The N-terminal Module of Thrombospondin-1 Interacts with the Link Domain of TSG-6 and Enhances Its Covalent Association with the Heavy Chains of Inter-α-trypsin Inhibitor

doi:10.1074/jbc.M500701200

The N-terminal Module of Thrombospondin-1 Interacts with the Link Domain of TSG-6 and Enhances Its Covalent Association with the Heavy Chains of Inter-α-trypsin Inhibitor^*

^‡Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892, ^§MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, and the ^∥Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706

** To whom correspondence should be addressed: Bldg. 10 Rm. 2A33, National Institutes of Health, 10 Center Dr. MSC1500, Bethesda, MD 20892-1500. Tel.: 301-496-6264; Fax: 301-402-0043; E-mail: droberts{at}helix.nih.gov.

Abstract

We recently found that leukocytes from thrombospondin-1 (TSP1)-deficient mice exhibit significant reductions in cell surface CD44 relative to those from wild type mice. Because TSG-6 modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We showed that recombinant full-length human TSG-6 (TSG-6Q) and the Link module of TSG-6 (Link_TSG6) bind ¹²⁵I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin results from its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca²⁺ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation dependence is due to conformational effects of calcium-binding to the C-terminal domains of TSP1. TSP1 enhances covalent modification of the inter-α-trypsin inhibitor by TSG-6 and transfer of its heavy chains to hyaluronan, suggesting a physiological function of TSP1 binding to TSG-6 in regulation of hyaluronan metabolism at sites of inflammation.

Footnotes

↵1 The abbreviations used are: TSP, thrombospondin; DPBS, Dulbecco's phosphate-buffered saline; HA, hyaluronan; IαI, inter-α-trypsin inhibitor; Link_TSG6, residues 36–133 of TSG-6 preprotein; NoC1, trimeric human thrombospondin-1 residues 1–356; TSG-6, tumor necrosis factor-α-stimulated gene-6; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HC, heavy chain.
↵2 S. A. Kuznetsova, S. O. Sharrow, J. Lawler, and D. D. Roberts, manuscript in preparation.
↵3 V. A. Higman and A. J. Day, unpublished data.
↵4 S. A. Kuznetsova and D. D. Roberts, unpublished results.
↵* This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. 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.
↵¶ Supported by Arthritis Research Campaign Grants M0625, 16119, and 16539 and the Medical Research Council.
- Received January 19, 2005.
- Revision received June 3, 2005.
The American Society for Biochemistry and Molecular Biology, Inc.