Identification of a major antigenic epitope on CNBr‐fragment 11 of type II collagen recognized by murine autoreactive B cells

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Abstract
Immunization of certain strains of mice with native type II collagen (CII) induces both development of arthritis and an antibody response to autologous CII. The autoantibody response in a high‐responder strain, the DBA/1 mouse, has been described earlier, and a number of monoclonal antibodies have been characterized for arthritogenicity and autoreactive binding to cartilage in vivo and in vitro. Here we map the antigenic epitope of one of these arthritogenic monoclonal antibodies (CII‐C1). It belongs to a group of antibodies recognizing the CNBr fragment α1(II)‐CB11 of CII. Using the enzyme‐linked immunosorbent assay technique, we show that the antibody reacts only with native, triplehelical CII, but not with other collagens. The antibody is able to stain specifically the CB11 fragment by immunoblotting, suggesting some partial renaturation of the CNBr fragment into triple‐helical structures after blotting. The binding site of CII‐C1 on CB11 was further focused by rotary shadowing of antibody‐labeled CII to a site 89 ± 8 nm from the amino end of CII, corresponding to the middle of CB11. This location was confirmed by cleavage of CB11 with trypsin, separation of the tryptic peptides by high‐performance liquid chromatography and dot‐blot analysis of the antigenic peptides with the CII‐C1 antibody. Sequencing of the single positive peptide located the antigenic epitope within the sequence GFAGQAGPAGAT‐GAPGRP (residues 316–333). Assuming 0.29 nm per residue, this corresponds to a position within 92–96.5 nm from NH2 terminal end of CII. Apart from glycine residues, which are not exposed on the triple‐helical structure, only two amino acid residues (F‐x‐y‐Q) are conserved in CII from different species but are not found in the triple‐helix of other collagens except type IV collagen. Therefore, this structure is likely to be of critical importance for the binding of the CII‐C1 antibody. Of potential importance is that this structure is also found in certain other arthritogenic proteins such as 65‐kDa mycobacterial protein, in CMV and EBV.