Journal zyxwv of Cellular Biochemistry zyxw 57:173-184 (1995) Heparan Sulfate Primed on p-D-Xylosides Restores Binding of Basic Fibroblast Growth Factor Hua-Quan Miao, Timothy A. Fritz, Jeffrey zyxwv 0. Esko, Joseph Zimmermann, Avner Yayon, and Israel Vlodavsky Department of Oncology, Hadassah University Hospital, Jerusalem 91 120 (H.-Q.M., I.V.), Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 761 00 (A.Y.), Israel; Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294 (T.A.F., J.D.E.); IBEX Technologies, Montreal, Canada zyx H4P 1 P7 zy (W.1 Abstract Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF).Mutant Chinese hamster ovary cells (pgsA-745)deficient in xylosyltransferase are unable to initiate glycosaminoglycan synthesis and hence can not bind bFGF to low- and high-affinity cell surface receptors. Exposure ofpgsA-745 cells to p-D-xylopyranosides containing hydrophobic aglycones resulted in restoration of bFGF binding in a manner similar to that induced by soluble heparin or by heparan sulfate (HS) normally associated with cell surfaces. Restoration of bFGF binding correlated with the ability of the p-D-xylosides to prime the synthesis of heparan sulfate. Thus, both heparan sulfate synthesis and bFGF receptor binding were induced by low concentrations (1 0-30 pM) of estradiol-p-D-xyloside and naphthyl-p-D-xyloside, but not by cisltrans-decahydro-2-naphthyl-p-D- xyloside, which at low concentration primes mainly chondroitin sulfate. The obligatory involvement of xyloside-primed heparan sulfate in restoration of bFGF-receptorbinding was also demonstrated by its sensitivity to heparinase treatment and by the lack of restoration activity in CHO cell mutants that lack enzymatic activities required to form the repeating disaccharide unit characteristic of heparan sulfate. Xyloside-primed heparan sulfate binds to the cell surface. Restora- tion of bFGF receptor binding was induced by both soluble and cell bound xyloside-primed heparan sulfate and was abolished in cells that were exposed to 0.5-1 .O M NaCl prior to the bFGF binding reaction. These results indicate that heparan sulfate chains produced on xyloside primers behave like heparan sulfate chains attached to cellular core proteins in terms of affinity for bFGF and ability to function as low-affinity sites in a dual receptor mechanism characteristic of bFGF and other heparin-binding growth promoting factors. zyxwv c 1995 Wiley-Liss, Inc Key words: heparin, proteoglycans, glycosaminoglycans, receptor-binding, heparinase, Chinese hamster ovary cell mutants Fibroblast growth factors (FGFs) are a family of at least eight structurally related polypep- tides characterized by their high affinity for heparin. They are highly mitogenic for meso- Abbreviations used: bFGF; basic fibroblast growth factor; BSA, bovine serum albumin; zyxwvutsrqp CHAPS, (3-[(3-chloramidopro- py1)-dimethylammonio]- l-propanesulfonate; CHO, Chinese hamster ovary; DMEM, Dulbecco’s modified Eagle’s me- dium; DX, zyxwvutsr cis zyxwvutsrqponml /trans-decahydro-2-naphthy1-~-D-xyIoside; D- xyl, D-xylose; EDX, 3-P-estradiol-P-D-xyloside; GAG, gly- cosaminoglycans; HEPES, N-(2-hydroxyethylpiperazine-N’- 2-ethanesulfonic acid); HS, heparan sulfate; HSPG, heparan sulfate proteoglycans; MeZSO, dimethyl sulfoxide; NX, 2-naphthyl- P-D-xyloside. Received February 24,1994; accepted June 10, 1994. Address reprint requests to Dr. Israel Vlodavsky, Depart- ment of Oncology, Hadassah Hospital, P.O.B. 12000, Jerusa- lem 91120, Israel. c 1995 Wiley-Liss, Inc. derm- and neuroectoderm-derived cells and are among the most potent inducers of neovascular- ization and mesoderm formation [Burgess and Maciag, 1989; Gospodarowicz, 1991; Folkman and Shing, 19921. Studies on the mode of action of basic fibroblast growth factor (bFGF) identi- fied a novel role for heparin and heparan sulfate (HS) in the formation of distinct bFGF-hepa- rinlHS complexes that are essential for bFGF receptor binding and activation mayon et al., 1991; Rapraeger et al., 1991; Ornitz et al., 19911. Recently, direct interaction of heparin with a specific sequence in the extracellular domain of the FGF receptor was also identified and sug- gested to be involved in FGF-induced cell prolif- eration [Kan et al., 19931. The crucial role of the cell surface HS was demonstrated by the finding