Molecular site specificity of pyridinoline and pyrrole cross-links in type I collagen of human bone.

Compared with soft tissue collagens, bone type I collagen displays a distinctive pattern of covalent cross-linking, with evidence of preferred sites of molecular interaction and a prominence of both immature, divalent cross-links and mature, trivalent cross-links in the adult tissue. In this study the site-specificity of the mature cross-links in human bone collagen was examined. Peptides containing fluorescent pyridinoline cross-links and Ehrlich's-reactive pyrrole cross-links were isolated from a bacterial collagenase digest of demineralized bone matrix. The digest was fractionated by molecular sieve chromatography, monitoring for peptide absorbance, pyridinoline fluorescence, pyrroles by Ehrlich's reagent, and immunoassay for cross-linked N-telopeptides. Individual cross-linked peptides were resolved by ion-exchange and reverse-phase HPLC. Structures were established by NH2-terminal microsequencing, cross-link analysis, electrospray mass spectrometry, and immunoassay. Two, about equally occupied, sites of pyridinoline cross-linking were identified, N-telopeptide to helix and C-telopeptide to helix. Pyrroles were alternative cross-linking products at the same sites, but concentrated (85%) at the N-telopeptide end. Only one combination of chains was cross-linked by pyridinolines at the C-telopeptide to helix site, [alpha1(I)C]2alpha1(I)helix. Several peptide combinations arose from the N-telopeptide to helix site, but the main source of pyridinolines was from the locus, alpha1(I)Nalpha2(I)Nalpha1(I)helix. Pyridinolines linking two alpha1(N) telopeptides were a minor component. Pyrroles were concentrated at the locus, alpha1(I)Nalpha2(I)Nalpha2(I)helix. The cross-link ratio of hydroxylysylpyridinoline to lysylpyridinoline differed between N-telopeptide and C-telopeptide sites, and between the individual interchain combinations. Cross-linked N-telopeptides accounted for two-thirds of the total lysylpyridinoline in bone. N-telopeptide pyridinoline fluorescence was quenched on chromatography, so that reliance on peptide fluorescence alone can underestimate the level of N-telopeptide pyridinoline cross-linking.