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. 2022 Dec 27;24(1):454.
doi: 10.3390/ijms24010454.

A Highly Sensitive Biomarker of Type II Collagen C-Terminal Pro-Peptide Associated with Cartilage Formation

Helena Port  1   2 , Anne-Christine Bay-Jensen  1 , Yi He  1 , Morten A Karsdal  1 , Thorbjørn Gantzel  3 , Christian S Thudium  1 , Signe Holm Nielsen  1
Affiliations

A Highly Sensitive Biomarker of Type II Collagen C-Terminal Pro-Peptide Associated with Cartilage Formation

Helena Port et al. Int J Mol Sci. .

Abstract

The type II collagen C-terminal pro-peptide is one of the most abundant polypeptides in cartilage. The purpose of this study was to develop a competitive chemiluminescence enzyme-linked immunosorbent assay, CALC2, targeting this pro-peptide as a marker of cartilage formation. Technical assay parameters were evaluated. CALC2 level was measured after in vitro cleavage of recombinant type II collagen with bone morphogenetic protein-1 (BMP-1) and treatment of ex vivo human osteoarthritis (OA) cartilage explant model (HEX) with insulin-like growth factor-1 (IGF-1). Serum CALC2 levels were assessed in 18 patients with rheumatoid arthritis (RA), 19 patients with ankylosing spondylitis (AS), and 18 age- and sex-matched controls in cohort 1 and 8 patients with OA and 14 age- and sex-matched controls in cohort 2. Type II collagen cleavage with BMP-1 increased the CALC2 level. IGF-1 treatment increased the CALC2 levels in HEX compared with the untreated explants (p < 0.05). Results were confirmed using Western blot analysis. CALC2 levels were decreased in the patients with RA and AS compared with the healthy controls (p = 0.01 and p = 0.02, respectively). These findings indicate that CALC2 may be a novel biomarker of type II collagen formation. However, further preclinical and clinical studies are required to validate these findings.

Keywords: C-terminal pro-peptide; ankylosing spondylitis; biomarker; cartilage; osteoarthritis; rheumatoid arthritis; type II collagen formation.

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Conflict of interest statement

A.-C.B.-J., Y.H., M.A.K., C.F.T. and S.H.N. are employeesand own stocks in Nordic Bioscience. The funders had no role in the design of the study; collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. All other authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1
CALC2 epitope location in type II collagen. Other biomarkers of type II collagen formation (PRO-C2, NPII, PIIANP, CPII, Pcol II-C, and PIICP) and degradation (T2CM, CTX-II, and C2M) are also represented.
Figure 2
Figure 2
Basic Local Alignment Search Tool sequence alignment of the targeted sequence for CALC2 in human with those in mouse, bovine, and rat. The sequence is marked in red.
Figure 3
Figure 3
Assay validation and proof of concept. (A), Specificity of the monoclonal CALC2 antibody. The monoclonal antibody reactivity towards the selection, truncated, elongated, and non-sense standard and coating peptides was tested. Data are shown as B/B0, where B represents the relative light units (RLU) at given concentrations, and B0 represents the RLU at 0 ng/mL peptide, and are presented as mean values. (B), In vitro cleavage of recombinant type II collagen with BMP-1. Recombinant type II collagen was incubated with BMP-1 for 24 h, and CALC2 levels were quantified (n = 3). Data are shown as mean ± standard deviation (SD).
Figure 4
Figure 4
Measurements of CALC2 levels in supernatant from OA HEX cultured for 35 days in serum-free Dulbecco’s modified Eagle medium/F12 medium with insulin-like growth factor 1 (IGF-1) and a control group without (w/o) treatment. (A) CALC2 levels over the culture period in the first study. (B) Validation of the Western blot analysis results for one patient in the first study. (C), CALC2 levels over the culture phases in the second study, baseline (day 0), early phase (days 3, 5, and 7), mid-phase (days 10, 12, and 14), and late phase (days 17, 19, and 21). The values in panels (A,C) are shown as mean ± SD, and treatments were compared using a Mann–Whitney test at each time point. Asterisks indicate the following: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 5
Figure 5
Serum CALC2 levels in the healthy donors and joint-related diseases investigated in the two cohorts. (A) CALC2 levels of cohort 1. (B) CALC2 levels of cohort 2. Data are shown as mean ± SD, and CALC2 levels are compared using Mann–Whitney tests. Asterisks indicate the following: * p < 0.05, ** p < 0.01.
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