The interaction between basic fibroblast growth factor and heparan sulfate can prevent the in vitro degradation of the glycosaminoglycan by Chinese hamster ovary cell heparanases.
Heparan sulfate proteoglycans on Chinese hamster ovary (CHO) cell surfaces can bind and internalize basic fibroblast growth factor (bFGF). We have investigated whether this interaction affects heparan sulfate catabolism in vitro by measuring the ability of partially purified CHO heparanase activities to degrade 35S-labeled heparan sulfate glycosaminoglycans in the absence or presence of bFGF. Our studies show that the presence of the growth factor prevents partially purified heparanases from degrading the nascent 81-kDa chains to short 6-kDa products, whether the glycosaminoglycan is free in solution or covalently bound to core proteins. A 30-60 molar excess of the growth factor is required to inhibit completely chain degradation by heparanases, implying that multiple bFGF molecules must be bound to the glycosaminoglycan to prevent heparanase-catalyzed catabolism. This hypothesis is supported by protection studies indicating that nascent CHO heparan sulfate glycosaminoglycans have at least four to eight bFGF binding sites/chain. It does not appear, however, that the growth factor inhibits heparanase-catalyzed degradation of the glycosaminoglycan by binding to the sequence cleaved by the enzyme. Both the nascent and short chains bind bFGF with similar affinity (Kd values of 27.0 +/- 3.5 and 38.9 +/- 5.1 nM, respectively), indicating that heparanase activities do not destroy the bFGF binding sites. Rather, our results suggest that the growth factor interferes sterically with heparanase action by binding the heparan sulfate chain at a sequence next to the cleavage site or at a secondary site recognized by the enzyme.
Tumova
S
S
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA.
Bame
K J
KJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D005966:Glucuronidase; D006025:Glycosaminoglycans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D008956:Models, Chemical; D010053:Ovary / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D000099175:Heparanase
D004229:Dithiothreitol; D016222:Fibroblast Growth Factor 2; D005966:Glucuronidase; D006025:Glycosaminoglycans; D006026:Glycoside Hydrolases; D006497:Heparan Sulfate; D013439:Sulfhydryl Reagents; D000099175:Heparanase