Premium
Cleavage site determination of dentin sialoprotein‐phosphophoryn precursor protein
Author(s) -
Ritchie Helena H,
Yang Robert T
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.277
Subject(s) - dmp1 , complementary dna , microbiology and biotechnology , mutant , chemistry , wild type , expression vector , biology , gene , biochemistry , recombinant dna , viral matrix protein
Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two noncollagenous proteins classically linked to dentin mineralization, but more recently found in bone, kidney and salivary glands. Point mutations in DSP‐PP gene are linked to Dentinogenesis imperfecta. These two proteins are derived from a single copy DSP‐PP gene. Because native PP has a DDPN N‐terminal sequence, we hypothesized that SMQG 447 |D 448 DPN is the specific cleavage site for generating DSP 430 and PP 240 . To test this hypothesis, our Objective was to generate DSP‐PP 240 containing mutated G 447 and follow the expression and processing of this mutated precursor protein. Methods: To achieve this goal, we used site‐mutagenesis to generate G 447 mutations in DSP‐PP 240 cDNA in pGEM7Z(+), which were subsequently subcloned into pVL1392 baculovirus vector for protein expression. Results: Wild type and mutated DSP‐PP 240 precursor proteins are secreted into the extracellular space in the baculovirus expression system. Using MS, MS/MS analysis on the recombinant PP sample, the N‐terminal sequence was determined to be DDPN. DSP‐PP 240 cDNA containing G 447 mutant yielded a DSP‐PP 240 precursor protein, which was not further processed into DSP 430 and PP 240 . Conclusion: G 447 |D 448 is the cleavage site for generating DSP 430 and PP 240 from the wild type precursor molecule. This work was supported by NIH DE11442‐9 to HHR.