Dynamic Processing of Recombinant Dentin-Sialoprotein-Phosphophoryn

Introduction: Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two most abundant noncollagenous proteins in dentin, which are derived from a single copy DSP-PP gene. It is commonly assumed that the DSP-PP gene is first transcribed into DSP-PP mRNAs, translated to become DSP-PP precursor proteins, then enzymatically cleaved to yield DSP and PP proteins found in dentin. Yet, dentin DSP protein has been estimated at 5-8% of the dentin NCP content and PP has been estimated to be >50% of dentin NCP content. To date, this discrepancy between the observed 1:6 DSP:PP dentin protein ratio and the expected 1:1 ratio has not been explained. A key-missing element in this story has been the inability to identify a DSP-PP precursor protein from any cell or tissue sample. Our Objective is to identify the DSP-PP precursor and track its processing. Methods: To achieve this goal, we utilized a baculovirus expression system to produce recombinant DSP-PP precursor proteins from a DSP-PP240 cDNA, which represents one of several endogenous DSP-PP transcripts that influence various tooth mineralization phases. N-terminal sequence, Mass spectral analyses and Western blot were used to determine the protein identities. Results: Our in vitro results demonstrate that DSP-PP240 precursor proteins are produced by this system. We found dynamic processing of DSP-PP protein and the purified DSP-PP240 precursor protein undergoes self-processing to yield both DSP and PP proteins. Using the NIH image J program, we determined the relative densities of recombinant DSP430 and PP240 from day 4 culture medium to be ~1:6. Conclusion: DSP-PP240 precursor protein was identified and the precursor protein undergoes dynamic self-processing to yield both DSP and PP proteins. In vitro DSP-PP processing results agree very well with the measured DSP/PP ratio in dentin tissue. This work was supported by NIH DE11442-9 to HHR.