Identification of Human Dentin Sialophosphoprotein (DSPP)-Derived Proteins

Objective: Dentin sialophosphoprotein (DSPP) cleavage products are the most abundant non-collagenous proteins in dentin. Mutations in DSPP are cause inherited dental malformations. Our objective in this study was to identify, isolate and characterize dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP) from human dentin.

Method: The root furcations from third molars extracted from a 17-year-old patient were pulverized and demineralized with HCl-formic acid solution (HF). DSPP-derived proteins were extracted from the HF insoluble material with acetic acid/NaCl solution (AN). Both HF and AN extracts were characterized by SDS-PAGE and Western blot analyses. The AN extracts were fractionated by reverse phase HPLC and selected components were characterized by Edman sequencing and amino acid analysis. We also tested whether DGP may function separately from DSP: porcine DSP-DGP complex was mixed in the collagen-hydroxyapatite (Col-HA) solution and later digested with porcine MMP20.

Result: Human DPP was isolated from both HF and AN extracts and appeared as a Stains-all positive doublet having an apparent molecular weight of 140-kDa. Human DGP migrated as a 19-kDa band, similar in size to porcine DGP. Edman sequencing of this 19-kDa band gave SGNRxI as its N-terminal protein sequence, demonstrating that the DGP cleavage site is the same in pig as in human. On Western analyses, we detected DSP positive smear bands over 250-kDa in both the HF and AN extracts. The results of in vitro study demonstrated that porcine DGP after the digestion by MMP20 was identified in HA-bound fraction.

Conclusion: DSP, DGP, and DPP have been identified and partially characterized from human tooth extracts. In vitro study suggested that DSP-DGP complex has an affinity for collagen, but after DSP-DGP was processed by MMP20, DGP showed affinity for hydroxyapatite. This study was funded in part by NIDCR grant DE18020.