Partial Blocking of Mouse DSPP Processing by Substitution of Gly451–Asp452 Bond Suggests the Presence of Secondary Cleavage Site(s)

Dentin sialophosphoprotein (DSPP) in the extracellular matrix of dentin is cleaved into dentin sialoprotein and dentin phosphoprotein, which originate from the NH₂-terminal and COOH-terminal regions of DSPP, respectively. In the proteolytic processing of mouse DSPP, the peptide bond at Gly⁴⁵¹–Asp⁴⁵² has been shown to be cleaved by bone morphogenetic protein 1 (BMP1)/Tolloid-like metalloproteinases. In this study, we generated transgenic mice expressing a mutant DSPP in which Asp⁴⁵² was substituted by Ala⁴⁵². Protein chemistry analyses of extracts from the long bone of these transgenic mice showed that the D452A substitution partially blocked DSPP processing in vivo. When the full-length form of mutant DSPP (designated “D452A-DSPP”) isolated from the transgenic mice was treated with BMP1 in vitro, a portion of the D452A-DSPP was cleaved, suggesting the presence of secondary peptide bond(s) that can be broken by BMP1. To identify the potential secondary DSPP cleavage site(s), site-directed mutagenesis was performed to generate nine DNA constructs expressing DSPP-bearing substitutions at potential scission sites. These different types of mutant DSPP made in eukaryotic cell lines were treated with BMP1 and the digestion products were assessed by Western immunoblotting. All of the mutant DSPP molecular species were partially cleaved by BMP1, giving rise to a protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to that of normal dentin sialoprotein. Taken together, we concluded that in addition to the peptide bond Gly⁴⁵¹–Asp⁴⁵², there must be a cryptic cleavage site or sites close to Asp⁴⁵² in the mouse DSPP that can be cleaved by BMP1.