Dentin sialoprotein is catalyzed by MMP-9 and its biological functions

Dentin sialophosphoprotein (Dspp) is a member of SIBLING gene family and encodes dentin sialoprotein (Dsp) and dentin phosphoprotein (Dpp). Dsp and Dpp are highly expressed in ameloblasts and predominantly in odontoblasts. Recent studies have shown that Dspp is catalyzed into several fragments including Dsp, Dpp and others by given proteases. Dsp and Dpp also play unique functions during tooth development, in particular amelogenesis. Objectives: This objective was to study whether Dsp interacts with protease(s) and cleaved by the enzyme (s) and to determine Dsp function during tooth development. Methods: Recombinant mouse Dsp (rDsp) cDNA was generated and subcloned into bacteria expression plasmid. The purified rDsp was incubated with several proteases in vitro. For mammalian cell expression, chimeric cop green fluorescent protein (copGFP)-Dsp gene construct was generated by inserting the mouse Dsp cDNA into pCDF-copGFP vector. The pCDF-copGFP-Dsp vector is able to express both copGFP and Dsp proteins by two CMV promoters, respectively. Mouse immortalized odontoblast-like and human kidney epithelial cells (HEK 293) were transiently transfected with this GFP-Dsp expression vector. Transfection efficiency was detected by fluorescent microscope. Results: Mouse rDsp construct was generated and this protein expressed in bacteria and purified. Purified rDsp was run onto 7% SDS-PAGE gels and confirmed with silver staining and Western blot assay. The rDsp protein interacts with matrix metalloproteinase-9 (MMP-9) by coimmunoprecipitation assay and cleaved by this enzyme in vitro. Furthermore, transient transfection assay showed that GFP-rDsp was highly expressed in HEK 293T and other cells. Conclusions: We cloned and expressed mouse rDsp protein. The rDsp interacts with MMP-9 and is cleaved by this enzyme in vitro. Secondly, using the chimeric expression vector, we can detect transfection efficiency of Dsp gene in the transfected cells. Biological function of Dsp with MMP-9 during tooth development is being investigated. This study was supported by NIDCR DE14484.