Abstract:
The covalent intermolecular cross-linking is essential in providing collagen matrices with physicochemical properties. Dehydro-histidinohydroxymerodesmosine is a major tetravalent collagen cross-link present in most non-mineralized tissues. Despite its abundance, this cross-link has been poorly characterized. The objectives of this study were to isolate the cross-linked peptides, determine the molecular loci and evaluate the state of glycosylation of the cross-link. The peripheral layer of the turkey leg tendon (never-mineralized collagenous tissue) was reduced with tritiated sodium borohydride, subjected to sequential enzymatic/chemical digestion and several cross-linked peptides were isolated by means of HPLC. Based on the amino-terminal sequence, amino acid composition and mass spectrometric analyses of the purified peptides, three molecular loci were identified. They are lysyl aldehyde (alpha1-9N) X lysyl aldehyde (alpha1-9N) X hydroxylysine (alpha1-930) X histidine (alpha2-935), lysyl aldehyde (alpha1-9N) X lysyl aldehyde (alpha2-6N) X hydroxylysine (alpha1-930) X histidine (alpha2-935) and lysyl aldehyde (alpha1-9N) X lysyl aldehyde (alpha2-6N) X hydroxylysine (alpha1-930) X histidine (alpha1-932). The data indicate that the aldol involved in the cross-link is derived from the amino-terminal telopeptidyl aldehydes, and that the hydroxylysine on an alpha1 chain (residue 930) and histidine from alpha2-935 is the preferred cross-linking site. The latter suggests a stereospecific packing of the type I collagen molecules in the fibril. In addition, chemical and mass spectroscopic analyses of the peptides and their acid/base hydrolysates revealed that the hydroxylysine residues (alpha1-930) that are involved in the formation of this complex cross-link were not glycosylated. These results suggest that the carbohydrate moiety on the alpha1-930 hydroxylysine may have a role in regulating the cross-link formation.
