Studies on the structure and chemistry of dentin collagen-phosphophoryn covalent complexes

Bovine and rat dentin contain aspartylphosphoseryl-enriched collagen-associated phosphoproteins which represent 1-2% of the mineral-free dry weight. These phosphophoryn moieties are not extracted by saturated neutral EDTA, pH 7.4, nor by guanidine hydrochloride-EDTA, pH 7.4. Cyanogen bromide degradation of the dentin matrix does release a high molecular weight fragment containing hydroxyprolyl, hydroxylysyl, prolyl, and glycyl residues as well as high concentrations of aspartyl and phosphoseryl residues, the amounts of which indicate a 50% collagen-50% phosphophoryn nature. Gel filtration and ion exchange chromatography under dissociative, denaturing conditions, as well as in the presence of disulfide bond reducing reagents failed to separate the collagen and phosphophoryn moieties. Hydroxyapatite, which selectively absorbs phosphophoryn, also failed to separate the collagenous component, leading to the conclusion that the moieties represented a covalent conjugate.³¹P NMR spectroscopy showed the bovine collagen-phosphophoryn complex to contain only phosphomonoesters similar to soluble phosphophoryn. Reduction with [³H]NaBH₄, followed by cross-link analysis, did not reveal any reduced aldimine cross-link amino acids. Of the 4 hydroxylysyl residues/1000 in the intact bovine collagen-phosphophoryn complex, one-fourth are periodate resistant, indicating either O-or N-substitution. The periodate-resistant hydroxylysyl residues are located in bacterial collagenase-sensitive regions, and it is likely that these represent hydroxylysine O-glycosides. These data suggest that: (a) the collagenous component of the conjugate derives from a glycosylated peptide, probably α2CB4, and (b) the association is covalent, but does not involve disulfides, phosphate-, hydroxylysine-, or reducible aldehyde-mediated covalent bonds.