 22 year-old was found to contain a high percent- data [27] that racemization of the adjacent Asp may be age of D-amino acids (Fig. 1). Indeed the levels of responsible. This was tested with four homologous MBP racemization of Asx (i.e. Asn + Asp) (Fig. 1a) were ap- (32–43) peptides. D-Asp and L-isoAsp or D-isoAsp on proximately 5 % by age 22 and thus are comparable to the C-terminal side of Arg inhibited digestion by tryspin, those found in human lens proteins [18], which do not whereas the L-Asp form was cleaved efficiently. to turn over and contain high levels of D-Asp by early adulthood [18]. In lens proteins, there is a rapid age- Glutamine dependent increase in racemization up to age ~20, after Deamidation of Gln is another age-related modifica- which levels increase much more slowly [9]. This sug- tion of proteins [19]. The deamidation of Gln147 143 152 gests that human MBP is a life-long protein although ( GVDAEGTLSK )asafunction of ageisshown further testing would be required to confirm this. in Fig. 4. The extent of deamidation of Gln147 in- Racemization data of MBP from MS patients revealed creased linearly with age in control MBP, although statistically significant increases in the overall extent of the values did not exceed 6 %. By contrast, in every racemization of Asx, Glx and Ser compared to controls case deamidation of Gln147 in MBP from MS pa- (Fig. 1a, b, c). On the basis of the number of Asn/Asp tients was greater than 6 % (Fig. 4). Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 4 of 12 Fig. 1 Racemization of a aspartic acid and asparagine (Asx), b glutamine and glutamic acid (Glx) and c serine (Ser) in MBP isolated from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽) as a function of age. Levels of D-Asx (p <0.01, Mann Whitney U), D-Glx (p = 0.016, Mann Whitney U) and D-Ser (p = 0.007, Mann Whitney U) were significantly elevated in MS patients. Age zero corresponds to MBP purified from goat cerebellum (▼) and was used as a control for artifactual racemi- zation during hydrolysis. All data are the mean ± SEM of three separate HPLC runs. Racemization in this, and subsequent figures, was expressed as a % . Controls, n = 15; multiple sclerosis patients n =8. In this DþL case, and Figs. 2, 3, 4, 5 statistical analysis was undertaken on combined controls vs combined MS patients Because many amino acid residues in adult MBP were found by proteomic analysis to be modified, in some cases more than one PTM was detected in a single tryptic 143 152 peptide. This is illustrated in MBP ( GVDAEGTLSK ), where, along with deamidation of Gln, Asp145 was also isomerized. D-isoAsp levels were found to be increased significantly in MS patients compared to controls for the Glu form of this peptide (Fig. 5). The L-isoAsp and D-Asp versions of the Glu version were not significantly different from the controls (data not shown). Arginine Deimination of Arg residues yields citrulline, an amino acid that is not cleaved by trypsin and therefore this PTM leads to missed cleavages during digestion [4]. Fifteen of the 19 Arg sites in MBP showed some degree of conversion to cit- rulline (Additional file 1: Table S3). Therefore, the relative quantification method used in this study took missed cleav- ages into account when determining the degree of age- related MBP modification (see Additional file 1: Figure S1). Deimination of Arg increased at some sites in an age- dependent manner. This is illustrated for Arg49 44 53 ( FFGGDRGAPK )that appeared to be converted linearly to citrulline (Fig. 6a). This peptide also displayed other MS-specific modifications. In MS samples, deimina- tion of Arg49 was accompanied by isomerisation of Asp48 44 53 ( FFGGD(Cit)GAPK ). In MS patients 11 ± 3 % of citrul- linated peptides were also racemized at Asp 48. This combination of racemization of Asp48 and dei- mination of Arg49 was found only in MBP from MS patients (Fig. 6b). Elevated deimination of Arg 65 and Arg122 wasalso detectedinMBP from MS patients (Additional file 1: Figure S1). Interaction between other sites of modification As noted, a number of tryptic peptides contained more than one PTM. Two such sites involving Asp145/Gln147 and Asp 48/Arg 49 were depicted in Figs. 5 and 6. Evidence for another such interplay was found for 14 25 Met21 and Asp22 ( YLATASTMDHAR ). Significant Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 5 of 12 Fig. 2 A major source of degradation of long-lived proteins is racemisation. This spontaneous process affects aspartate, asparagine and serine residues in unstructured regions of these proteins. Illustrated is the mechanism responsible for Asp and Asn racemization oxidation of Met21 was detected in all control MBP of Met 21 increased linearly with age in control MBP samples (Fig. 7a). Since Met can oxidise artifactually and again minimal oxidation was detected in MS pa- during extraction or digestion [24], and no precautions tients. IsoAsp22 levels were consistently higher in this were taken specifically to minimize oxidation, this PTM Met peptide from MS patients (Fig. 7c). Other authors was initially disregarded. However, other considerations have described oxidation of Met 21 in MBP with specu- suggest that Met oxidation in MBP from controls may lation that it may be linked to nearby phosphorylation of be real. Firstly, very little oxidation of Met21 was found Ser or Thr [22]. in MBP from MS patients treated in exactly the same manner. Secondly, in controls, the levels increased as a MS specific sites of MBP modification function of age. Thirdly, when Met sulfoxide levels were A summary of MBP sites where PTMs differed signifi- monitored in the same tryptic peptide where racemiza- cantly in controls and MS patients is shown in Fig. 8a. tion of Asp22 was also present (Fig. 7b), a similar pat- Mapping of these sites onto a proposed structure of tern of Met sulfoxide formation was noted. In this case, MBP [3] (Fig. 8b) showed that these sites were clustered as was found with the L-Asp isoform (Fig. 7a), oxidation into two distinct regions. Site A incorporated the most Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 6 of 12 Fig. 3 Racemization of Asp34 in MBP from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽). Conversion of L-Asp34 was measured using the tryptic peptide HRDTGILDSIGR to (a)HR(D-isoAsp)TGILDSIGR and (b)HR(L-isoAsp)T- GILDSIGR. Elevated racemization of Asp34 was detected in multiple sclerosis patients for both isoforms: HR(L-isoAsp)TGILDSIGR (p <0.001, Mann–Whitney-U) and HR(D-isoAsp)TGILDSIGR (p = 0.002, Mann– Whitney-U). c An example of selected ion chromatographs from the tryptic digest of MBP from a control (66y) and a multiple sclerosis patient (48y). The percentage of modification was determined by the ion intensities of (HRDTGILDSIGR)/(HRDTGILDS IGR + HRDTGILDSIGR) × 100. Controls, n = 10; multiple sclerosis patients n =8 abundant site of racemization in MS patients (Asp34) with an estimated 32 % of this residue racemized. Site B contained a dual modification; racemization at Asp48 together with citrulline 49. Asp48 and citrulline 49 were present in each of the MS patients but were not detected in controls. This intriguing finding may suggest that PTMs within two exposed patches of MBP could be involved in provoking an immune response that ultim- ately results in MS. Discussion This study has revealed the diversity and extent of modi- fications present in MBP from the normal adult human brain. In addition, MBP