Biochem. J. (2007) 403
(553–563) (Printed in Great Britain)
Cell surface chondroitin sulfate glycosaminoglycan in melanoma: role in the activation of pro-MMP-2 (pro-gelatinase A)
Joji IIDA*†1, Krista L. WILHELMSON*, Janet NG*, Peter LEE‡, Charlotte MORRISON§, Eric TAM§, Christopher M. OVERALL§ and James B. McCARTHY*†
*Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, U.S.A., †Cancer Center, University of Minnesota, Minneapolis, MN 55455, U.S.A., ‡Department of Surgery, University of Minnesota, Minneapolis, MN 55455, U.S.A., and §Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
We previously reported that CS (chondroitin sulfate) GAG (glycosaminoglycan), expressed on MCSP (melanoma-specific CS proteoglycan), is important for regulating MT3-MMP [membrane-type 3 MMP (matrix metalloproteinase)]-mediated human melanoma invasion and gelatinolytic activity in vitro. In the present study, we sought to determine if CS can directly enhance MT3-MMP-mediated activation of pro-MMP-2. Co-immunoprecipitation studies suggest that MCSP forms a complex with MT3-MMP and MMP-2 on melanoma cell surface. When melanoma cells were treated with bDX (p-nitro-b-D-xylopyranoside) to inhibit coupling of CS on the core protein, both active form and proform of MMP-2 were no longer co-immunoprecipitated with either MCSP or MT3-MMP, suggesting a model in which CS directly binds to MMP-2 and presents the gelatinase to MT3-MMP to be activated. By using recombinant proteins, we determined that MT3-MMP directly activates pro-MMP-2 and that this activation requires the interaction of the C-terminal domain of pro-MMP-2 with MT3-MMP. Activation of pro-MMP-2 by suboptimal concentrations of MT3-MMP is also significantly enhanced in the presence of excess C4S (chondroitin 4-sulfate), whereas C6S (chondroitin 6-sulfate) or low-molecular-mass hyaluronan was ineffective. Affinity chromatography studies using CS isolated from aggrecan indicate that the catalytic domain of MT3-MMP and the C-terminal domain of MMP-2 directly bind to the GAG. Thus the direct binding of pro-MMP-2 with CS through the C-domain would present the catalytic domain of pro-MMP-2 to MT3-MMP, which facilitates the generation of the active form of MMP-2. These results suggest that C4S, which is expressed on tumour cell surface, can function to bind to pro-MMP-2 and facilitate its activation by MT3-MMP-expressing tumour cells to enhance invasion and metastasis.
Key words: chondroitin sulfate, matrix metalloproteinase (MMP), membrane-type 3 MMP (MT3-MMP), proteoglycan, sulfation, tissue inhibitor of metalloproteinases-2 (TIMP-2).
Abbreviations used: APMA, 4-aminophenylmercuric acetate; BAP, Escherichia coli bacterial alkaline phosphatase; bDX, p-nitro-b-D-xylopyranoside; ConA, concanavalin A; CS, chondroitin sulfate; CDMMP-2, C-terminal domain of MMP-2; C4S, chondroitin 4-sulfate; ECL, enhanced chemiluminescence; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan; HRP, horseradish peroxidase; IDV, individual values; IPTG, isopropyl b-D-thiogalactoside; MCSP, melanoma-specific CS proteoglycan; MMP, matrix metalloproteinase; MT-MMP, membrane-type MMP; TIMP-2, tissue inhibitor of metalloproteinases-2.
1To whom correspondence should be addressed (email iidax002@tc.umn.edu).
Received 2 August 2006/2 January 2007; accepted 12 January 2007
Published as BJ Immediate Publication 12 January 2007, doi:10.1042/BJ20061176
© The Authors Journal compilation © 2007 The Biochemical Society, London
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