BMP-1 cleaves Dentin Sialophosphoprotein (DSPP) to generate Dentin Phosphoprotein (DPP)

Dentin sialophosphoprotein (DSPP) is critical for proper dentin biomineralization. In humans, genetic defects in DSPP cause dentin dysplasia type II, and dentinogenesis imperfecta type II and type III. DSPP is processed by proteases into smaller subunits. In the initial cleavage, DSPP is hydrolyzed to generate dentin phosphoprotein (DPP) and dentin sialoprotein–dentin glycoprotein (DSP-DGP) complex. Objectives: To test the hypothesis that bone morphogenetic protein 1 (BMP-1), an endopeptidase of the astacin M12A family, is present in the pre-dentin/dentin extracellular matrix, and is uniquely able to catalyze DSPP to generate DPP. Methods: We designed a fluorescence resonance energy transfer (FRET)-DSPP peptide containing the initial cleavage site in DSPP: Abz-YEFDGKSMQGDDPN(Lys)Dnp. BMP-1 enzyme was isolated from developing porcine molars (in vivo BMP-1) and was characterized by Western blotting and by its ability to cleave known BMP-1 substrates. FRET-DSPP was incubated with in vivo BMP-1, MMP-20, KLK4, and by the commercially available enzymes plasminogen, rhBMP-1, rhMMP-2, and rhMMP-9. The peptide cleavage products were isolated by reverse-phase HPLC monitored by UV and fluorescence detectors, and were characterized by N-terminal sequencing analysis or by LCMSMS. Results: BMP-1 was isolated from dentin extracts. The FRET-DSPP peptide was cleaved by rhBMP-1, in vivo BMP-1, plasminogen and KLK4. The cleavage sites by rhBMP-1 and in vivo BMP-1 were at the appropriate site for DSPP initial processing, but plasminogen or KLK4 cleaved at different sites in the FRET-DSPP peptide. DPP isolated from in vivo did not cleave the FRET-DSPP peptide. Conclusions: We demonstrate that BMP-1 is present in the pre-dentin matrix and uniquely cleaves before the DDPN sequence at the amino-terminus of DPP. We conclude that DSPP is cleaved by BMP-1 to generate DPP and DSP-DGP during odontogenesis. This study is supported by NIDCR grants DE018020.