Identification and processing of dentin sialoprotein-phosphophoryn precursor protein

Introduction: Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two noncollagenous proteins classically linked to dentin, but more recently found in bone, kidney and salivary glands. These two proteins are derived from a single copy DSP-PP gene. We hypothesize that the DSP-PP gene is first transcribed into DSP-PP mRNAs, which later undergo processing to yield the DSP and PP proteins. To test this hypothesis, 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 may influence various tooth mineralization phases. N-terminal sequence identification, mass spectral analyses and Western blots were used to determine the protein identities. Results: Our in vitro results demonstrated that DSP-PP240 precursor proteins are secreted into the extracellular space in the baculovirus expression system. This work for the first time identified the presence of DSP-PP240 precursor protein. We found dynamic processing of DSP-PP protein since 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: A recombinant DSP-PP240 precursor protein was identified in baculovirus culture medium suggesting that it is secreted into the extracellular space where this precursor protein undergoes autocleavage to generate DSP and PP. In addition, in vitro DSP-PP processing results agree very well with the measured 1:6 DSP/PP ratio in dentin tissue. This work was supported by NIH DE11442-9 to HHR.