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Protein Modification by Deamidation Indicates Variations in Joint Extracellular Matrix Turnover*

  • Jonathan B. Catterall
    Affiliations
    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710
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  • Ming F. Hsueh
    Affiliations
    Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710
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  • Thomas V. Stabler
    Affiliations
    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710
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  • Christopher R. McCudden
    Affiliations
    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710
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  • Michael Bolognesi
    Affiliations
    Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina 27710
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  • Robert Zura
    Affiliations
    Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina 27710
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  • Joanne M. Jordan
    Affiliations
    Departments of Thurston Arthritis Research Center, University of North Carolina, Chapel Hill, North Carolina 27514

    Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514

    Department of Orthopaedics, University of North Carolina, Chapel Hill, North Carolina 27514
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  • Jordan B. Renner
    Affiliations
    Departments of Thurston Arthritis Research Center, University of North Carolina, Chapel Hill, North Carolina 27514

    Department of Radiology, University of North Carolina, Chapel Hill, North Carolina 27514
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  • Sheng Feng
    Affiliations
    Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina 27710 and
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  • Virginia B. Kraus
    Correspondence
    To whom correspondence should be addressed: Box 3416, Duke University Medical Center, Durham, NC 27710. Tel.: 919-681-6652; Fax: 919-684-8907
    Affiliations
    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

    Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

    Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina 27710
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant 5P30 AG028716 from NIA (Claude D. Pepper Older Americans Independence Centers), Grant P01 AR050245 from NIAMS, Grant 5-P60-AR49465 from NIAMS (Multidisciplinary Clinical Research Center), and Grant 5-P60-AR-3070 from NIAMS (Multipurpose Arthritis and Musculoskeletal Diseases Center 1). This work was also supported by Centers for Disease Control and Prevention/Association of Schools of Public Health Grants S043 and S3486. We acknowledge use of tissues procured by the National Disease Research Interchange (NDRI) with support from National Institutes of Health Grant 5 U42 RR006042.
    This article contains supplemental Fig. 1 and Tables 1 and 2.
Open AccessPublished:December 16, 2011DOI:https://doi.org/10.1074/jbc.M111.249649
      As extracellular proteins age, they undergo and accumulate nonenzymatic post-translational modifications that cannot be repaired. We hypothesized that these could be used to systemically monitor loss of extracellular matrix due to chronic arthritic diseases such as osteoarthritis (OA). To test this, we predicted sites of deamidation in cartilage oligomeric matrix protein (COMP) and confirmed, by mass spectroscopy, the presence of deamidated (Asp64) and native (Asn64) COMP epitopes (mean 0.95% deamidated COMP (D-COMP) relative to native COMP) in cartilage. An Asp64, D-COMP-specific ELISA was developed using a newly created monoclonal antibody 6-1A12. In a joint replacement study, serum D-COMP (p = 0.017), but not total COMP (p = 0.5), declined significantly after replacement demonstrating a joint tissue source for D-COMP. In analyses of 450 participants from the Johnston County Osteoarthritis Project controlled for age, gender, and race, D-COMP was associated with radiographic hip (p < 0.0001) but not knee (p = 0.95) OA severity. In contrast, total COMP was associated with radiographic knee (p < 0.0001) but not hip (p = 0.47) OA severity. D-COMP was higher in soluble proteins extracted from hip cartilage proximal to OA lesions compared with remote from lesions (p = 0.007) or lesional and remote OA knee (p < 0.01) cartilage. Total COMP in cartilage did not vary by joint site or proximity to the lesion. This study demonstrates the presence of D-COMP in articular cartilage and the systemic circulation, and to our knowledge, it is the first biomarker to show specificity for a particular joint site. We believe that enrichment of deamidated epitope in hip OA cartilage indicates a lesser repair response of hip OA compared with knee OA cartilage.

