In vitro and in vivo association of dentin phosphophoryn with alpha1CB6 peptide of type I collagen.

A small number of molecules of phosphophoryns, dentin phosphoproteins, are associated to collagen tightly with the maturation of dentin. As an in vitro model of the process, we examined non-covalent association of phosphophoryns and collagen CNBr peptides. Cyanogen bromide peptides of type I collagen were separated with electrophoresis and transferred to a membrane, which was probed with labeled phosphophoryns. Phosphophoryns were bound preferentially to alpha1CB6 peptide. Another experiment using a cleavable crosslinking agent also demonstrated the affinity between phosphophoryns and the alpha1CB6 region of collagen. The matrix-bound fraction of phosphophoryns was solubilized by CNBr digestion of bovine dentin, and was partially purified. Compositional analysis revealed that the fraction was composed of association products of phosphophoryns and collagen at the ratio of 2:3. Considering the high molecular weight of the fraction, one phosphophoryn molecule should be associated with several collagen peptides. The fraction reacted with the antibody against alphalCB6 peptide. When the fraction was digested with lysyl endopeptidase, several peptides that coincided with peptides from alpha1CB6 were released. One of the peptides was sequenced and identified to be a peptide with Asp 975 of the alpha1(I) chain as an amino terminal residue. The alpha1CB6 peptide may be involved in the matrix-bound fraction, although involvement of other collagen peptides cannot be ruled out. Phosphophoryns may associate with collagen at the alpha1CB6 region at first, and then be immobilized on it, possibly by covalent crosslinking, with maturation of dentin. Binding of several alpha1CB6 peptides to a single phosphophoryn molecule is possible in current 3-dimensional models of collagen packing in mineralized tissue, which allows alignment of several hole zones in register.