Dentin sialophosphoprotein (DSPP) is critical for proper human dentin formation, as DSPP mutations have been identified in kindreds with type II dentin dysplasia (DD-II) and dentinogenesis imperfecta types II and III (DGI-II; DGI-III), and because Dspp^-/- mice display tooth defects that resemble human DGI-III. Shortly after DSPP is synthesized by odontoblasts, it is cleaved into three structural domains: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). Porcine DSP contains putative N-glycosylations (9), O-glycosylations (>3), and glycan attachments (3). To date, only one putative glycosylation site (Asn¹⁵⁵) has been determined experimentally. Objectives: to characterize the structural properties of DSP. Methods: DSP was isolated from surgically extracted unerupted pig third molars and digested with pronase. Pronase degrades most of the protein, but cannot cleave the peptide backbone close to bulky posttranslational modifications. The cleavage products were separated by size exclusion chromatography, and the phenol-sulfuric acid-positive fraction (indicating carbohydrate attachments) was fractionated by RP-HPLC. Each fraction was characterized by Edman sequencing. Results: Protein sequences were obtained for six of the glycopeptides: 1) GxGDDEGEE, x = S²⁶⁵; 2) EDVGxA, x = N¹⁷⁰; 3) EETGVxSGGSGA, x = T²³¹; 4) EETGVxSGGSG, x = T²³¹; 5) GGxSSSAR, x = N⁹²; and 6) TxPPGEGEI, x = T²¹⁵. In each case, the blank cycle (x) corresponded to a potential glycosylation or glycan attachment site. Conclusions: Whether or not eight potential carbohydrate attachment sites are glycosylated in porcine DSP was determined. Ser²⁶⁵ is, but Ser²⁴⁵ is not, a glycosaminoglycan attachment site. Asn⁹² and Asn¹⁷⁰ are both N-linked glycosylation sites. Thr²¹⁵ and Thr²³¹ are, but Thr²¹⁴ and Thr²²⁸ are not, O-linked glycosylation sites. Better characterization of the structure of DSP is important to gain insight into function in forming dentin. This study is supported by NIDCR grants DE12769 and DE15846.

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