      Introduction

      As proteins age they undergo nonenzymatic post-translational modifications. When aged proteins are intracellular, they can be repaired or the protein replaced (
      • Cloos P.A.
      • Christgau S.
      Nonenzymatic covalent modifications of proteins. Mechanisms, physiological consequences, and clinical applications.
      ); however, in the extracellular milieu, where no repair mechanisms exist, nonenzymatic modifications can accumulate in a time-dependent manner in proteins whose turnover is slow. Accumulation of these modifications in long lived proteins can potentially alter both their structural and functional properties and provide insights into turnover rates at which proteins are replaced through net synthesis and degradation processes.
      One form of nonenzymatic protein modification, deamidation, is believed to be a mechanism of amino acid damage and aging in numerous proteins (
      • McCudden C.R.
      • Kraus V.B.
      Biochemistry of amino acid racemization and clinical application to musculoskeletal disease.
      ) and a variety of tissues (
      • McCudden C.R.
      • Kraus V.B.
      Biochemistry of amino acid racemization and clinical application to musculoskeletal disease.
      ,
      • Robinson N.E.
      • Robinson A.B.
      ). Protein deamidation has been demonstrated to interfere with protein function of interleukin-1β (
      • Daumy G.O.
      • Wilder C.L.
      • Merenda J.M.
      • McColl A.S.
      • Geoghegan K.F.
      • Otterness I.G.
      Reduction of biological activity of murine recombinant interleukin-1β by selective deamidation at asparagine 149.
      ), soluble CD4 (
      • Teshima G.
      • Porter J.
      • Yim K.
      • Ling V.
      • Guzzetta A.
      Deamidation of soluble CD4 at asparagine 52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120.
      ), angiogenin (
      • Deverman B.E.
      • Cook B.L.
      • Manson S.R.
      • Niederhoff R.A.
      • Langer E.M.
      • Rosová I.
      • Kulans L.A.
      • Fu X.
      • Weinberg J.S.
      • Heinecke J.W.
      • Roth K.A.
      • Weintraub S.J.
      Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage.
      ), and Bcl-xL (
      • Daumy G.O.
      • Wilder C.L.
      • Merenda J.M.
      • McColl A.S.
      • Geoghegan K.F.
      • Otterness I.G.
      Reduction of biological activity of murine recombinant interleukin-1β by selective deamidation at asparagine 149.
      ,
      • Teshima G.
      • Porter J.
      • Yim K.
      • Ling V.
      • Guzzetta A.
      Deamidation of soluble CD4 at asparagine 52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120.
      ,
      • Deverman B.E.
      • Cook B.L.
      • Manson S.R.
      • Niederhoff R.A.
      • Langer E.M.
      • Rosová I.
      • Kulans L.A.
      • Fu X.
      • Weinberg J.S.
      • Heinecke J.W.
      • Roth K.A.
      • Weintraub S.J.
      Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage.
      ,
      • Hallahan T.W.
      • Shapiro R.
      • Strydom D.J.
      • Vallee B.L.
      Importance of asparagine 61 and asparagine 109 to the angiogenic activity of human angiogenin.
      ) and may also incite autoimmunity (
      • Moss C.X.
      • Matthews S.P.
      • Lamont D.J.
      • Watts C.
      Asparagine deamidation perturbs antigen presentation on class II major histocompatibility complex molecules.
      ). Nonenzymatic deamidation involves the conversion of Asn to Asp or the conversion of Gln to Glu in a spontaneous manner without enzymes (
      • Robinson N.E.
      • Robinson A.B.
      ). During nonenzymatic Asn deamidation, the side chain amine group is lost, causing formation of a succinimide ring. The succinimide ring is unstable and susceptible to hydrolysis at the imide to form Asp in either an α-Asp or β-Asp (isomerized) form depending upon which side of the imide hydrolysis occurs. Things are further complicated as the α-carbon atom in the succinimide ring can undergo racemization allowing the formation of both levorotatory and dextrorotatory optical isomers after hydrolysis. Similarly, Gln deamidation involves formation of a ring structure (glutarimide), where loss of the functional amine group leads to the formation of the acidic residue Glu in again both the levorotatory and dextrorotatory forms of α- or β-Glu. Asn deamidation occurs roughly twice as fast as Gln deamidation because of the less favorable 6-membered glutarimide ring structure. Particular hot spots for deamidation are predicted to exist based upon factors such as steric hindrance and protein context (
