Intermolecular crosslinking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament

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Abstract
A trypsin digest of denatured NaB3H4-reduced native bovine periodontal ligament was prepared and fractionated by gel filtration and cellulose ion-exchange column chromatography. Prior to trypsin digestion, a complete acid hydrolysate was subjected to analyses for nonreducible stable and reducible intermolecular cross-links. Minute amounts of the former and significant amounts of the reduced cross-links dihydroxylysinonorleucine (1.1 mol/mol of collagen), hydroxylysinonorleucine (0.9 mol/mol of collagen), and histidinohydroxymerodemosine (0.6 mol/mol of collagen) were found. The covalent intermolecular cross-linked two-chained peptides that were isolated were subjected to amino acid and sequence analyses. The structures for the different two-chained linked peptides were .alpha.1CB4-5(76-90)[Hyl-87] .times. .alpha.1CB6-(993-22c)[Lysald-16c], .alpha.-1CB4-5(76-90)[Hyl-87] .times. .alpha.1CB6(993-22c)[Hylald-16c], .alpha.2CB4(76-90)[Hyl-87] .times. .alpha.1CB6(993-22c)[Lys-ald-16c], and .alpha.2CB4(76-90)[Hyl-87] .times. .alpha.1CB6(993-22c)[Hylald-16c]. The cross-link in each peptide was glycosylated. This is the first characterization by sequence analysis of a cross-link involving Hyl-87 in an .alpha.2 chain in collagen. A stoichiometric conversion of residue 16c aldehyde to an intermolecular cross-link in each of the COOH-terminal nonhelical peptide regions of both .alpha.1 chains in a molecule of type I collagen was found. The ratio of .alpha.1 to .alpha.2 intermolecularly cross-linked chains involved was 3.3:1, indicating a stereospecific three-dimensional molecular packing of type I collagen molecules in bovine periodontal ligament.