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J. Biol. Chem., Vol. 281, Issue 50, 38235-38243, December 15, 2006

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Dentin Sialophosphoprotein Is Processed by MMP-2 and MMP-20 in Vitro and in Vivo^*

Yasuo Yamakoshi, Jan C-C. Hu, Takanori Iwata^¶, Kazuyuki Kobayashi^||, Makoto Fukae^||, and James P. Simmer¹

From the Departments of Biologic and Materials Sciences and Orthodontics and Pediatric Dentistry, University of Michigan Dental Research Laboratory, Ann Arbor, Michigan 48108, the ^¶Department of Hard Tissue Engineering, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan, and the ^||Department of Periodontics and Endodontics, and Makoto Fukae Department of Biochemistry, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230, Japan

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 Thr²⁰⁰, Ser³³⁰, Val³⁵³, Leu³⁶⁰, Ile³⁶², Ser³⁷⁷, Ser⁴⁰⁸, and Asp⁴⁵⁸. 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.

Received for publication, August 14, 2006

^* This investigation was supported by NIDCR, National Institutes of Health, United States Public Health Service Research Grants DE12769, DE15846, and DE11301. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: University of Michigan Dental Research Laboratory, 1210 Eisenhower Pl., Ann Arbor, MI 48108. Tel.: 734-975-9318; Fax: 734-975-9329; E-mail: jsimmer{at}umich.edu.

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