      • Robinson N.E.
      • Robinson A.B.
      ), as well as peptide sequence (for instance, dipeptides that deamidate more readily than others include Gly-Asn, Asn-Gly, and Gln-Gly (
      • Robinson N.E.
      • Robinson A.B.
      )).
      One of the tissues in the body most susceptible to accumulation of nonenzymatic protein modifications is cartilage. This is due to the slow turnover rate of many cartilage proteins; for instance, cartilage aggrecan has a predicted half-life of 25 years (
      • Maroudas A.
      • Bayliss M.T.
      • Uchitel-Kaushansky N.
      • Schneiderman R.
      • Gilav E.
      Aggrecan turnover in human articular cartilage. Use of aspartic acid racemization as a marker of molecular age.
      ), whereas cartilage collagens have predicted half-lives of at least 120 years (
      • Maroudas A.
      • Palla G.
      • Gilav E.
      Racemization of aspartic acid in human articular cartilage.
      ,
      • Verzijl N.
      • DeGroot J.
      • Thorpe S.R.
      • Bank R.A.
      • Shaw J.N.
      • Lyons T.J.
      • Bijlsma J.W.
      • Lafeber F.P.
      • Baynes J.W.
      • TeKoppele J.M.
      Effect of collagen turnover on the accumulation of advanced glycation end products.
      ). Nothing at present is known of the biological effects of these amino acid changes in cartilage, thus representing a significant knowledge gap. We hypothesized that the nonenzymatic modifications that accumulate with cartilage aging could be used to systemically monitor onset and degree of extracellular matrix loss during osteoarthritis (OA).
      The abbreviations used are: OA
      osteoarthritis
      BAJA
      Biomarkers and Joint Arthroplasty study
      COMP
      cartilage oligomeric matrix protein
      Gdn-HCl
      guanidine HCl
      JoCo OA
      Johnston County Osteoarthritis Project
      KL grade
      Kellgren-Lawrence grade of OA severity
      mAb
      monoclonal antibody
      OPD
      o-phenylenediamine dihydrochloride
      D-COMP
      deamidated COMP.
      To test this hypothesis, we identified a novel protein modification due to deamidation in cartilage oligomeric matrix protein (COMP). COMP is a noncollagenous glycoprotein and a member of the thrombospondin family of extracellular calcium-binding proteins that was initially isolated from cartilage (
      • Hedbom E.
      • Antonsson P.
      • Hjerpe A.
      • Aeschlimann D.
      • Paulsson M.
      • Rosa-Pimentel E.
      • Sommarin Y.
      • Wendel M.
      • Oldberg A.
      • Heinegård D.
      Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage.
      ). Although primarily expressed in cartilage, COMP is also expressed in tendons, meniscus, and synovial membranes. The carboxyl-terminal globular domain of COMP binds to collagens I and II (
      • Rosenberg K.
      • Olsson H.
      • Mörgelin M.
      • Heinegård D.
      Cartilage oligomeric matrix protein shows high affinity zinc-dependent interaction with triple helical collagen.
      ). The amino terminus of COMP oligomerizes to form a pentamer of five identical subunits of 110 kDa (
      • Mörgelin M.
      • Heinegård D.
      • Engel J.
      • Paulsson M.
      Electron microscopy of native cartilage oligomeric matrix protein purified from the Swarm rat chondrosarcoma reveals a five-armed structure.
      ), creating a pore that is believed to bind chloride and vitamin D3 (
      • Ozbek S.
      • Engel J.
      • Stetefeld J.
      Storage function of cartilage oligomeric matrix protein. The crystal structure of the coiled-coil domain in complex with vitamin D(3).
      ).
      We raised novel monoclonal antibodies (mAb) and developed an ELISA that specifically distinguished and quantified the deamidated form of the epitope from the total amount of the epitope (amidated and deamidated). We further demonstrated the presence of deamidated COMP (D-COMP) in articular cartilage and the systemic circulation, an enrichment of this epitope specifically in hip articular cartilage, and a correlation of this epitope in the systemic circulation with hip OA severity.

