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

Abstract

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 35 S-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 (K d 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.Heparan sulfate proteoglycans (HSPGs), 1 molecules composed of heparan sulfate (HS) glycosaminoglycan chains covalently linked to a protein core, are ubiquitously present on cell surfaces and in extracellular matrix and basement membranes (1-3). Their expression appears to be developmentally regulated (4, 5) and cell-specific (6). Proteoglycans are implicated in a number of cellular processes, including cell adhesion, migration, differentiation, and proliferation (for review, see Refs. 7 and 8). Most of the identified proteoglycan functions are attributed to the interaction of the glycosaminoglycan chain with a protein ligand, such as lipoprotein lipase, fibronectin, or various members of heparin-binding growth factor family (9). The last has received a great deal of attention after the presence of heparin or heparan sulfate had been shown to be prerequisite for high affinity binding of basic fibroblast growth factor (bFGF) to its cell surface fibroblast growth factor receptor (FGFR) (10 -12). It was thought originally that binding to HS might change the growth factor conformation so that it can be recognized by the FGFR; however, cocrystallization of bFGF with heparin oligosaccharides demonstrated no structural changes in the growth factor upon the binding (13). Since heparin can form a ternary complex with bFGF and FGFR (14 -16), it has been proposed that the proteoglycans function as a coreceptor for ...

Aβ(1–40) Prevents Heparanase-catalyzed Degradation of Heparan Sulfate Glycosaminoglycans and Proteoglycans in Vitro

“…The pH of the running buffer was pH 7.0 to dissociate any A␤(1-40)-HS complexes. The column was calibrated with heparin, HS, and chondroitin sulfate polysaccharides of known molecular mass (19). By our estimation, nascent CHO HS chains are 81 Ϯ 5.4 kDa, and the short, degraded glycosaminoglycans are 6.0 Ϯ 1.1 kDa (19).…”

“…The TSK 4000 column was equilibrated and run identically to the TSK 3000 column used in the glycosaminoglycan degradation assay. It has also been calibrated with glycosaminoglycans of known molecular mass (19).…”

“…The shorter HS chains do not appear to bind to A␤(1-40) as well as the longer glycosaminoglycans; however, this may be an artifact based on the size difference. Nascent CHO HS chains are approximately 14 times longer than the short, heparanase products (13,19), thus the binding of one long HS molecule will retain more 35 S radioactivity on the membrane than the binding of one short glycosaminoglycan chain.…”

“…Heparanase activities, partially purified from CHO cells, degrade long 81-kDa HS chains to short 6-kDa glycosaminoglycans (19). The short glycosaminoglycan products can be separated from the substrate based on their solubility in the detergent CPC; long HS chains precipitate in the detergent, whereas the short heparanase-degraded glycosaminoglycans remain soluble.…”

“…Experiments by Snow et al (1) suggest that both interactions are necessary for the formation of fibrillar A␤-amyloid, since coinfusion of A␤ and HSPGs into rat brain produced deposits, whereas coinfusion of A␤ and HS glycosaminoglycans did not. This is somewhat surprising, since in vitro, HS chains alone can cause both A␤ and A␤(1-40) to aggregate (3), and heparin converts A␤ (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) into macrofibers (2). It may be that in vivo, stabilization of the aggregates and decreased amyloid susceptibility to proteolysis require A␤ to interact with both the core protein and HS chains.…”