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J. Biol. Chem., Vol. 283, Issue 50, 35023-35032, December 12, 2008

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Identification and Role of the Homodimerization Interface of the Glycosylphosphatidylinositol-anchored Membrane Type 6 Matrix Metalloproteinase (MMP25)^*

Huiren Zhao¹, Anjum Sohail¹, Qing Sun¹, Qicun Shi, Seaho Kim, Shahriar Mobashery, and Rafael Fridman²

From the Department of Pathology, Wayne State University and Proteases and Cancer Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201 and the Department of Chemistry and Biochemistry and Walther Cancer Research Center, University of Notre Dame, Notre Dame, Indiana 46556

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M_r 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys⁵³² is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys⁵³⁰ and Cys⁵³⁴ form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of -1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.

Received for publication, August 22, 2008 , and in revised form, October 16, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant CA-61986 (to R. F.). 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 These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Pathology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1218; Fax: 313-577-8180; E-mail: rfridman{at}med.wayne.edu.

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