      DISCUSSION

      To our knowledge, this is the first demonstration that a cartilage extracellular matrix component, measured systemically, demonstrated specificity for OA severity at a particular joint site. We believe this reflects a fundamental underlying difference between hip and knee cartilage turnover and repair responses in OA. We observed no association between D-COMP serum levels and chronological age in non-OA subjects. This is not surprising as the half-life for generating our epitope is predicted by the Robinson and Robinson algorithm (
      • Robinson N.E.
      • Robinson A.B.
      ) to be between 39 and 50 years. Assuming COMP has a turnover rate similar to the predicted half-life of 25 years for aggrecan (
      • Maroudas A.
      • Bayliss M.T.
      • Uchitel-Kaushansky N.
      • Schneiderman R.
      • Gilav E.
      Aggrecan turnover in human articular cartilage. Use of aspartic acid racemization as a marker of molecular age.
      ), then accumulation of D-COMP with age in healthy cartilage would be prevented by normal cartilage turnover.
      We demonstrated in our post-joint arthroplasty BAJA study of 14 patients, a significant decrease in the concentrations of our new and novel D-COMP biomarker after the removal of the affected arthritic joints. The greatest decrease in D-COMP was observed for the hip, although the knee showed a much more modest decrease in D-COMP levels. As OA does not usually affect only a single joint, and even normal joint turnover would be expected to contribute to the overall serum D-COMP levels, replacement of a single joint led, as expected, to a decline but not a disappearance of D-COMP from the serum. In contrast, we did not observe any significant decrease in total COMP levels after joint arthroplasty. These results with total COMP are consistent with observations made previously by Sharif et al. (
      • Sharif M.
      • Kirwan J.R.
      • Elson C.J.
      • Granell R.
      • Clarke S.
      Suggestion of nonlinear or phasic progression of knee osteoarthritis based on measurements of serum cartilage oligomeric matrix protein levels over five years.
      ) who demonstrated that total COMP actually increased, rather than decreased, for at least 6 months following joint replacement. Other reports have demonstrated total COMP production by osteoblasts (
      • Leslie M.
      • Fang C.
      • Carlson C.
      • Tulli H.
      • Perris R.
      • DiCesare P.
      Expression of cartilage oligomeric matrix protein (COMP) by human embryonic and adult osteoblasts.
      ) and the potential for elevated total COMP during the period of reactive bone repair following joint arthroplasty. We believe that D-COMP measured systemically reflects degradation of mature aged cartilage; this is in contrast to native or total (the majority of which is native nondeamidated) COMP epitope in the systemic circulation that we posit reflects, in part, high turnover of newly synthesized tissue, or so-called “frustrated repair” (
      • Sofat N.
      Analyzing the role of endogenous matrix molecules in the development of osteoarthritis.
      ), or new COMP production from a reparative bone response occurring after joint replacement.
      In the much larger JoCo OA cohort, we were better able to refine our understanding of the utility of D-COMP as a biomarker. In this cohort, we observed that D-COMP was highly significantly correlated with hip OA severity but not with knee OA severity. We also observed that total COMP was strongly associated with knee OA but not with hip OA severity. Of note, in the JOCO cohort sample, the greatest increase in serum D-COMP occurred with the transition from the KL0-1 to the KL2 level of hip OA severity. This would suggest that D-COMP may be of particular value as an early indicator of hip pathology and OA.
      The association of total COMP with knee OA progression is well established (
      • Clark A.G.
      • Jordan J.M.
      • Vilim V.
      • Renner J.B.
      • Dragomir A.D.
      • Luta G.
      • Kraus V.B.
      Serum cartilage oligomeric matrix protein reflects osteoarthritis presence and severity. The Johnston County Osteoarthritis Project.
      ,
      • Golightly Y.M.
      • Marshall S.W.
      • Kraus V.B.
      • Renner J.B.
      • Villaveces A.
      • Casteel C.
      • Jordan J.M.
      Biomarkers of incident radiographic knee osteoarthritis. Do they vary by chronic knee symptoms.
      ,
      • Hunter D.J.
      • Li J.
      • LaValley M.
      • Bauer D.C.
      • Nevitt M.
      • DeGroot J.
      • Poole R.
      • Eyre D.
      • Guermazi A.
      • Gale D.
      • Felson D.T.