from MS patients displayed several sites where the covalent alteration differed sig- nificantly from that of normal individuals. Racemization was found to be a widespread PTM of MBP, with some sites present specifically in MS patients. In particular racemization of L-Asp to the three other Fig. 4 Deamidation of Gln147 in MBP from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽). Deamidation of Gln147 was measured using the tryptic peptide GVDAQGTSK (i.e. GVDAQGTSK to GVDAEGTSK). Deamidation was significantly greater in the multiple sclerosis patients (p < 0.001, Mann–Whitney-U.). Deamidation increased with age in controls (R = 0.571, p = 0.011) Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 7 of 12 Fig. 6 Deimination of Arg49 coupled with racemization of Asp48 in MBP from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽). a For the L-Asp version (FFGGD(Cit)GAPK), no statistically significant difference was found in the levels of citrulline between controls and multiple sclerosis Fig. 5 Deamidation of Gln147 coupled with racemization of Asp145 patients, but linear regression analysis revealed a significant increase in MBP from controls (●) and multiple sclerosis (MS) patients suffering 2 in the amount of citrulline with age in control samples (R = 0.664, from SPMS (○) PPMS (◊) and RRMS (▽). a Conversion of L-Asp145 p = 0.004). b Conversion of L-Asp48 to the other Asp isomers in the to the D-isoAsp form in the deamidated peptide (GVDAEGTSK). tryptic peptide deiminated at Arg49 (FFGGD(Cit)GAPK). An increase Racemization of Asp145 to D-isoAsp GV(D-isoAsp)AEGTSK was in racemization of Asp48 in the deiminated peptide was seen in significantly greater in multiple sclerosis patients (p < 0.001, multiple sclerosis patients (p <0.001, Mann–Whitney-U). Racemization Mann–Whitney-U). There was no significant difference for the in this case refers to combined D-Asp, D-isoAsp and L-isoAsp, since the other Asp isomers; GV(D-Asp)AEGTSK and GV(L-isoAsp)AEGTSK. isomers were not separated under these conditions. The The percentage of modification was calculated using the ion percentage of modification was determined by the ion intensities of intensities of (GV(D-isoAsp)AEGTSK)/(GVDAEGTSK) × 100. b Selected (FFGGD(Cit)GAPK))/(FFGGD(Cit)GAPK + FFGGD(Cit)GAPK) × 100. ion chromatograph from the tryptic digest of MBP from a control (66y) Controls n = 10; multiple sclerosis patients n =8 and an multiple sclerosis patient (48y). Controls n = 10; multiple sclerosis patients n =8 methylation and deamidation e.g. [16, 22], age-related Asp isomers (see Fig. 2) was an abundant modification and MS-related changes have not been previously re- and conversion of L-Asp to isoAsp was characterized ported. Analysis of human MBP for MS-related modi- at several sites. One particular PTM; isoAsp48 in com- fications in this study, show that it is essential to bination with citrulline 49 was detected only in MS evaluate such PTMs in relation to the background of patients (Fig. 6a). age-related changes. At other sites, isoAsp was present in MS patients at Formation of isoAsp at several sites is likely to be levels significantly higher than those of the controls significant in terms of the conversion of MBP to a novel (Asp34 and Asp82). Although MBP has been investi- antigenic form that could potentially act to trigger an gated for PTMs such as citrullination, phosphorylation, immune response. This is because others have shown Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 8 of 12 Fig. 7 Oxidation of Met22 coupled with racemization of Asp23 in MBP from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽). a When all Asp versions of (YLATASTMDHAR) were included, Met oxidation in the controls was significantly greater than in multiple sclerosis patients (p < 0.001, Mann–Whitney-U). b Oxidation of Met22 and racemization of Asp23 to isoAsp in YLATASTMDHAR. In the Met-oxidized peptide, racemization of Asp23 (YLATAST(MetSO)(isoAsp)HAR) was greater in the control samples (p = 0.008, Mann–Whitney-U). IsoAsp in this case refers to combined D- and L-isoAsp, since the isomers were not separated under these conditions. c Racemization of Asp23 to isoAsp(YLAT ASTM(isoAsp)HAR) in the absence of Met oxidation. Levels of isoAsp were significantly higher in multiple sclerosis patients (p < 0.001, Mann–Whitney-U). The percentage of modification was determined by the ion intensities of (Modified YLATASTMDHAR)/ (YLATASTMDHAR + YLATASTMDHAR) × 100. Controls n = 10; multiple sclerosis patients n =8 that replacement of L-Asp by an isoAsp in a peptide converts it to an immunogen [9, 29]. If, as in the case of MBP where isoAsp 48 is adjacent to another known immunogenic amino acid, citrulline (49), then this site may be particularly antigenic. This dual modification was detected only in MS patients (Fig. 8). It should how- ever be noted that we cannot definitively conclude that the MS -specific sites of modification detected in this study are the cause of MS; their formation may be a consequence of the disease. This is currently a subject of further investigations. The human body contains a number of long-lived proteins and their degradation may contribute to age- related diseases [36–38]. Rodent studies using a diet of labelled amino acids showed that MBP was a stable pro- tein [35]. In the case of human MBP, the amino acid racemization data alone (Fig. 1) suggest that it, like lens proteins, is a life-long protein [26]. The protein data correlate with recent cellular data [43]. For example, the final number of oligodendrocytes in the human brain is attained by age ~9 and, once formed, they undergo little turn over. In addition mature oligodendrocytes myeli- nate axons very poorly [40]. Thus any turnover of carbon in myelin that may be associated with an in- crease in white matter volume [6] appears to involve changes in lipid, while the myelin proteins are retained. PTMs such as deamidation and racemization docu- mented for MBP (Fig. 8) are consistent with those ex- pected for susceptible amino acids in proteins that reside for years in the body. Due to the sheer number of modifications of different types, the structure of MBP will inevitably be altered in adult myelin compared with that when it was first synthesized. For example, deimina- tion alters the net charge on MBP and this will reduce its binding to the negatively-charged head groups of phospholipids. This could lead to localized disruption of myelin [16]. Deamidation, even at just one site, can lead to significant protein denaturation [12] and racemization Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 9 of 12 Fig. 8 (See legend on next page.) Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 10 of 12 (See figure on previous page.) Fig. 8 A summary of the sites of modification detected in human MBP. a A histogram of the percentage modification at particular sites for controls (■) and multiple sclerosis (MS) patients (□). The underlined residues in bold correspond to the site of modification in MBP. (+) total Asp racemization in peptide YLATASTMDHAR with and without Met oxidation; (#) total deamidation of Gln147 incorporating all isomers of Asp GVDAEGTLSK. [^] total Asp145 racemization incorporating the Gln and Glu versions of peptide GVDAEGTLSK. Values for Met sulfoxide 21 were not plotted since the levels increased significantly with age. All values are the mean of all ages ± SEM. Asterisks represent level of significance (* p ≤ 0.05, ** ≤ 0.01 and *** ≤ 0.001, Mann–Whitney-U). b A model of MBP highlighting the residues that differ significantly in multiple sclerosis. The amino acid residues in blue correspond to the unmodified conformation, those in magenta illustrate the changes in conformation in multiple sclerosis. With the exception of TAHYGSLPQK, all the modifications are clustered within two zones as illustrated; site A) contains six and site B) contains three modified residues. At each of these amino acid residues, the extent of modification was found to be significantly different in multiple sclerosis patients compared with controls. Sites of Asp racemization labeled as D-Asp in 6b, include all Asp isomers (i.e. D-Asp, L-isoAsp and D-isoAsp). In the case of TQDENPVVHFFK only the D-isoAsp version was significantly different of amino acids is also likely to lead to unfolding [17]. In aspect could involve suppression of the immune system this study we found a number of sites of racemization. and components that modulate it, such as vitamin D, or it With regard to the impact of extensive PTMs on might include the masking of altered sites on MBP; for ex- conformation, it should be emphasized that the MBP ample, chaperones could act to minimize T-cell responses. structure shown in Fig. 8b is a model [3]. The majority In this regard, αB-crystallin expression is increased in MS of MBP is unstructured and this accords with the fact lesions [2]. Given that MBP is highly modified by early that PTMs, such as racemization, identified here are adulthood, it will be important in the future to investigate typically localized to unstructured regions [19, 20, 41]. how such a newly generated ‘non-self‘protein is prevented If PTMs of MBP are indeed responsible for inducing from eliciting an antigenic response in controls. It is likely MS, their age-dependent profile can account for an that other major myelin proteins like proteolipid protein otherwise puzzling observation i.e. that MS often begins and myelin oligodendrocyte glycoprotein will be modified in the fourth decade of life. It is clear from the graphs with age, since these are also long-lived [35, 36] and have (Figs. 3, 4, 5a, 6 and 7) that by the age of 30, MBP has also been implicated in MS [14, 15]. Detailed proteomic undergone a plethora of PTMs. Every amino acid change analysis of these proteins may therefore yield other poten- effectively introduces a “non-self” motif into the protein; tial MS-related epitopes. thus each one, or a combination of several, could poten- Our proteomic data revealed specific sites of modifica- tially generate an immune response. There are precedents tion in MBP that were common to all MS patients. One for amino acid racemization, in particular isoAsp forma- conclusion is that these sites may be particularly antigenic. tion, eliciting an immune reaction, e.g. autoimmunity to Other PTM sites have been reported previously [22], e.g. histone H2B in systemic lupus erythematosus [8, 10]. an increase in methylation of Arg107, deimination of Arg There is still much to be understood about the detailed at several sites, and a reduction of phosphorylation in MS molecular architecture of myelin and questions remain to [22]. The majority of sites of Arg deamination in our study be answered in relation to the part played by MBP. In rela- match those reported previously [22]. Deimination at tion to this, if MBP is an intracellular protein, how could some sites was age dependent (Fig. 6a), but in most cases it act as a trigger for an immune response? The literature the amount of citrulline in MBP from MS patients did not is not clear as to whether all of MBP is indeed intracellu- differ significantly from control MBP Sites of racemization lar. In addition, if indeed all of MBP were originally intra- are generated by spontaneous processes which occur more cellular e.g. in childhood, it is also possible that changes rapidly in unstructured regions of a protein. For MS that occur with age could lead to partial myelin break- patients to display elevated D-isoAsp in some locations down and therefore exposure of MBP to the immune suggests that MBP may exist in a different conformation system. Previous research has shown that MBP fragments in diseased myelin. can be presented by MHC activating CD8+ cells [44], and In a recent review [28], Mahad and colleagues consid- it is well known that in mouse models of MS, demyelin- ered that MS could be viewed either as a classic auto- ation can be induced by injection of MBP fragments [1]. A immune disease (the so called “outside-in hypothesis”)or number of studies support a role for MBP in the progres- as a disease triggered by a foreign, e.g., viral antigen (the sion of the MS [5, 31, 32]. so called “inside-out hypothesis”). Our observations pro- Since the brains of all people contain MBP that is highly vide a means to meld these apparently separate mecha- modified by age 20, it is conceivable that the reason some nisms into one. The formation of MS-specific PTMs of people develop MS, while others do not, can be traced MBP via spontaneous decomposition mechanisms may either to the specific types of PTMs and/or the way a effectively convert an abundant neural protein into a subsequent immune response is modulated. This latter ‘non-self antigen’. The recent report of a lymphatic Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 11 of 12 drainage system in the CNS [25] opens up new possibil- 5. Berger T, Rubner P, Schautzer F, Egg R, Ulmer H, Mayringer I, Dilitz E, Deisenhammer F, Reindl M. Antimyelin antibodies as a predictor of clinically ities of how non-self antigens may be detected by the definite multiple sclerosis after a first demyelinating event. N Engl J Med. immune system. 2003;349:139–45. doi:10.1056/NEJMoa022328. 6. Blumenfeld-Katzir T, Pasternak O, Dagan M, Assaf Y. Diffusion MRI of structural brain plasticity induced by a learning and memory task. PLoS Conclusions One. 2011;6:e20678. The finding herein that specific sites of PTM in MS 7. Chevalier D, Allen BG. Purification of myelin basic protein from bovine brain. patients are localized in two zones of MBP suggests that Protein Expr Purif. 2000;18:229–34. 8. Doyle HA, Aswad DW, Mamula MJ. Autoimmunity to isomerized histone these regions may be involved in antigen recognition by H2B in systemic lupus erythematosus. Autoimmunity. 2012;46:6–13. the body’s immune surveillance machinery. This discov- 9. Doyle HA, Gee RJ, Mamula MJ. Altered immunogenicity of isoaspartate ery unlocks the possibility of selectively masking such containing proteins. Autoimmunity. 2007;40:131–7. 10. Doyle HA, Mamula MJ. Autoantigenesis: the evolution of protein sites on MBP using small molecules. If this hypothesis modifications in autoimmune disease. Curr Opin Immunol. 2012;24:112–8. can be verified, it may lead to the development of a new 11. Doyle HA, Zhou J, Wolff MJ, Harvey BP, Roman RM, Gee RJ, Koski RA, class of drugs that could potentially inhibit the onset of Mamula MJ. Isoaspartyl post-translational modification triggers anti-tumor T and B lymphocyte immunity. J Biol Chem. 2006;281:32676–83. MS, as well as help in modulating the immune response 12. Flaugh SL, Mills IA, King J. Glutamine deamidation destabilizes human of patients who already have developed the disease. γD-crystallin and lowers the kinetic barrier to unfolding. J Biol Chem. 2006;281:30782–93. 