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.
{"title":"DSP-PP C-Terminal Conservation Is Crucial for Accurate DSP-PP Precursor Cleavage.","authors":"Ko-Chien Wu, Helena H Ritchie","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>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-PP<sub>240</sub> recombinant protein, a variant form of rat DSP-PP. Previous evidence stated that DSP-PP<sub>240</sub> 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: SMQG<sup>447</sup>|D<sup>448</sup>DPN. 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 <i>in vitro</i>. Taken together, these evidence supports our hypothesis that the C-terminal peptides D<sup>686</sup>D<sup>687</sup> actively participates in controlling DSP-PP cleavage and that C-terminal conservation is critical for proper DSP-PP precursor cleavage by TLR1 and BMP1.</p>","PeriodicalId":93743,"journal":{"name":"Dentistry (Lisle, Ill.)","volume":"2 4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9248859/pdf/nihms-1002092.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40466382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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