Identification of hydroxypyridinium crosslinking sites in type II collagen of bovine articular cartilage

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Abstract
In mature cartilage, collagen fibrils are strengthened by covalent intermolecular bonds provided by 2-hydroxypyridinium cross-linking residues. To determine the location of these trifunctional cross-links within the type II collagen molecule, CNBr peptides were analyzed from pepsin-soluble collagen and from guanidine hydrochloride insoluble collagen of bovine articular cartilage. The presence of hydroxypyridinium residues in collagen .alpha. chains and CNBr-derived peptides was detected by their characteristic natural fluorescence. Quantitatively, .apprx. 1 in 3 .alpha. chains from pepsin-soluble collagen contained a hydroxypyridinium residue. Its distribution in the chains was limited to 2 CNBr peptides, which were purified by column chromatography on CM-cellulose and Bio-Gel P-30 followed by slab-gel electrophoresis in sodium dodecyl sulfate-polyacrylamide. The composition and properties of the 2 peptides indicated that the main component of 1 was .alpha.1(II)-CB9,7 and of the other .alpha.1(II)CB12. Two amino-terminal telopeptides were cross-linked by hydroxylysylpyridinoline to .alpha.1(II)CB9,7 and 2 carboxy-terminal telopeptides to .alpha.1(II)CB12. The properties of fluorescent CNBr peptides isolated from digests of insoluble cartilage collagen supported this conclusion. Cleavage of the 3-hydroxypyridinium ring by UV light was exploited to confirm the identity of the cross-linked peptides. On UV irradiation, 1 cross-linked peptide released .alpha.1(II)CB9,7, and the other, .alpha.1(II)CB12. Evidently, there are only 2 hydroxypyridinium cross-linking sites within the triple-helical region of the type II collagen molecule, probably placed symmetrically at opposite ends at residues 87 and 930, where telopeptide aldehydes are known to react to form the initial head to tail intermolecular bonds.