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-alpha-trypsin inhibitor.
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.
Kuznetsova
Svetlana A
SA
Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892-1500, USA.
Day
Anthony J
AJ
Mahoney
David J
DJ
Rugg
Marilyn S
MS
Mosher
Deane F
DF
Roberts
David D
DD
D000510:Alpha-Globulins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000510:Alpha-Globulins; D002413:Cations, Divalent; D015815:Cell Adhesion Molecules; D011994:Recombinant Proteins; C072273:TNFAIP6 protein, human; D019700:Thrombospondin 1; C513057:Tnfaip6 protein, mouse; C011877:inter-alpha-inhibitor; D006820:Hyaluronic Acid; D006493:Heparin