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.
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