1103 DSP-PP Precursor Protein Cleavage by Tolloid-related-1 and Bone Morphogenetic Protein-1

Friday, March 23, 2012: 3:30 p.m. - 4:45 p.m.
Presentation Type: Poster Session
H.H. RITCHIE1, C.T. YEE1, X. TANG2, and R.S. FULLER1, 1Cariology, Restorative Sciences and Endodontics, University of Michigan, Ann Arbor, MI, 2Endodontology, School of Stomatology, Nanjing University Medical Center, Nanjing University, Nanjing, China
Objective: Dentin Sialoprotein (DSP) and phosphophoryn (PP) proteins, both critical to dentin mineralization, are synthesized from a single transcript and translated into a DSP-PP precursor protein, which is later believed to be cleaved into DSP and PP proteins in the dentin matrix.  The cleavage mechanism continues to be controversial because of the difficulty of identifying DSP-PP precursor proteins in mammalian cell lines and dentin matrix. 

Method: Using Sf9 insect cells infected with a recombinant baculovirus encoding the splice variant DSP-PP240, which secrete large quantities of the precursor protein.

Result: we have identified an endogenous Sf9 proteolytic activity that selectively cleaves DSP-PP240 precursor protein into DSP and PP240.  This DSP-PP processing activity is secreted by Sf9 cells during the first 3 days after infection but is diminished thereafter. Analysis of Sf9 mRNA sequences combined with RT-PCR enabled us to identify the partial cDNA sequence of a Sf9 cell tolloid-related-1 protein.  Within the partial sequence, Sf9 tolloid-related-1 protein exhibits 65% peptide sequence identity to Drosophilia melanogaster tolloid but 78% peptide sequence identity to the Drosophila tolloid-related-1/tolkin protein. Tolloid-related-1 protein mRNA expression in Sf9 cells infected with the DSP-PP240 baculovirus decreased from day 1 to day 4 after infection, paralleling the observed decrease in DSP-PP processing activity after infection.  Among human homologs, BMP1 is most similar to Sf9 tolloid-related-1 protein.

Conclusion: Human BMP1 was found to process DSP-PP precursor protein in a dose-dependent manner.  Our data strongly suggest that human DSP-PP precursor cleavage requires BMP1 for the production of mature DSP and PP proteins in the dentin matrix.

This abstract is based on research that was funded entirely or partially by an outside source: NIH DE11442 and DE18901

Keywords: Dentin Sialoprotein,phosphophoryn