Porcine Dentin Phosphoprotein: Polymorphisms, Phosphorylations, and Glycosylations

Dentin phosphoprotein (DPP) is the C-terminal domain of dentin sialophosphoprotein (DSPP). DPP isolated from 40 g of porcine dentin powder migrates as a cluster of three or four distinct bands at 100-kDa on SDS-PAGE. Objectives: To determine the structural reasons for DPP size heterogeneity. Methods: Porcine molars at the crown formation stage were surgically extracted from 22 pigs and the soft tissue and enamel removed. The remaining material was pulverized in a jaw crusher. The dentin powder from each pig was sequentially extracted with guanidine (G), acetic acid (A) and acetic acid with sodium chloride (AN). DPP was isolated from AN extract by size exclusion chromatography followed by RP-HPLC. DPP bands having different mobilities on SDS-PAGE were characterized by N-terminal sequencing. DPP was dephosphorylated and deglycosylated, and compared to the native protein on SDS-PAGE. DPP phosphorylation levels were measured in two ways: by colorimetric assay after dephosphorylation with acid phosphatase and by HPLC analysis of phosphoamino acids following acid hydrolysis. To identify glycosylation sites, DPP was digested with pronase and the products were isolated by size exclusion chromatography and analyzed for the presence of glycopeptides using a phenol-sulfuric acid assay. Glycosylation positive peptides were further purified by HPLC and characterized by N-terminal sequencing. Results: DPP from individual pigs showed either one or two distinct DPP bands on SDS-PAGE. DPP contains 150 phosphates per molecule and is glycosylated in its N- and C-terminal domains. Conclusion: DPP is a highly phosphorylated glycoprotein. DPP heterogeneity is not due to variations in proteolytic processing or variations in its phosphorylation or glycosylation levels. We conclude that DPP size heterogeneity is due to allelic variations in the length of the DPP coding regions, and are not due to variations in its posttranslational modifications. This study is supported by NIDCR grant DE015846.