Key proteolytic cleavage site and full-length form of DSPP

J Dent Res. 2010 May;89(5):498-503. doi: 10.1177/0022034510363109. Epub 2010 Mar 23.

Abstract

It is known that dentin sialophosphoprotein (DSPP) is processed into NH(2)- 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 Asp(452), a cleavage site residue, was replaced by Ala(452). 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 Asp(452) by Ala(452) completely blocks the cleavage of mouse DSPP in the transfected cells, indicating that the NH(2)-terminal peptide bond of Asp(452) 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alanine / genetics
  • Amino Acids / analysis
  • Animals
  • Aspartic Acid / genetics
  • Bone Morphogenetic Protein 1 / pharmacology
  • Carbon Dioxide / analysis
  • Cell Line
  • Dental Pulp / chemistry
  • Dentin / chemistry
  • Extracellular Matrix Proteins / analysis*
  • Extracellular Matrix Proteins / genetics
  • Free Radicals / analysis
  • Genetic Vectors / genetics
  • Humans
  • Mass Spectrometry
  • Mice
  • Mice, Inbred C57BL
  • Mice, Inbred Strains
  • Molecular Weight
  • Mutation / genetics
  • Odontoblasts / chemistry
  • Peptide Fragments / analysis*
  • Phosphoproteins / analysis*
  • Phosphoproteins / genetics
  • Plasmids / genetics
  • Rats
  • Recombinant Proteins
  • Sialoglycoproteins / analysis*
  • Sialoglycoproteins / genetics
  • Tandem Mass Spectrometry
  • Transfection

Substances

  • Amino Acids
  • Extracellular Matrix Proteins
  • Free Radicals
  • Peptide Fragments
  • Phosphoproteins
  • Recombinant Proteins
  • Sialoglycoproteins
  • dentin sialophosphoprotein
  • Carbon Dioxide
  • carboxyl radical
  • Aspartic Acid
  • BMP1 protein, human
  • Bone Morphogenetic Protein 1
  • Alanine