13. Geiger T, Clarke S. Deamidation, isomerization, and racemization at Additional file asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J Biol Chem. 1987;262:785–94. Additional file 1: Sites of Asp, Asn, Ser and Gln deamidation / racemisation. 14. Genain CP, Cannella B, Hauser SL, Raine CS. Identification of autoantibodies In addition to the sites of modification described, other sites of modification associated with myelin damage in multiple sclerosis. Nat Med. 1999;5:170–5. were detected in MBP. Some differences between MS patients and controls 15. Greer JM, Pender MP. Myelin proteolipid protein: an effective autoantigen and for Asp, Asn, Ser and Gln are summarised. (DOCX 1.84 mb) target of autoimmunity in multiple sclerosis. J Autoimmun. 2008;31:281–7. 16. Harauz G, Ishiyama N, Hill CMD, Bates IR, Libich DS, Farès C. Myelin basic protein—diverse conformational states of an intrinsically unstructured Acknowledgements protein and its roles in myelin assembly and multiple sclerosis. Micron. This work was supported by a grant from the National Health and Medical 2004;35:503–42. Research Council of Australia (NHMRC #1008667). Tissues were obtained with 17. Heck SD, Siok CJ, Krapcho KJ, Kelbaugh PR, Thadeio PF, Welch MJ, Williams appropriate ethical approvals from the New South Wales Tissue Resource RD, Ganong AH, Kelly ME, Lanzetti AJ, et al. Functional consequences of Centre at the University of Sydney, supported by the NHMRC, Schizophrenia posttranslational isomerization of Ser46 in a calcium channel toxin. Science. Research Institute and the National Institute of Alcohol Abuse and 1994;266:1065–8. Alcoholism (NIAAA): NIH R24AA012725. We thank Terry Lachlan for his 18. Hooi M, Truscott R. Racemisation and human cataract. d-Ser, d-Asp/Asn invaluable help with amino acid analysis. and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses. Age. 2011;33:131–41. Authors’ contributions 19. Hooi MYS, Raftery MJ, Truscott RJW. Age-dependent deamidation of All authors read and approved the final manuscript. MF performed glutamine residues in human γS crystallin: deamidation and unstructured experimental analyses and data evaluation; MR performed LC/MS analyses; regions. Protein Sci. 2012;21:1074–9. SH undertook statistical analyes. RT and MF conceived the project and wrote 20. Hooi MYS, Raftery MJ, Truscott RJW. Age-dependent racemization of serine the manuscript. residues in a human chaperone protein. Protein Sci. 2013;22:93–100. 21. Hooi MYS, Raftery MJ, Truscott RJW. Racemization of two proteins over Competing interests our lifespan: deamidation of asparagine 76 in γS crystallin is greater in The authors declare that they have no competing interest. cataract than in normal lenses across the age range. Invest Ophthalmol Vis Sci. 2012;53:3554–61. Author details 22. Kim JK, Mastronardi FG, Wood DD, Lubman DM, Zand R, Moscarello MA. Illawarra Health and Medical Research Institute, University of Wollongong, Multiple sclerosis: an important role for post-translational modifications of Wollongong, NSW 2522, Australia. School of Medicine, University of myelin basic protein in pathogenesis. Mol Cell Proteomics. 2003;2:453–62. Wollongong, Wollongong, NSW 2522, Australia. Bioanalytical Mass 23. Larocca JN, Norton WT. Isolation of Myelin. Current Protocols in Cell Biology. Spectrometry Facility, University of New South Wales, Sydney, NSW 2052, Hoboken: John Wiley & Sons; 2001. Australia. 24. Liu H, Ponniah G, Neill A, Patel R, Andrien B. Accurate determination of protein methionine oxidation by stable isotope labeling and LC-MS analysis. Received: 7 June 2016 Accepted: 15 July 2016 Anal Chem. 2013;85:11705–9. doi:10.1021/ac403072w. 25. Louveau A, Smirnov I, Keyes TJ, Eccles JD, Rouhani SJ, Peske JD, Derecki NC, Castle D, Mandell JW, Lee KS, et al. 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Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

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Springer Journals
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Copyright © 2016 by The Author(s).
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Biomedicine; Neurosciences; Pathology; Neurology
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2051-5960
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10.1186/s40478-016-0348-x
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27519525
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Abstract

Multiple sclerosis (MS) is associated with breakdown of the myelin sheath that coats neurons in the central nervous system. The cause of MS is not known, although the pathogenesis involves destruction of myelin by the immune system. It was the aim of this study to examine the abundant myelin protein, myelin basic protein (MBP), to determine if there are sites of modification that may be characteristic for MS. MBP from the cerebellum was examined from controls and MS patients across the age range using mass spectrometry and amino acid analysis. Amino acid racemization data indicated that myelin basic protein is long-lived and proteomic analysis of MBP showed it to be highly modified. A common modification of MBP was racemization of Asp and this was significantly greater in MS patients. In long-lived proteins, L-Asp and L-Asn can racemize to three other isomers, D-isoAsp, L-isoAsp and D-Asp and this is significant because isoAsp formation in peptides renders them immunogenic. Proteomic analysis revealed widespread modifications of MBP with two surface regions that are altered in MS. In particular, isoAsp was significantly elevated at these sites in MS patients. The generation of isoAsp could be responsible for eliciting an immune response to modified MBP and therefore be implicated in the etiology of MS. Introduction age-related post-translational modifications (PTMs) were MS has long been thought to be an autoimmune disease. localized to unstructured regions [19] and racemization, Injection of brain homogenates into animals results in which involves conversion of L- to D-amino acid re- demyelination resembling that seen in MS [33] and a sidues, was found to be the most abundant type of body of data, including the presence of antibodies to PTM [39]. D-amino acids at some sites were present myelin components in MS patients, supports a role at levels that exceeded the amount of the precursor for an autoimmune response in the genesis of human L-amino acid [21]. MS [32, 42]. In humans, major proteins from cataract lenses are MBP accounts for 35 % of myelin protein and is characterized by specific racemization sites that differ intrinsically unstructured [16] Recently, MBP and other from those in comparable age-matched normal lenses proteins in myelin were shown to be long-lived [35]. [18, 20, 21]. This finding implies that pathways of Over time, long-lived proteins degrade and this time- protein degradation in the body may possibly determine dependent deterioration has been well studied in the disease outcome. In long-lived proteins, Asp and Asn lens, a tissue with no protein turnover. In lens proteins, residues are particularly sensitive to racemization, undergoing a spontaneous cyclisation reaction that leads to the formation of L-Asp, D-Asp, L-isoAsp and D- * Correspondence: rjwt@uow.edu.au isoAsp (see Fig. 2). isoAsp residues are typically present Illawarra Health and Medical Research Institute, University of Wollongong, in the highest amounts [13]. Wollongong, NSW 2522, Australia Full list of author information is available at the end of the article © 2016 The Author(s). 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. Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 2 of 12 Modifications on this scale inevitably have conse- by SDS-PAGE (Additional file 1: Figure S2). Since quences for protein structure and can lead to a func- PTMs, such as those associated with aging, can cause tional decline of the protein [34]. Recently it has been major alterations to the properties of proteins, MBP shown that the formation of isoAsp can also lead to was not separated into isoforms (charge isomers) prior altered immunogenicity of peptides/proteins, inducing to proteomic analyses [22]. both T and B cell immunity [11]. Thus, like citrulline formation from Arg residues, conversion of Asp/Asn to Amino acid analysis isoAsp, can potentially induce an immune response to D-amino acid content of MBP samples was determined self-antigens [10]. In the current study we examined the by amino acid analysis as described [18]. Three separate hypothesis that since MBP is long-lived and unstruc- runs were carried out for each sample on an Agilent tured it, like lens proteins, may also undergo significant 1100 HPLC system. covalent alterations. For the reasons outlined above, particular reference was paid to Asp/Asn racemizaton to Capillary LC mass spectrometry isoAsp. If specific PTMs such as these were found in the Capillary LC mass spectrometry was undertaken as MBP from MS patients, then they could play a role in described [21]. Briefly, MBP (~50 μg) was digested with the etiology of MS by provoking an immune response to sequence grade trypsin (1 μg) (Promega) for 16 h at 37 ° the selectively modified myelin. C. Peptides were desalted and concentrated using a Ziptip (0.6 μL, C18 resin; Millipore) and freeze dried. Materials and methods The lyophilised peptides were re-suspended in formic Tissue samples acid:heptafluorobutyric acid:water (0.1:0.05:98.85). Tan- Cerebellum samples from control (n = 21) and MS pa- dem mass spectra were acquired after LC using a Thermo tients (n = 8) were obtained from the New South Wales LTQ Orbitrap as described [21]. Tissue Resource Centre at the University of Sydney with Data were searched against the Swiss-Prot database approval from the University of Wollongong ethics com- with a range of PTMs using MASCOT (Matrix Science, mittee (Ethics #11/267). MS patients were diagnosed as UK), with enzyme specificity set to trypsin. Peptide follows: four with secondary progressive MS (ages 65, tolerance: 1 ppm; fragment tolerance: 0.6 Da with 1 68, 48, 60), two with relapsing remitting MS (ages 70 missed cleavage. The following PTMs were listed as and 72) and two with primary progressive MS (ages 36 variable modifications: deamidation (N,Q,R), oxidation and 62). All MS samples displayed microscopically small (H,W,M), methylation (R) and phosphorylation (S,T). lesions with the exception of the 68 year-old patient. Routinely greater than 80 % sequence coverage of MBP Control and MS samples were analysed separately, blind was observed. To confirm assignments, tandem mass with respect to age and severity of MS. Further details spectrometric fragmentation of each peak was per- are provided in Additional file 1: Tables S1 and S2. formed and synthetic peptides incorporating the par- ticular modified amino acid (see below for list of Homogenization of human brain tissue commercial standards) were run using the same Frozen cerebellum (Gyri of the posterior lobe) from con- method to confirm identity by comparison of retention trols (105 +/− 11 mg) and MS patients (115 +/− 13 mg) time and MS/MS. was pulverized then homogenized as detailed in Norris et. al. [30] After homogenization, each sample was Data analysis transferred to a 5 mL glass tube and centrifuged (1000 g, The doubly charged ions [FFGGDR m/z = 349.66, FFG 10 min) at 4 °C. Myelin was enriched by use of a sucrose GD(Cit)GAPK m/z = 526.76, GVDAQGTLSK m/z = gradient as described in Larocca and Norton [23]. 488.25, YLATASTMDHAR m/z = 668.82, TAHYGSLP QK m/z = 551.28 and TAHYGSLPEK m/z = 551.78] and Purification of MBP triply charged ions [TQDENPVVHFFK m/z = 487.57 and Myelin basic protein was enriched with modifications to HRDTGILDSIGR m/z = 447.24] for MBP-derived tryptic the protocol of Chevalier and Allen [7]. Briefly, the peptides were utilised for relative quantification. Their enriched myelin fraction was re-suspended in 50 mM intensities from the extracted ion chromatogram (XIC) Tris buffer (pH 7.4) and centrifuged (21,000 g, 20 min). were determined using Xcalibur software. Peak areas of The supernatant was discarded and the pellet re- each peptide were calculated using a smoothing method extracted. The pellet was re-suspended in 50 mM [Gaussian, 7 points]. The MS/MS spectrum of each pep- acetic acid (1 mL) and centrifuged (21,000 g,20min) tide was matched to the XIC, ensuring that the peak and the pellet re-extracted with 50 mM acetic acid. areas used corresponded to that of the matched peptide. The acetic acid extracts were combined and freeze The percentage of modification was calculated by using: dried. Greater than 90 % purity of MBP was confirmed [Modified/(Modified + Non- modified)] × 100. Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 3 of 12 Peptide standards residues present in MBP, on average, approximately one FFGGDR (MBP tryptic peptide 44–49), FFGGDRGAPK Asp residue in every MBP polypeptide is racemized MS and FFGGD(Cit)GAPK (MBP tryptic peptide 44–53), patients. In order to pinpoint the exact sites of modifica- GVDAQGTLSK, (MBP tryptic peptide 143–152), HRD tion of MBP, samples were treated with trypsin and the TGILDSIGR (MBP tryptic peptide 32–43), YLATAST peptides characterized by capillary liquid chromatog- MDHAR (MBP tryptic peptide 14–25), TAHYGSLPQK raphy tandem mass spectrometry (LC-MS). (MBP tryptic peptide 66–75), TAHYGSLPEK (MBP tryptic peptide 66–75) and TQDENPVVHFFK MBP MBP from MS patients differs from controls (tryptic peptide 80–91) were synthesized by GLS Bio- To determine the exact sites of modification, MBP chem (Shanghai, China). FFGGDR, FFGGDRGAPK, FFG was digested with trypsin, which cleaves only at ar- GD(Cit)GAPK, HRDTGILDSIGR, YLATASTMDAR, TQ ginine and lysine residues, and the peptide mixture DENPVVHFFK and GVDAQGTLSK were synthesized examined by LC–MS. LC enables the separation of with aspartic acid in four structural isomers, i.e., L-aspartic racemized forms of each peptide in MBP and tandem acid, L-isoaspartic acid, D-aspartic acid, or D-isoaspartic MS/MS fragments each peptide giving its sequence acid. TAHYGSLPQK was synthesized with L- and D- and sites of modification. Initial proteomic data versions of Ser. showed that the degree of PTM of all MBP samples, both in terms of the number of sites modified, as well Statistical analysis as the extent of modification, was considerable. At Statistical analysis was performed using SPSS Statistics several sites in MBP there were significant differences (version 19, IBM Corp. NY, USA) and R (version 3.1.1). in the degree of PTM between controls and MS pa- Comparison of controls and MS patients was made tients. Individual sites are discussed below with a using a Mann Whitney U test with a significance level of more comprehensive analysis of all detected PTMs p = 0.05. Changes to controls with age were analysed by provided in Additional file 1. linear regression. Prior to performing linear regression, normality of the dependent variable was assessed by Aspartic acid examining the histograms of the standardised