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

Abstract

Bovine and rat dentin contain aspartylphosphoseryl-enriched collagen-associated phosphoproteins which represent 1-2% of the mineralfree 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. 3~p 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 collagenphosphophoryn 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 Oglycosides. These data suggest that: (a) the collagenous component of the conjugate derives from a glycosylated peptide, probably c~2CB4, and (b) the Present address: Send offprint request~s to Dr. Arthur Veis at above address. association is covalent, but does not involve disulfides, phosphate-, hydroxylysine-, or reducible aldehyde-mediated covalent bonds.

Ultrastructural localization and gradient of activity of alkaline phosphatase activity during rodent odontogenesis

“…In the matrix, strong binders of calcium, such as proteoglycans in the ground substance and phosphoproteins convalently linked to collagen [9], may need to be broken down by phosphatase activity to liberate Ca ions, whereas pyrophosphatase activity near these sites may be required to break down serum inorganic pyrophosphate, an inhibitor of mineralization, to liberate PO]-ions. Consistent with this hypothesis, the first activity was observed as a pattern of small dots of lead deposit similar in distribution to that of cross-linking proteoglycans and glycoproteins in forming matrix as revealed by ruthenium red and phosphotungstic acid staining [10,11].…”

Systematic purification of free and matrix-bound phosphophoryns of bovine dentin: Presence of matrix-bound phosphophoryn as a distinct molecular entity

“…However, it is thought to be covalent bonding, as claimed by Lee and Veis [4], since artificial crosslink products behaved similarly to matrixbound phosphophoryn in DEAE-cellulose chromatography ( Fig. 2A and Fig.…”

“…This organic phosphate-containing component was believed to be a complex of phosphophoryn and collagen [3][4][5]. Lee and Veis [4] suggested that the association between phosphophoryn and collagen was covalent in nature, whereas Linde et al [6] claimed that the association was noncovalent and probably electrostatic bonding.…”

Presence of lysinoalanine and histidinoalanine in bovine dentin phosphoprotein

“…However, it has been known that the decalcified insoluble matrix of the bovine dentin collagen contains a small amount (about 1-2%) of phosphoprotein which firmly attached to the main component, collagen (3). In addition to this boundtype phosphoprotein, the same species of phosphoprotein is present as a soluble form in the EDTA extract of this tissue (4,5).…”