      Cartilage markers and their association with cartilage loss on magnetic resonance imaging in knee osteoarthritis: the Boston Osteoarthritis Knee Study.
      ,
      • Sowers M.F.
      • Karvonen-Gutierrez C.A.
      • Yosef M.
      • Jannausch M.
      • Jiang Y.
      • Garnero P.
      • Jacobson J.
      Longitudinal changes of serum COMP and urinary CTX-II predict X-ray defined knee osteoarthritis severity and stiffness in women.
      ,
      • Tseng S.
      • Reddi A.H.
      • Di Cesare P.E.
      Cartilage Oligomeric Matrix Protein (COMP). A biomarker of arthritis.
      ). A few studies have investigated total COMP in the setting of hip OA (
      • Chaganti R.K.
      • Kelman A.
      • Lui L.
      • Yao W.
      • Javaid M.K.
      • Bauer D.
      • Nevitt M.
      • Lane N.E.
      Change in serum measurements of cartilage oligomeric matrix protein and association with the development and worsening of radiographic hip osteoarthritis.
      ,
      • Conrozier T.
      • Saxne T.
      • Fan C.S.
      • Mathieu P.
      • Tron A.M.
      • Heinegård D.
      • Vignon E.
      Serum concentrations of cartilage oligomeric matrix protein and bone sialoprotein in hip osteoarthritis. A one-year prospective study.
      ,
      • Kelman A.
      • Lui L.
      • Yao W.
      • Krumme A.
      • Nevitt M.
      • Lane N.E.
      Association of higher levels of serum cartilage oligomeric matrix protein and N-telopeptide cross-links with the development of radiographic hip osteoarthritis in elderly women.
      ,
      • Dragomir A.D.
      • Kraus V.B.
      • Renner J.B.
      • Luta G.
      • Clark A.
      • Vilim V.
      • Hochberg M.C.
      • Helmick C.G.
      • Jordan J.M.
      Serum cartilage oligomeric matrix protein and clinical signs and symptoms of potential pre-radiographic hip and knee pathology.
      ). Three studies observed that higher levels of baseline COMP predicted the development of hip radiographic OA (
      • Chaganti R.K.
      • Kelman A.
      • Lui L.
      • Yao W.
      • Javaid M.K.
      • Bauer D.
      • Nevitt M.
      • Lane N.E.
      Change in serum measurements of cartilage oligomeric matrix protein and association with the development and worsening of radiographic hip osteoarthritis.
      ,
      • Kelman A.
      • Lui L.
      • Yao W.
      • Krumme A.
      • Nevitt M.
      • Lane N.E.
      Association of higher levels of serum cartilage oligomeric matrix protein and N-telopeptide cross-links with the development of radiographic hip osteoarthritis in elderly women.
      ,
      • Dragomir A.D.
      • Kraus V.B.
      • Renner J.B.
      • Luta G.
      • Clark A.
      • Vilim V.
      • Hochberg M.C.
      • Helmick C.G.
      • Jordan J.M.
      Serum cartilage oligomeric matrix protein and clinical signs and symptoms of potential pre-radiographic hip and knee pathology.
      ). One of these studies suggested that high baseline COMP levels were associated with a reduction in radiographic progression (
      • Chaganti R.K.
      • Kelman A.
      • Lui L.
      • Yao W.
      • Javaid M.K.
      • Bauer D.
      • Nevitt M.
      • Lane N.E.
      Change in serum measurements of cartilage oligomeric matrix protein and association with the development and worsening of radiographic hip osteoarthritis.
      ). One small study found an association between hip JSN and increasing serum COMP levels over a 1-year follow up period (
      • Conrozier T.
      • Saxne T.
      • Fan C.S.
      • Mathieu P.
      • Tron A.M.
      • Heinegård D.
      • Vignon E.
      Serum concentrations of cartilage oligomeric matrix protein and bone sialoprotein in hip osteoarthritis. A one-year prospective study.
      ), and another larger study found no association of total serum COMP with hip OA but a significant association with knee OA osteophyte (
      • Kraus V.B.
      • Kepler T.B.
      • Stabler T.
      • Renner J.
      • Jordan J.
      First qualification study of serum biomarkers as indicators of total body burden of osteoarthritis.
      ). We believe the unique ability of our D-COMP biomarker to correlate with hip but not knee OA severity demonstrates the utility and value of studying post-translational modifications in cartilage proteins as biomarkers of OA.
      To better understand the underlying biological basis for D-COMP as a hip OA but not knee OA biomarker, we investigated the concentrations of D-COMP in cartilage extracts from OA hips and knees. We hypothesized that D-COMP would be increased in cartilage with a low turnover rate and that biologically older tissues would have a higher D-COMP/total COMP ratio. We observed a significantly higher mean D-COMP/total COMP ratio in OA hip cartilages compared with OA knee cartilages. Total COMP concentrations between hip and knee cartilage were not significantly different confirming that the differences observed in the ratios were due to a higher proportion of D-COMP in hip cartilage. These data strongly suggest that COMP, and by inference the cartilage extracellular matrix in an OA hip joint, is turned over (net of catabolism and anabolism) at a much lower rate than cartilage in an OA knee joint. Conversely, this would indicate that knees are more robust at repairing ongoing degradation than hips.
