Key Proteolytic Cleavage Site and Full-length Form of DSPP

Abstract

It is known that dentin sialophosphoprotein (DSPP) is processed into NH₂- and COOH-terminal fragments, but its key cleavage site has not been identified, nor has its full-length form been discovered. The objectives of this study were to identify the key cleavage site during DSPP processing and to search for full-length DSPP in vivo. We generated a construct encoding DSPP, in which Asp⁴⁵², a cleavage site residue, was replaced by Ala⁴⁵². The pulp-odontoblast complex and dentin were extracted, chromatographically separated, and assessed by Stains-All staining, Western immunoblotting, and mass spectrometry. These studies showed that the substitution of Asp⁴⁵² by Ala⁴⁵² completely blocks the cleavage of mouse DSPP in the transfected cells, indicating that the NH₂-terminal peptide bond of Asp⁴⁵² is essential for the initiation of DSPP proteolytic processing. The results of this study revealed the presence of full-length DSPP and its processed fragments in extracts from the pulp/odontoblast and dentin.