Identification of hydroxypyridinium cross-linking sites in type II collagen of bovine articular cartilage.
In mature cartilage, collagen fibrils are strengthened by covalent intermolecular bonds provided by 3- 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, about one in three alpha chains from pepsin-soluble collagen was found to contain a hydroxypyridinium residue. Its distribution in the chains was limited to two 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 two peptides indicated that the main component of one was alpha 1(II)- CB9 ,7 and of the other alpha 1(II) CB12 . It was suspected that two amino-terminal telopeptides were cross-linked by hydroxylysylpyridinoline to alpha 1(II) CB9 ,7 and two 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, one cross-linked peptide released alpha 1(II) CB9 ,7, and the other, alpha 1(II) CB12 . The findings indicate there are only two 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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002358:Cartilage, Articular / Q000032:analysis*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000302:isolation & purification*; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis; D011725:Pyridines / Q000032:analysis*
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011725:Pyridines; C023500:3-hydroxypyridine; D003094:Collagen; D010434:Pepsin A; D003488:Cyanogen Bromide