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J. Biol. Chem., Vol. 280, Issue 41, 34441-34446, October 14, 2005

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Plasmin- and Thrombin-accelerated Shedding of Syndecan-4 Ectodomain Generates Cleavage Sites at Lys¹¹⁴–Arg¹¹⁵ and Lys¹²⁹–Val¹³⁰ Bonds^*

Annette Schmidt1, Frank Echtermeyer, Anthony Alozie, Kerstin Brands, and Eckhart Buddecke

From the Leibniz-I Institute of Arteriosclerosis Research, Department of Physiological Chemistry and Pathobiochemistry, University of Muenster, D-48149 Muenster, Germany

Syndecans are transmembranous heparan sulfate proteoglycans abundant in the surface of all adherent mammalian cells and involved in vital cellular functions. In this study, we found syndecan-1, -2, -3, and -4 to be constitutively expressed by human umbilical vein endothelial cells. The exposure of the ectodomains of syndecan-1 and -4 to the cell surface and their constitutive shedding into the extracellular compartment was measured by immunoassays. In the presence of plasmin and thrombin, shedding was accelerated and monitored by detection and identification of ³⁵S-labeled proteoglycans. To elucidate the cleavage site of the syndecan ectodomains, we used a cell-free in vitro system with enzyme and substrate as the only reactants. For this purpose, we constructed recombinant fusion proteins of the syndecan-1 and -4 ectodomain together with maltose-binding protein and enhanced yellow fluorescent protein as reporter proteins attached to the N and C termini via oligopeptide linkers. After protease treatment of the fusion proteins, the electrophoretically resolved split products were sequenced and cleavage sites of the ectodomain were identified. Plasmin generated cleavage sites at Lys¹¹⁴Arg¹¹⁵ and Lys¹²⁹Val¹³⁰ in the ectodomain of syndecan-4. In thrombin proteolysates of the syndecan-4 ectodomain, the cleavage site Lys¹¹⁴Arg¹¹⁵ was also identified. The cleavage sites for plasmin and thrombin within the syndecan-4 ectodomain were not present in the syndecan-1 ectodomain. Cleavage of the syndecan-1 fusion protein by thrombin occurred only at a control cleavage site (ArgGly) introduced into the linker region connecting the ectodomain with the enhanced yellow fluorescent protein. Because both plasmin and thrombin are involved in thrombogenic and thrombolytic processes in the course of the pathogenesis of arteriosclerosis, the detachment of heparan sulfate-bearing ectodomains could be relevant for the development of arteriosclerotic plaques and recruitment of mononuclear blood cells to the plaque.

Received for publication, February 18, 2005 , and in revised form, June 28, 2005.

^* This work was financially supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 492, Project B12) (to F. E.). 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: Institute of Arteriosclerosis Research, Dept. of Molecular Cardiology, Domagkstr. 3, D-48149 Muenster, Germany. Tel.: 49-251-835-8626; Fax: 49-251-835-8628; E-mail: annschm{at}uni-muenster.de.

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