Abstract
C-telopeptide of type II collagen (CTX-II) has been shown to be a highly relevant biomarker of cartilage degradation in human rheumatic diseases, if measured in synovial fluid or urine. However, serum or plasma CTX-II have not been demonstrated to have any clinical utility to date. Here, we describe the GPDPLQ1237 ELISA which targets the EKGPDPLQ↓ neo-epitope, an elongated version of the CTX-II neo-epitope (EKGPDP↓), speculated to be a blood-precursor of CTX-II generated by the cysteine protease cathepsin K. Human osteoclast cartilage resorption cultures as well as oncostatin M and tumour necrosis factor α-stimulated bovine cartilage explant cultures were used to validate GPDPLQ1237 biologically by treating the cultures with the cysteine protease inhibitor E-64 and/or the matrix metalloproteinase (MMP) inhibitor GM6001 to assess the potential contributions of these two protease classes to GPDPLQ1237 release. Cartilage resorption-derived GPDPLQ1237 release was inhibited by E-64 (72.1% inhibition), GM6001 (75.5%), and E-64/GM6001 (91.5%), whereas CTX-II release was inhibited by GM6001 (87.0%) but not by E-64 (5.5%). Cartilage explant GPDPLQ1237 and CTX-II release were both fully inhibited by GM6001 but were not inhibited by E-64. No clinically relevant GPDPLQ1237 reactivity was identified in human serum, plasma, or urine from healthy donors or arthritis patients. In conclusion, the GPDPLQ1237 biomarker is released during osteoclast-derived cysteine protease- and MMP-mediated cartilage degradation in vitro, whereas CTX-II release is mediated by MMPs and not by cysteine proteases, as well as from MMP-mediated cartilage degradation under a pro-inflammatory stimulus. These findings suggest that GPDPLQ1237 may be relevant in diseases with pathological osteoclast activity and cartilage degradation. Further studies are required to validate the neo-epitope in human samples.
Highlights
Peptide identifier Biotinylated peptide Standard peptide Elongated +1 peptide Elongated +2 peptide Truncated −1 peptide Truncated −2 peptide Immunogenic peptide
In this study we investigated the release of this neo-epitope from non-calcified articular cartilage, in in vitro models of cartilage degradation derived from osteoclasts[36] or inflammation[24], using the novel competitive GPDPLQ1237 enzyme-linked immunosorbent assay (ELISA)
E-64/GM6001 combined did not result in further inhibition of CTX-II release (61.1 ± 11.6% inhibition, P = 0.246). These findings indicate that the release of GPDPLQ1237 and CTX-II neo-epitopes from articular cartilage undergoing osteoclast-mediated degradation are not mediated by the same proteases; while we demonstrated that both matrix metalloproteinase (MMP) and cysteine proteases contribute to GPDPLQ1237 release during osteoclastic cartilage resorption, only MMPs contributed to CTX-II release
Summary
Peptide identifier Biotinylated peptide Standard peptide Elongated +1 peptide Elongated +2 peptide Truncated −1 peptide Truncated −2 peptide Immunogenic peptide. SF collection is more complicated and inconvenient than collecting either serum, plasma, or urine and is a more complex biological matrix to work with. Osteoclasts play important roles in diseases with progressive joint destruction, for bone erosion in diarthrodial joints in RA30–34. Their role in cartilage alterations—such as those seen in OA—is poorly understood, albeit strongly implicated[35]. An elongated CTX-II neo-epitope (EKGPDPLQ↓) has been previously described in patent literature[26,27,28], where it has been suggested to be generated by cathepsin K-mediated cleavage of calcified cartilage, to be osteoclast-specific, and to be present in blood while being absent or only present in trace amounts in urine and SF. The validated assay was used to test for GPDPLQ1237 reactivity in human serum, plasma, and urine
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