title: Dentin sialophosphoprotein is processed by MMP-2 and MMP-20 in vitro and in vivo.
abstract: Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper tooth dentin formation. During dentinogenesis, DSPP is proteolytically cleaved into smaller subunits. These cleavages are proposed activation steps, and failure to make these cleavages is a potential cause of developmental tooth defects. We tested the hypothesis that dentin-resident matrix metalloproteinases catalyze the cleavages that process DSPP. We defined the exact DSPP cleavages that are catalyzed by proteases during crown formation by isolating DSPP-derived proteins from developing porcine molars and characterizing their N-terminal sequences and apparent size on SDS-PAGE and Western blots. The in vivo DSPP cleavage sites were on the N-terminal sides of Thr(200), Ser(330), Val(353), Leu(360), Ile(362), Ser(377), Ser(408), and Asp(458). The initial DSPP cleavage is between dentin glycoprotein (DGP) and dentin phosphoprotein (DPP), generating dentin sialoprotein (DSP)/DGP and DPP. Gelatin and casein zymograms identified MMP-2, MMP-20, and KLK4 in the dentin extracts. MMP-2 and MMP-20 were purified from over 150 g of porcine dentin powder and incubated with DSP-DGP and DPP. These enzymes show no activity in further cleaving DPP. MMP-20 cleaves DSP-DGP to generate DSP and DGP. MMP-20 also cleaves DSP at multiple sites, releasing N-terminal DSP cleavage products ranging in size from 25 to 38 kDa. MMP-2 makes multiple cleavages near the DSP C terminus, releasing larger forms of DGP, or "extended DGPs." Exact correspondence between DSPP cleavage sites that occur in vivo and those generated in vitro demonstrates that MMP-2 and MMP-20 process DSPP into smaller subunits in the dentin matrix during odontogenesis.
authors.affiliation: Departments of Biologic and Materials Sciences, University of Michigan Dental Research Laboratory, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yamakoshi
authors.forename: Jan C-C
authors.initials: JC
authors.lastname: Hu
authors.forename: Takanori
authors.initials: T
authors.lastname: Iwata
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Makoto
authors.initials: M
authors.lastname: Fukae
authors.forename: James P
authors.initials: JP
authors.lastname: Simmer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D053519:Matrix Metalloproteinase 20 / Q000378:metabolism*; D008969:Molecular Sequence Data; D012795:Sialoglycoproteins / Q000378:metabolism*; D013552:Swine
chemical_list: D012795:Sialoglycoproteins; D053519:Matrix Metalloproteinase 20; D020778:Matrix Metalloproteinase 2