Hyaluronate binding properties of versican.
We have previously cloned a large chondroitin sulfate proteoglycan (versican) from human fibroblasts. The primary sequence shows that the N terminus contains sequence homology with known hyaluronate-binding molecule, suggesting that versican can bind hyaluronate. To test this hypothesis we have reconstructed a full-length versican cDNA and a versican cDNA fragment encoding the N terminus and have transfected Chinese hamster ovary cells and mouse 3T3 fibroblasts, respectively, with these constructs. The transfected Chinese hamster ovary cells make a proteoglycan shown to be versican by enzymatic and immunologic analysis. No corresponding proteoglycan was seen in the control cells. Using hyaluronate affinity chromatography, we show that recombinant versican specifically binds hyaluronate and does not bind to heparin or chondroitin sulfate. The transfected fibroblasts make a 78-kDa truncated form of versican that also binds hyaluronate and does not bind the related polysaccharides, showing that the hyaluronate binding activity resides at the N terminus of versican. The binding of versican to hyaluronate is substrate-concentration dependent and time dependent and can be competed with unlabeled versican. The dissociation constant for versican binding to hyaluronate was determined to be 4 x 10(-9) M.
LeBaron
R G
RG
La Jolla Cancer Research Foundation, California 92037.
Zimmermann
D R
DR
Ruoslahti
E
E
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism*; D015151:Immunoblotting; D007700:Kinetics; D037181:Lectins, C-Type; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D053675:Versicans
D011508:Chondroitin Sulfate Proteoglycans; D037181:Lectins, C-Type; D011993:Recombinant Fusion Proteins; C508322:VCAN protein, human; C508323:Vcan protein, mouse; D053675:Versicans; D006820:Hyaluronic Acid; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase