Friedrich et al. Acta Neuropathologica Communications (2016) 4:83 DOI 10.1186/s40478-016-0348-x RESEARCH Open Access Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset? Michael G. Friedrich1,2*, Sarah E. Hancock1,2, Mark J. Raftery3 and Roger J. W. Truscott1* 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: [email protected] 1Illawarra Health and Medical Research Institute, University of Wollongong, isoAsp (see Fig. 2). isoAsp residues are typically present Wollongong, NSW 2522, Australia in the highest amounts [13]. 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).