Structural analysis of cross-linking domains in cartilage type XI collagen: Insights on polymeric assembly

Abstract

The collagen framework of hyaline cartilage is based on copolymers of types II, IX, and XI collagens. Previous studies have established specific covalent interactions between types II and IX collagens. The present study examined cross-linking sites in type XI collagen to define better the full heteropolymeric assembly. Pepsin-solubilized type XI collagen was purified from fetal bovine cartilage. The cross-linking amino acids in the preparation were primarily divalent, borohydride-reducible structures; pyridinoline residues were essentially absent. Individual α1(XI), α2(XI), and α3(XI) chains were resolved by high performance liquid chromatography. Telopeptides still attached by cross-links to helical sites were released by periodate oxidation and identified by microsequencing. Analysis of cross-linked peptides isolated from trypsin digests of each α-chain identified the attachment helical sites for the telopeptides. A high degree of interchain specificity was evident in the cross-linking between type XI collagen molecules. The dominant cross-links were between N-telopeptides and the COOH terminus of the triple-helix, consistent with a head-to-tail interaction of molecules staggered by 4D (D = 67 nm) periods. In addition, α1(II) C-telopeptide was linked to the amino-terminal site of the α1-(XI) triple helix. In summary, the results show that type XI collagen molecules are primarily cross-linked to each other in cartilage, implying that a homopolymer is initially formed. Links to type II collagen are also indicated, consistent with an eventual cofibrillar assembly. Analysis of cartilage extracts showed that all three chains, α1(XI), α2(XI), and α3(XI), had at least in part retained their N-propeptides in cartilage matrix and that the α3(XI) chain was the IIB splicing variant product of the COL2A1 gene. Of particular note was the finding that the N-telopeptide cross-linking site in both α1-(XI) and α2(XI) is located amino-terminal to the putative N-propeptidase cleavage site. This structural feature provides a potential mechanism for the proteolytic depolymerization of type XI collagen by proteases that can cleave between the cross-link and the triple helix (e.g. stromelysin).