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.
Keywords: Dentin Sialoprotein,phosphophoryn