Friday, March 23, 2012: 3:30 p.m. - 4:45 p.m.
Presentation Type: Poster Session
This abstract is based on research that was funded entirely or partially by an outside source:
Objective: It has been difficult to study the DSP-PP (Dentin Sialoprotein-Phosphoryn) precursor protein in cell lines and in tissues because this precursor protein is extremely unstable. In mammalian cells, many research groups aimed to produce sufficient amounts of DSP-PP precursor protein for cleavage studies. Most of these attempts resulted in extremely low yields of DSP-PP precursor proteins that do not allow amino acid sequence analysis of the cleavage products. Thus to date there is no direct evidence showing that DSP-PP precursor protein was cleaved at a defined site to generate mature DSP and PP. We have previously demonstrated that Sf9 cells can provide an abundant source of DSP-PP240
precursor proteins. The purpose of this project is to (1) determine the exact cleavage site of Sf9 cell recombinant DSP-PP240
precursor protein, to (2) examine the wild type DSP-PP240
zymogram profile, and to (3) investigate the mutated DSP-PP240
Method: Sf9 cells were infected with wild type viral DSP-PP240 cDNA or mutated DSP-PP240 cDNA and the recombinant precursor proteins were isolated with TCA precipitation, neutralized and resuspended in 0.1 M EDTA. The recombinant proteins were loaded onto PAGE gel to obtain for MS or MS/MS analyses or onto PAGE gelatin gels to test proteolytic activity.
Result: We obtained the N-terminal 34 amino acid sequence (i.e., DDPNSSDESNGSDGSDDANSESAIENGNHGDASY). Interestingly, these mature proteins are stable in the condition medium for over one month at 28°C. Wild type DSP-PP240 showed proteolytic activity in gelatin gels but mutated DSP-PP240 protein did not show proteolytic activity.
Conclusion: The recombinant PP240 protein generated by the baculovirus expression system can be used to study DSP-PP240 precursor protein cleavage. The difference in zymograms between wild type and mutated DSP-PP240 proteins leads to several possible models to account for this phenomenon.
Keywords: Dentin Sialoprotein,Phosphophoryn