      In the hip, we also observed significantly higher concentrations of D-COMP and D-COMP/total Comp ratio in lesional OA cartilage when compared with remote regions. The elevated concentrations of D-COMP at hip OA lesions further support the serum biomarker observation that D-COMP was associated with hip OA. Moreover, this suggests that lesional cartilage is biologically older than the cartilage remote from the lesion and is consistent with lower rates of COMP synthesis at sites of hip OA lesions. For comparison, we included non-OA and age-matched cartilage collected as cadaveric tissue or shortly after trauma at the time of surgical repair. We found no significant difference between non-OA hip and knee cartilage for either D-COMP or total COMP. However, the D-COMP/total COMP ratio of OA lesional hip cartilage was significantly higher than non-OA cartilage suggesting that cartilage at the lesion is older. In contrast, we observed the opposite in the knee, with significantly lower than normal D-COMP/total COMP ratios in the OA tissue. As we were unable to find any evidence of enhanced cross-linking of older COMP in the knee OA cartilage, we believe these data support the hypothesis that serum D-COMP reflects cartilage turnover and that hip OA tissue is older (low repair response) than non-OA hip, whereas the knee OA tissue is younger (high repair response) than non-OA.
      Although higher concentrations of D-COMP at hip lesions could potentially be explained by accelerated D-COMP production, we believe this to be unlikely as there was no evidence for accelerated production of D-COMP at knee OA lesions, which would be expected to be exposed to very similar catabolic conditions during cartilage loss. Radiolabeled proteoglycan studies in cartilage have shown that cartilage matrix turnover is greatest in the superficial zone whereas the matrix becomes older and the protein turnover rate decreases deeper into the cartilage (
      • Maroudas A.
      Glycosaminoglycan turnover in articular cartilage.
      ). Therefore, we favor a model wherein gradual erosion of the cartilage at hip OA lesions leads, in the relative absence of synthetic repair, to the loss of the superficial and medial cartilage layers, leaving behind the older deeper zones.
      In summary, we were able to predict a deamidation event in the cartilage matrix molecule COMP, demonstrate its presence in human cartilage, produce specific mAbs to this modification, and develop an ELISA to study its utility as a biomarker in OA. We identified a D-COMP modification that is not only a novel biomarker in OA but also a biomarker with specificity for hip OA, which, to the best of our knowledge, is the first OA biomarker specific to a particular joint site. Based upon our studies of COMP in cartilage, we believe we can explain the specificity of our D-COMP biomarker through a lower COMP protein synthesis in hip OA when compared with knee OA and higher rate of knee cartilage extracellular matrix turnover. The clear differences in D-COMP concentrations at remote OA hip and knee cartilage are consistent with different biological aging rates in these two large joint sites in OA due in part to less COMP synthesis in hips than knees. Hip OA cartilage lesions, in particular, were differentially enriched for D-COMP. This is compatible both with loss of the more rapidly regenerating superficial cartilage layers and inadequate COMP synthesis, resulting in biologically older remaining deep layers of hip OA cartilage. We believe that D-COMP warrants further study as a marker of hip disease to determine more clearly its utility in a patient setting, although currently we would envision its use as a longitudinal marker to follow disease progression within a subject. Examination is also warranted to determine its ability to detect occult or pre-radiographic hip disease.

      Acknowledgments

      We thank David Quintana for assistance with the BAJA sample collection. We also thank the Duke Proteomics Core Facility (Drs. Erik Soderblom and Arthur Moseley) for their expert assistance and Dr. Vladimir Vilim for the gift of 17-C10 antibody and purified COMP from human cartilage.

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