Dentin Sialophosphoprotein is Processed by MMP-2 and MMP-20

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts, and is critical for proper tooth dentin formation. During dentinogenesis DSPP is proteolytically cleaved into smaller subunits. These cleavages are proposed activation steps and failure to make these cleavages is a potential cause of developmental tooth defects. Objectives: to test the hypothesis that dentin-resident matrix metalloproteinases process DSPP. Methods: We isolated DSPP-derived proteins from developing porcine molars and characterized their N-terminal sequences by Edman degradation and their apparent sizes by SDS-PAGE and Western blotting. Dentin resident proteases were identified by gelatin and casein zymography and by Western blotting. MMP-2 and MMP-20 were purified from over 150 grams of porcine dentin powder and used to digest the major DSPP-derived protein in vitro. The in vitro cleavage products were characterized by N-terminal sequencing, SDS-PAGE, and Western blot analyses. Results: The in vivo DSPP cleavage sites were on the N-terminal sides of Thr200, Ser330, Val353, Leu360, Ile362, Ser377, Ser408, and Asp458. The initial DSPP cleavage is between dentin glycoprotein (DGP) and dentin phosphoprotein (DPP), generating DSP-DGP and DPP. Gelatin and casein zymograms identified MMP-2, MMP-20, and KLK4 in dentin extracts. MMP-2 and MMP-20 were incubated with DSP-DGP and DPP. These enzymes showed no activity against DPP. MMP-20 cleaved DSP-DGP to generate dentin sialoprotein (DSP) and DGP. MMP-20 also cleaved DSP at multiple sites, releasing N-terminal DSP cleavage products ranging from 25- to 38-kDa. MMP-2 makes multiple cleavages near the DSP C-terminus, releasing larger forms of DGP, or “extended DGPs”. Conclusions: The exact correspondence between DSPP cleavage sites that occur in vivo and those generated in vitro demonstrate that MMP-2 and MMP-20 process DSPP into smaller subunits in the dentin matrix during odontogenesis. This study is supported by NIDCR grants DE 12769 and 15846.