Interactions of thrombospondin with sulfated glycolipids and proteoglycans of human melanoma cells.
Human melanoma cell spreading on thrombospondin substrates and chemotaxis in a gradient of soluble thrombospondin requires the amino-terminal heparin/sulfatide-binding domain of thrombospondin. Some melanoma cell lines attach but do not spread or respond in chemotaxis assays. Sulfated glycoconjugates produced by melanoma cells that could mediate these activities were identified by metabolic labeling with [35S] sulfate and tested for their ability to bind thrombospondin. Heparan sulfate proteoglycans that bind thrombospondin are made by both spreading and non-spreading melanoma cell lines. Thrombospondin binds with high affinity to a high molecular weight heparan sulfate proteoglycan, but not to the major chondroitin sulfate. The active heparan sulfate proteoglycan can be partially purified by affinity chromatography on thrombospondin-agarose or hydrophobic interaction with octyl-Sepharose. Thrombospondin binding requires the amino-terminal domain and is inhibited by monoclonal antibody A2.5 or fucoidan. Binding activity is lost following degradation of the proteoglycan with heparatinase or nitrous acid. [35S]Sulfate labels several melanoma cell glycolipids including galactosylceramide-I3-sulfate, lactosyl ceramide-II3-sulfate, and sulfated glucuronosylparagloboside. The latter glycolipid was detected in three cell lines that spread on thrombospondin but not in the nonspreading C32 melanoma cells. Thrombospondin binds to the isolated glycolipid, and the glycolipid and an antibody to this structure inhibit cell spreading on thrombospondin substrates. Thus, the presence of glycoconjugates with terminal nonreducing glucuronosyl 3-sulfate correlates with melanoma cell spreading on thrombospondin, whereas expression of heparan sulfate proteoglycans that bind thrombospondin does not.
Roberts
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Laboratory of Structural Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D002846:Chromatography, Affinity; D006017:Glycolipids / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology; D011509:Proteoglycans / Q000378:metabolism*; D019699:Thrombospondins
D011508:Chondroitin Sulfate Proteoglycans; D006017:Glycolipids; D006023:Glycoproteins; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D019699:Thrombospondins; C011117:sulfoglycolipids