residuals L-Asp and L-Asn residues in long-lived proteins can and non-normal data were transformed where required. undergo age-related racemization via an intramolecular condensation involving a succinimide [13]. Hydrolysis of Results the succinimide produces four structural isomers: L- Adult human MBP is extensively racemized Asp, D-Asp, L-isoAsp and D-isoAsp [13] (see Fig. 2). Racemization involves the conversion of an L-amino The formation of D-Asp, D-isoAsp and L-isoAsp from 32 43 acid to a D-amino acid and is a defining feature of long- L-Asp34 in MBP ( HRDTGILDSIGR ) as a function of lived proteins e.g. [13, 18]. It significantly affects protein age is illustrated in Fig. 3. For each of the abnormal Asp structure and can lead to the formation of epitopes that isomers there was a significantly greater amount present the body recognizes as being foreign [8]. Initial experi- in MBP from MS patients. Of particular importance, ments established the overall degree of racemization of neither D-isoAsp or nor L-isoAsp was detected in the MBP using acid hydrolysis followed by separation of the control samples. It should be noted that this peptide L- and D-forms of individual amino acids by HPLC [18]. contains an internal Arg33 residue that trypsin would All samples showed substantial racemization, even MBP normally cleave. We suspected on the basis of previous from a 22 year-old was found to contain a high percent- data [27] that racemization of the adjacent Asp may be age of D-amino acids (Fig. 1). Indeed the levels of responsible. This was tested with four homologous MBP racemization of Asx (i.e. Asn + Asp) (Fig. 1a) were ap- (32–43) peptides. D-Asp and L-isoAsp or D-isoAsp on proximately 5 % by age 22 and thus are comparable to the C-terminal side of Arg inhibited digestion by tryspin, those found in human lens proteins [18], which do not whereas the L-Asp form was cleaved efficiently. to turn over and contain high levels of D-Asp by early adulthood [18]. In lens proteins, there is a rapid age- Glutamine dependent increase in racemization up to age ~20, after Deamidation of Gln is another age-related modifica- which levels increase much more slowly [9]. This sug- tion of proteins [19]. The deamidation of Gln147 143 152 gests that human MBP is a life-long protein although ( GVDAEGTLSK )asafunction of ageisshown further testing would be required to confirm this. in Fig. 4. The extent of deamidation of Gln147 in- Racemization data of MBP from MS patients revealed creased linearly with age in control MBP, although statistically significant increases in the overall extent of the values did not exceed 6 %. By contrast, in every racemization of Asx, Glx and Ser compared to controls case deamidation of Gln147 in MBP from MS pa- (Fig. 1a, b, c). On the basis of the number of Asn/Asp tients was greater than 6 % (Fig. 4). Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 4 of 12 Fig. 1 Racemization of a aspartic acid and asparagine (Asx), b glutamine and glutamic acid (Glx) and c serine (Ser) in MBP isolated from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽) as a function of age. Levels of D-Asx (p <0.01, Mann Whitney U), D-Glx (p = 0.016, Mann Whitney U) and D-Ser (p = 0.007, Mann Whitney U) were significantly elevated in MS patients. Age zero corresponds to MBP purified from goat cerebellum (▼) and was used as a control for artifactual racemi- zation during hydrolysis. All data are the mean ± SEM of three separate HPLC runs. Racemization in this, and subsequent figures, was expressed as a % . Controls, n = 15; multiple sclerosis patients n =8. In this DþL case, and Figs. 2, 3, 4, 5 statistical analysis was undertaken on combined controls vs combined MS patients Because many amino acid residues in adult MBP were found by proteomic analysis to be modified, in some cases more than one PTM was detected in a single tryptic 143 152 peptide. This is illustrated in MBP ( GVDAEGTLSK ), where, along with deamidation of Gln, Asp145 was also isomerized. D-isoAsp levels were found to be increased significantly in MS patients compared to controls for the Glu form of this peptide (Fig. 5). The L-isoAsp and D-Asp versions of the Glu version were not significantly different from the controls (data not shown). Arginine Deimination of Arg residues yields citrulline, an amino acid that is not cleaved by trypsin and therefore this PTM leads to missed cleavages during digestion [4]. Fifteen of the 19 Arg sites in MBP showed some degree of conversion to cit- rulline (Additional file 1: Table S3). Therefore, the relative quantification method used in this study took missed cleav- ages into account when determining the degree of age- related MBP modification (see Additional file 1: Figure S1). Deimination of Arg increased at some sites in an age- dependent manner. This is illustrated for Arg49 44 53 ( FFGGDRGAPK )that appeared to be converted linearly to citrulline (Fig. 6a). This peptide also displayed other MS-specific modifications. In MS samples, deimina- tion of Arg49 was accompanied by isomerisation of Asp48 44 53 ( FFGGD(Cit)GAPK ). In MS patients 11 ± 3 % of citrul- linated peptides were also racemized at Asp 48. This combination of racemization of Asp48 and dei- mination of Arg49 was found only in MBP from MS patients (Fig. 6b). Elevated deimination of Arg 65 and Arg122 wasalso detectedinMBP from MS patients (Additional file 1: Figure S1). Interaction between other sites of modification As noted, a number of tryptic peptides contained more than one PTM. Two such sites involving Asp145/Gln147 and Asp 48/Arg 49 were depicted in Figs. 5 and 6. Evidence for another such interplay was found for 14 25 Met21 and Asp22 ( YLATASTMDHAR ). Significant Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 5 of 12 Fig. 2 A major source of degradation of long-lived proteins is racemisation. This spontaneous process affects aspartate, asparagine and serine residues in unstructured regions of these proteins. Illustrated is the mechanism responsible for Asp and Asn racemization oxidation of Met21 was detected in all control MBP of Met 21 increased linearly with age in control MBP samples (Fig. 7a). Since Met can oxidise artifactually and again minimal oxidation was detected in MS pa- during extraction or digestion [24], and no precautions tients. IsoAsp22 levels were consistently higher in this were taken specifically to minimize oxidation, this PTM Met peptide from MS patients (Fig. 7c). Other authors was initially disregarded. However, other considerations have described oxidation of Met 21 in MBP with specu- suggest that Met oxidation in MBP from controls may lation that it may be linked to nearby phosphorylation of be real. Firstly, very little oxidation of Met21 was found Ser or Thr [22]. in MBP from MS patients treated in exactly the same manner. Secondly, in controls, the levels increased as a MS specific sites of MBP modification function of age. Thirdly, when Met sulfoxide levels were A summary of MBP sites where PTMs differed signifi- monitored in the same tryptic peptide where racemiza- cantly in controls and MS patients is shown in Fig. 8a. tion of Asp22 was also present (Fig. 7b), a similar pat- Mapping of these sites onto a proposed structure of tern of Met sulfoxide formation was noted. In this case, MBP [3] (Fig. 8b) showed that these sites were clustered as was found with the L-Asp isoform (Fig. 7a), oxidation into two distinct regions. Site A incorporated the most Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 6 of 12 Fig. 3 Racemization of Asp34 in MBP from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽). Conversion of L-Asp34 was measured using the tryptic peptide HRDTGILDSIGR to (a)HR(D-isoAsp)TGILDSIGR and (b)HR(L-isoAsp)T- GILDSIGR. Elevated racemization of Asp34 was detected in multiple sclerosis patients for both isoforms: HR(L-isoAsp)TGILDSIGR (p <0.001, Mann–Whitney-U) and HR(D-isoAsp)TGILDSIGR (p = 0.002, Mann– Whitney-U). c An example of selected ion chromatographs from the tryptic digest of MBP from a control (66y) and a multiple sclerosis patient (48y). The percentage of modification was determined by the ion intensities of (HRDTGILDSIGR)/(HRDTGILDS IGR + HRDTGILDSIGR) × 100. Controls, n = 10; multiple sclerosis patients n =8 abundant site of racemization in MS patients (Asp34) with an estimated 32 % of this residue racemized. Site B contained a dual modification; racemization at Asp48 together with citrulline 49. Asp48 and citrulline 49 were present in each of the MS patients but were not detected in controls. This intriguing finding may suggest that PTMs within two exposed patches of MBP could be involved in provoking an immune response that ultim- ately results in MS. Discussion This study has revealed the diversity and extent of modi- fications present in MBP from the normal adult human brain. In addition, MBP from MS patients displayed several sites where the covalent alteration differed sig- nificantly from that of normal individuals. Racemization was found to be a widespread PTM of MBP, with some sites present specifically in MS patients. In particular racemization of L-Asp to the three other Fig. 4 Deamidation of Gln147 in MBP from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽). Deamidation of Gln147 was measured using the tryptic peptide GVDAQGTSK (i.e. GVDAQGTSK to GVDAEGTSK). Deamidation was significantly greater in the multiple sclerosis patients (p < 0.001, Mann–Whitney-U.). Deamidation increased with age in controls (R = 0.571, p = 0.011) Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 7 of 12 Fig. 6 Deimination of Arg49 coupled with racemization of Asp48 in MBP from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽). a For the L-Asp version (FFGGD(Cit)GAPK), no statistically significant difference was found in the levels of citrulline between controls and multiple sclerosis Fig. 5 Deamidation of Gln147 coupled with racemization of Asp145 patients, but linear regression analysis revealed a significant increase in MBP from controls (●) and multiple sclerosis (MS) patients suffering 2 in the amount of citrulline with age in control samples (R = 0.664, from SPMS (○) PPMS (◊) and RRMS (▽). a Conversion of L-Asp145 p = 0.004). b Conversion of L-Asp48 to the other Asp isomers in the to the D-isoAsp form in the deamidated peptide (GVDAEGTSK). tryptic peptide deiminated at Arg49 (FFGGD(Cit)GAPK). An increase Racemization of Asp145 to D-isoAsp GV(D-isoAsp)AEGTSK was in racemization of Asp48 in the deiminated peptide was seen in significantly greater in multiple sclerosis patients (p < 0.001, multiple sclerosis patients (p <0.001, Mann–Whitney-U). Racemization Mann–Whitney-U). There was no significant difference for the in this case refers to combined D-Asp, D-isoAsp and L-isoAsp, since the other Asp isomers; GV(D-Asp)AEGTSK and GV(L-isoAsp)AEGTSK. isomers were not separated under these conditions. The The percentage of modification was calculated using the ion percentage of modification was determined by the ion intensities of intensities of (GV(D-isoAsp)AEGTSK)/(GVDAEGTSK) × 100. b Selected (FFGGD(Cit)GAPK))/(FFGGD(Cit)GAPK + FFGGD(Cit)GAPK) × 100. ion chromatograph from the tryptic digest of MBP from a control (66y) Controls n = 10; multiple sclerosis patients n =8 and an multiple sclerosis patient (48y). Controls n = 10; multiple sclerosis patients n =8 methylation and deamidation e.g. [16, 22], age-related Asp isomers (see Fig. 2) was an abundant modification and MS-related changes have not been previously re- and conversion of L-Asp to isoAsp was characterized ported. Analysis of human MBP for MS-related modi- at several sites. One particular PTM; isoAsp48 in com- fications in this study, show that it is essential to bination with citrulline 49 was detected only in MS evaluate such PTMs in relation to the background of patients (Fig. 6a). age-related changes. At other sites, isoAsp was present in MS patients at Formation of isoAsp at several sites is likely to be levels significantly higher than those of the controls significant in terms of the conversion of MBP to a novel (Asp34 and Asp82). Although MBP has been investi- antigenic form that could potentially act to trigger an gated for PTMs such as citrullination, phosphorylation, immune response. This is because others have shown Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 8 of 12 Fig. 7 Oxidation of Met22 coupled with racemization of Asp23 in MBP from controls (●) and multiple sclerosis (MS) patients suffering from SPMS (○) PPMS (◊) and RRMS (▽). a When all Asp versions of (YLATASTMDHAR) were included, Met oxidation in the controls was significantly greater than in multiple sclerosis patients (p < 0.001, Mann–Whitney-U). b Oxidation of Met22 and racemization of Asp23 to isoAsp in YLATASTMDHAR. In the Met-oxidized peptide, racemization of Asp23 (YLATAST(MetSO)(isoAsp)HAR) was greater in the control samples (p = 0.008, Mann–Whitney-U). IsoAsp in this case refers to combined D- and L-isoAsp, since the isomers were not separated under these conditions. c Racemization of Asp23 to isoAsp(YLAT ASTM(isoAsp)HAR) in the absence of Met oxidation. Levels of isoAsp were significantly higher in multiple sclerosis patients (p < 0.001, Mann–Whitney-U). The percentage of modification was determined by the ion intensities of (Modified YLATASTMDHAR)/ (YLATASTMDHAR + YLATASTMDHAR) × 100. Controls n = 10; multiple sclerosis patients n =8 that replacement of L-Asp by an isoAsp in a peptide converts it to an immunogen [9, 29]. If, as in the case of MBP where isoAsp 48 is adjacent to another known immunogenic amino acid, citrulline (49), then this site may be particularly antigenic. This dual modification was detected only in MS patients (Fig. 8). It should how- ever be noted that we cannot definitively conclude that the MS -specific sites of modification detected in this study are the cause of MS; their formation may be a consequence of the disease. This is currently a subject of further investigations. The human body contains a number of long-lived proteins and their degradation may contribute to age- related diseases [36–38]. Rodent studies using a diet of labelled amino acids showed that MBP was a stable pro- tein [35]. In the case of human MBP, the amino acid racemization data alone (Fig. 1) suggest that it, like lens proteins, is a life-long protein [26]. The protein data correlate with recent cellular data [43]. For example, the final number of oligodendrocytes in the human brain is attained by age ~9 and, once formed, they undergo little turn over. In addition mature oligodendrocytes myeli- nate axons very poorly [40]. Thus any turnover of carbon in myelin that may be associated with an in- crease in white matter volume [6] appears to involve changes in lipid, while the myelin proteins are retained. PTMs such as deamidation and racemization docu- mented for MBP (Fig. 8) are consistent with those ex- pected for susceptible amino acids in proteins that reside for years in the body. Due to the sheer number of modifications of different types, the structure of MBP will inevitably be altered in adult myelin compared with that when it was first synthesized. For example, deimina- tion alters the net charge on MBP and this will reduce its binding to the negatively-charged head groups of phospholipids. This could lead to localized disruption of myelin [16]. Deamidation, even at just one site, can lead to significant protein denaturation [12] and racemization Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 9 of 12 Fig. 8 (See legend on next page.) Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 10 of 12 (See figure on previous page.) Fig. 8 A summary of the sites of modification detected in human MBP. a A histogram of the percentage modification at particular sites for controls (■) and multiple sclerosis (MS) patients (□). The underlined residues in bold correspond to the site of modification in MBP. (+) total Asp racemization in peptide YLATASTMDHAR with and without Met oxidation; (#) total deamidation of Gln147 incorporating all isomers of Asp GVDAEGTLSK. [^] total Asp145 racemization incorporating the Gln and Glu versions of peptide GVDAEGTLSK. Values for Met sulfoxide 21 were not plotted since the levels increased significantly with age. All values are the mean of all ages ± SEM. Asterisks represent level of significance (* p ≤ 0.05, ** ≤ 0.01 and *** ≤ 0.001, Mann–Whitney-U). b A model of MBP highlighting the residues that differ significantly in multiple sclerosis. The amino acid residues in blue correspond to the unmodified conformation, those in magenta illustrate the changes in conformation in multiple sclerosis. With the exception of TAHYGSLPQK, all the modifications are clustered within two zones as illustrated; site A) contains six and site B) contains three modified residues. At each of these amino acid residues, the extent of modification was found to be significantly different in multiple sclerosis patients compared with controls. Sites of Asp racemization labeled as D-Asp in 6b, include all Asp isomers (i.e. D-Asp, L-isoAsp and D-isoAsp). In the case of TQDENPVVHFFK only the D-isoAsp version was significantly different of amino acids is also likely to lead to unfolding [17]. In aspect could involve suppression of the immune system this study we found a number of sites of racemization. and components that modulate it, such as vitamin D, or it With regard to the impact of extensive PTMs on might include the masking of altered sites on MBP; for ex- conformation, it should be emphasized that the MBP ample, chaperones could act to minimize T-cell responses. structure shown in Fig. 8b is a model [3]. The majority In this regard, αB-crystallin expression is increased in MS of MBP is unstructured and this accords with the fact lesions [2]. Given that MBP is highly modified by early that PTMs, such as racemization, identified here are adulthood, it will be important in the future to investigate typically localized to unstructured regions [19, 20, 41]. how such a newly generated ‘non-self‘protein is prevented If PTMs of MBP are indeed responsible for inducing from eliciting an antigenic response in controls. It is likely MS, their age-dependent profile can account for an that other major myelin proteins like proteolipid protein otherwise puzzling observation i.e. that MS often begins and myelin oligodendrocyte glycoprotein will be modified in the fourth decade of life. It is clear from the graphs with age, since these are also long-lived [35, 36] and have (Figs. 3, 4, 5a, 6 and 7) that by the age of 30, MBP has also been implicated in MS [14, 15]. Detailed proteomic undergone a plethora of PTMs. Every amino acid change analysis of these proteins may therefore yield other poten- effectively introduces a “non-self” motif into the protein; tial MS-related epitopes. thus each one, or a combination of several, could poten- Our proteomic data revealed specific sites of modifica- tially generate an immune response. There are precedents tion in MBP that were common to all MS patients. One for amino acid racemization, in particular isoAsp forma- conclusion is that these sites may be particularly antigenic. tion, eliciting an immune reaction, e.g. autoimmunity to Other PTM sites have been reported previously [22], e.g. histone H2B in systemic lupus erythematosus [8, 10]. an increase in methylation of Arg107, deimination of Arg There is still much to be understood about the detailed at several sites, and a reduction of phosphorylation in MS molecular architecture of myelin and questions remain to [22]. The majority of sites of Arg deamination in our study be answered in relation to the part played by MBP. In rela- match those reported previously [22]. Deimination at tion to this, if MBP is an intracellular protein, how could some sites was age dependent (Fig. 6a), but in most cases it act as a trigger for an immune response? The literature the amount of citrulline in MBP from MS patients did not is not clear as to whether all of MBP is indeed intracellu- differ significantly from control MBP Sites of racemization lar. In addition, if indeed all of MBP were originally intra- are generated by spontaneous processes which occur more cellular e.g. in childhood, it is also possible that changes rapidly in unstructured regions of a protein. For MS that occur with age could lead to partial myelin break- patients to display elevated D-isoAsp in some locations down and therefore exposure of MBP to the immune suggests that MBP may exist in a different conformation system. Previous research has shown that MBP fragments in diseased myelin. can be presented by MHC activating CD8+ cells [44], and In a recent review [28], Mahad and colleagues consid- it is well known that in mouse models of MS, demyelin- ered that MS could be viewed either as a classic auto- ation can be induced by injection of MBP fragments [1]. A immune disease (the so called “outside-in hypothesis”)or number of studies support a role for MBP in the progres- as a disease triggered by a foreign, e.g., viral antigen (the sion of the MS [5, 31, 32]. so called “inside-out hypothesis”). Our observations pro- Since the brains of all people contain MBP that is highly vide a means to meld these apparently separate mecha- modified by age 20, it is conceivable that the reason some nisms into one. The formation of MS-specific PTMs of people develop MS, while others do not, can be traced MBP via spontaneous decomposition mechanisms may either to the specific types of PTMs and/or the way a effectively convert an abundant neural protein into a subsequent immune response is modulated. This latter ‘non-self antigen’. The recent report of a lymphatic Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 Page 11 of 12 drainage system in the CNS [25] opens up new possibil- 5. Berger T, Rubner P, Schautzer F, Egg R, Ulmer H, Mayringer I, Dilitz E, Deisenhammer F, Reindl M. Antimyelin antibodies as a predictor of clinically ities of how non-self antigens may be detected by the definite multiple sclerosis after a first demyelinating event. N Engl J Med. immune system. 2003;349:139–45. doi:10.1056/NEJMoa022328. 6. Blumenfeld-Katzir T, Pasternak O, Dagan M, Assaf Y. Diffusion MRI of structural brain plasticity induced by a learning and memory task. 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Published: Aug 12, 2016

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