Selection of COS cell mutants defective in the biosynthesis of heparan sulfate proteoglycan.
A simple procedure using human basic fibroblast growth factor (FGF) was utilized for the selection of COS cell mutants with defects in the biosynthesis or expression of heparan sulfate proteoglycan (HSPG). Our approach was based on the strong binding affinity exhibited by COS cells to human basic FGF that had been adsorbed to plastic dishes. Cell binding to basic FGF could be inhibited by heparin and heparan sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, or hyaluronic acid, suggesting that the cell binding involved an interaction between basic FGF and cell surface heparin-like molecules. COS cells were treated with ethyl methanesulfonate and four stable mutants were subsequently isolated that did not bind strongly to basic FGF adsorbed to plastic. These mutants cell lines (CM-2, CM-8, CM-9, and CM-15) exhibited significantly reduced 35SO4 incorporation into HS (40-70% depending on the cellular pool analyzed). In one of these cell lines, CM-15, the incorporation of [6-3H]glucosamine into HS was unaltered, suggesting that the extent of oligosaccharide polymerization was equivalent to that observed for the wild-type cells. Structural analysis revealed that N-sulfated glucosamine residues were present much less frequently in HS derived from these cells as compared with that derived from wild-type cells. Furthermore, CM-15 was found to be three-fold deficient in HS N-sulfotransferase activity, but contained wild-type levels of HS O-sulfotransferase activities.(ABSTRACT TRUNCATED AT 250 WORDS)
Ishihara
M
M
Glycomed Inc., Alameda, California 94501.
Kiefer
M C
MC
Barr
P J
PJ
Guo
Y
Y
Swiedler
S J
SJ
D000818:Animals; D002460:Cell Line; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis / Q000302:isolation & purification; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000096:biosynthesis*; D007700:Kinetics; D009154:Mutation; D011509:Proteoglycans / Q000096:biosynthesis*; D011865:Radioisotope Dilution Technique; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D016222:Fibroblast Growth Factor 2; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D017468:Receptors, Fibroblast Growth Factor; D013431:Sulfates; D015238:Sulfotransferases; D013462:Sulfur Radioisotopes