1104 Zymograms of wild type DSP-PP240 and mutated recombinant precursor proteins

Friday, March 23, 2012: 3:30 p.m. - 4:45 p.m.
Presentation Type: Poster Session
C.T. YEE1, R.S. FULLER1, and H.H. RITCHIE2, 1Cariology, Restorative Sciences and Endodontics, University of Michigan, Ann Arbor, MI, 2Caries, Restorative Sciences and Endodontics, University of Michigan, Ann Arbor, MI
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 zymogram profile.

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.

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

Keywords: Dentin Sialoprotein,Phosphophoryn