title: The efficiency of dentin sialoprotein-phosphophoryn processing is affected by mutations both flanking and distant from the cleavage site.
abstract: Normal dentin mineralization requires two highly acidic proteins, dentin sialoprotein (DSP) and phosphophoryn (PP). DSP and PP are synthesized as part of a single secreted precursor, DSP-PP, which is conserved in marsupial and placental mammals. Using a baculovirus expression system, we previously found that DSP-PP is accurately cleaved into DSP and PP after secretion into medium by an endogenous, secreted, zinc-dependent Sf9 cell activity. Here we report that mutation of conserved residues near and distant from the G(447)↓D(448) cleavage site in DSP-PP(240) had dramatic effects on cleavage efficiency by the endogenous Sf9 cell processing enzyme. We found that: 1) mutation of residues flanking the cleavage site from P(4) to P(4)' blocked, impaired, or enhanced DSP-PP(240) cleavage; 2) certain conserved amino acids distant from the cleavage site were important for precursor cleavage; 3) modification of the C terminus by appending a C-terminal tag altered the pattern of processing; and 4) mutations in DSP-PP(240) had similar effects on cleavage by recombinant human BMP1, a candidate physiological processing enzyme, as was seen with the endogenous Sf9 cell activity. An analysis of a partial TLR1 cDNA from Sf9 cells indicates that residues that line the substrate-binding cleft of Sf9 TLR1 and human BMP1 are nearly perfectly conserved, offering an explanation of why Sf9 cells so accurately process mammalian DSP-PP. The fact that several mutations in DSP-PP(240) significantly modified the amount of PP(240) product generated from DSP-PP(240) precursor protein cleavage suggests that such mutation may affect the mineralization process.
authors.affiliation: Department of Cariology, Restorative Sciences and Endodontics, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Yang
authors.forename: Glendale L
authors.initials: GL
authors.lastname: Lim
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Dong
authors.forename: Arthur M
authors.initials: AM
authors.lastname: Lee
authors.forename: Colin T
authors.initials: CT
authors.lastname: Yee
authors.forename: Robert S
authors.initials: RS
authors.lastname: Fuller
authors.forename: Helena H
authors.initials: HH
authors.lastname: Ritchie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D055395:Bone Morphogenetic Protein 1 / Q000378:metabolism; D002460:Cell Line; D029721:Drosophila Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D005786:Gene Expression Regulation; D007313:Insecta; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012795:Sialoglycoproteins / Q000737:chemistry*; D000099311:Dentin Sialophosphoprotein
chemical_list: D055395:Bone Morphogenetic Protein 1; D029721:Drosophila Proteins; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein; C017630:phosphophoryn; C097491:Tok protein, Drosophila