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-alpha-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.
Highlights
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
To determine which domain of TSG-6 mediates binding of TSP1, we examined binding of TSP1 to the HA binding domain of TSG-6 (Link_TSG6). 125I-TSP1 bound to immobilized Link_TSG6 in a similar temperature- and dose-dependent manner as to TSG-6Q (Fig. 1B)
Binding of NoC1 to Link_TSG6 was slightly higher in the absence of cations (Fig. 4B). These results indicate that the divalent cation dependence observed for full-length TSP1 binding to TSG-6Q is not because of direct effects of divalent cations on either TSG-6 or its interaction with the N-modules of TSP1; NMR titration studies have shown that there is no specific binding of Ca2ϩ ions to the TSG-6 Link module
Summary
30899 –30908, 2005 Printed in U.S.A. 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-␣-trypsin Inhibitor*. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Tel.: 301-496-6264; Fax: 301-402-0043; E-mail: droberts@ helix.nih.gov One such extracellular matrix protein that is induced by specific inflammatory signals is thrombospondin-1 (TSP1) We report that TSP1 is a specific ligand for TSG-6 This interaction is mediated by the N-module of TSP1 and the Link domain of TSG-6. Binding is regulated by divalent cations and is strongly inhibited by the interaction of heparin with the N-module of TSP1 but not by HA binding to the Link domain of TSG-6. We further show that TSP1 binding modulates the ability of TSG-6 to covalently modify I␣I
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