Separate and overlapping specificities in rheumatoid arthritis antibodies binding to citrulline- and homocitrulline-containing peptides related to type I and II collagen telopeptides

Abstract

Introduction: Our objective was to find out if there are antibodies binding to homocitrulline-containing type I and II collagen carboxyterminal telopeptides in sera of patients with rheumatoid arthritis (RA), and if these antibodies cross-react with citrulline and homocitrulline in the same peptide sequence.

Methods: A total of 72 RA and 72 control sera were analyzed for binding using enzyme-linked immunosorbent assay to citrulline- or homocitrulline-containing type I and II collagen carboxyterminal telopeptides, as well as to cyclic citrullinated peptide (CCP) and to mutated citrullinated vimentin (MCV). Specificities of the antibodies were tested using inhibition-ELISA.

Results: Of the RA sera, 39 (54%) and 41 (57%) were positive for binding to CCP and MCV, respectively. Further, 34 (47%) and 30 (42%) of the patients had specific antibodies binding to and being inhibited by citrulline-containing type I collagen telopeptides and by citrulline-containing type II collagen carboxyterminal telopeptides, respectively. The corresponding figures regarding homocitrulline-containing type I and homocitrulline-containing type II collagen telopeptides were 16 (22%) and 14 (19%). Most of the patients, who were seropositive for citrullinated peptides, showed binding in multiple assays. A total of 10 (14%) RA patients were positive for all the tested peptide pairs, while 28 (39%) of them had antibodies that contained overlapping specifities between citrulline and homocitrulline in the same peptide sequence.

Conclusions: Antibodies to both citrulline and homocitrulline containing type I and II collagen telopeptides can be found in sera of RA patients. These antibodies are not constant from one RA patient to another, but contain separate or overlapping specificities within the same peptide sequence varying between individuals. Our results suggest some relationship between citrulline and homocitrulline-recognizing antibodies, since homocitrulline antibodies exist mainly in individuals seropositive to anti-CCP and anti-MCV.

Figure 1

Illustration of the specific binding (inhibition-%) in RA patients (n = 72) and in controls (n = 72). Mean is shown as a solid line and ± 2SD is shown by dotted lines. For abbreviations, see Table 1. RA, rheumatoid arthritis; SD, standard deviation.

Figure 2

Overview of the antibody specifities in controls (panels A and C) and RA patients (panels B and D) and RA patients’ CCP and MCV results. In panels A and B, antibodies specific to CitI are depicted in dark columns/blue and those specific for HcitI in light columns/green. In panels C and D, antibodies to CitII are presented in dark columns/blue and those to HcitII in light column/green. The values shown are percent of inhibition by respective soluble peptides to binding to the similar immobilized peptide. Panel E shows the binding of RA patient sera to the antigens of the CCP and MCV assays, shown as arbitrary units defined by each manufacturer. CCP, cyclic citrullinated protein; Cit, citrulline; Hcit, homocitrulline; MCV, mutated citrullinated vimentin; RA, rheumatoid arthritis.