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J. Biol. Chem., Vol. 278, Issue 36, 34700-34708, September 5, 2003

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Evidence for the Proteolytic Processing of Dentin Matrix Protein 1

IDENTIFICATION AND CHARACTERIZATION OF PROCESSED FRAGMENTS AND CLEAVAGE SITES^*

Chunlin Qin  , Jan C. Brunn , Richard G. Cook ¶ ||, Ralph S. Orkiszewski ||, James P. Malone **, Arthur Veis ** and William T. Butler 

From the Department of Basic Sciences, The University of Texas-Houston Health Science Center, Dental Branch, Houston, Texas 77030, the ^¶Department of Immunology and the ^||Protein Chemistry Core Laboratory, Baylor College of Medicine, Houston, Texas 77030, and the ^**Department of Cell and Molecular Biology, Northwestern University, Feinberg Medical School, Chicago, Illinois 60611

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH₂-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe¹⁷³ and Ser¹⁸⁰ and were thus COOH termini of 37K fragments. Two peptides were from the NH₂ termini of 57K fragments, starting at Asp²¹⁸ and Asp²²². These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe¹⁷³–Asp¹⁷⁴, Ser¹⁸⁰–Asp¹⁸¹, Ser²¹⁷–Asp²¹⁸, and Gln²²¹–Asp²²², forming eight fragments. The uniformity of cleavages at the NH₂-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.

Received for publication, May 20, 2003 , and in revised form, June 16, 2003.

^* This work was supported by National Institutes of Health Grants DE 05092 (to W. T. B.) and AR 13921 and DE 01374 (to A. V.). 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.

To whom correspondence should be addressed: Dept. of Basic Sciences, University of Texas-Houston Health Science Center, Dental Branch, 6516 M.D. Anderson Blvd., DBB, Rm. 4.133, Houston, TX 77030. Tel.: 713-500-4583; Fax: 713-500-4500; E-mail: Chunlin.Qin{at}uth.tmc.edu.

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