Dynamic Processing of Recombinant Dentin Sialoprotein-Phosphophoryn Protein

Abstract

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. Although this suggests that the DSP-PP gene is first transcribed into DSP-PP mRNAs, which later undergo processing to yield the DSP and PP proteins, this mechanism has not yet been demonstrated because of the inability to identify a DSP-PP precursor protein from any cell or tissue sample. To study this problem, 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. Our in vitro results demonstrate that DSP-PP240 precursor proteins are produced by this system and are capable of self-processing to yield both DSP and PP proteins. We further demonstrated that purified recombinant DSP-PP240, purified recombinant PP240, and the native highly phosphorylated protein (equivalent to the PP523 isoform) have proteolytic activity. These newly identified tissue proteases may play key roles in tissue modeling during organogenesis. 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. Although this suggests that the DSP-PP gene is first transcribed into DSP-PP mRNAs, which later undergo processing to yield the DSP and PP proteins, this mechanism has not yet been demonstrated because of the inability to identify a DSP-PP precursor protein from any cell or tissue sample. To study this problem, 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. Our in vitro results demonstrate that DSP-PP240 precursor proteins are produced by this system and are capable of self-processing to yield both DSP and PP proteins. We further demonstrated that purified recombinant DSP-PP240, purified recombinant PP240, and the native highly phosphorylated protein (equivalent to the PP523 isoform) have proteolytic activity. These newly identified tissue proteases may play key roles in tissue modeling during organogenesis. Dentin sialoprotein (DSP) 2The abbreviations used are: DSP, dentin sialoprotein; PP, phosphophoryn; DE, dentin extract; HP, highly phosphorylated protein; CAPS, 3-(cyclohexylamino)propanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MMP, matrix metalloprotease.2The abbreviations used are: DSP, dentin sialoprotein; PP, phosphophoryn; DE, dentin extract; HP, highly phosphorylated protein; CAPS, 3-(cyclohexylamino)propanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MMP, matrix metalloprotease. and phosphophoryn (PP) are the two most abundant noncollagenous proteins (NCPs) in dentin. In rats, DSP and PP coding sequences are derived from DSP-PP transcripts (1Ritchie H.H. Kim L.-H. J. Biol. Chem. 1996; 271: 21695-21698Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 2Ritchie H.H. Kim L.-H. Biochem. Biophys. Res. Commun. 1997; 231: 425-428Crossref PubMed Scopus (41) Google Scholar), a finding confirmed in the mouse (3MacDougall M. Kim D. Luan X. Nydegger J. Feng J. Gu T.T. J. Biol. Chem. 1997; 272: 835-842Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) and in human DSP-PP transcripts (4Gu K. Kim S. Ritchie H. Clarkson B. Rutherford R. Eur. J. Oral Sci. 2000; 108: 35-42Crossref PubMed Scopus (72) Google Scholar). Immunohistochemistry and in situ studies showed that D