DSP-PP C-Terminal Conservation Is Crucial for Accurate DSP-PP Precursor Cleavage

نویسندگان

  • Ko-Chien Wu
  • Helena H Ritchie
چکیده

Dentin Sialoprotein (DSP) and Phosphophoryn (PP), acidic proteins critical to dentin mineralization, are translated from a single transcript as a DSP-PP precursor that undergoes proteolytic processing to generate DSP and PP. Because of the difficulty in obtaining large amounts of DSP-PP, we used a Sf9-baculovirus expression system to yield large amounts of DSP-PP240 recombinant protein, a variant form of rat DSP-PP. Previous evidence stated that DSP-PP240 produced by baculovirus-infected Sf9 cells can be cleaved accurately into DSP and PP by the endogenous processing enzyme Sf9 Tolloid-Related 1 (TLR1), a homolog for human Bone Morphogenic Protein 1 (BMP1) and the proposed protease to cleave DSP-PP in human. It was also discovered via mass spectrometric analysis that the specific cleavage occurred at the site: SMQG447|D448DPN. In addition, we reported that any mutations within the DSP-PP P4 to P4’cleavage site can block, impair or accelerate DSP-PP cleavage, which suggest that its BMP1 cleavage site is highly conserved to regulate its cleavage efficiency. Furthermore, mutations outside of the DSP-PP P4 to P4’ cleavage site can impair or accelerate DSP-PP cleavage. Here, we investigate the role of the highly conserved DSPP C-terminal region in DSP-PP cleavage. We generated a DSP-PP C-terminal mutation by substituting the terminal two aspartate residues for two histamine residues (DD/HH-DSP-PP). To test the impact of the DD/HH mutant on DSP-PP cleavage, we used the Sf9 expression system’s endogenous TLR1 and exogenous recombinant BMP1. The DD/HH mutation was shown to block DD/HH-DSP-PP cleavage into DSP and PP by both TLR1 and BMP1 in vitro. Taken together, these evidence supports our hypothesis that the C-terminal peptides D686D687 actively participates in controlling DSP-PP cleavage and that C-terminal conservation is critical for proper DSP-PP precursor cleavage by TLR1 and BMP1.

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تاریخ انتشار 2018