BIOCHEMICAL-BASES OF THE INTERACTION OF HUMAN BASIC FIBROBLAST GROWTH-FACTOR WITH GLYCOSAMINOGLYCANS - NEW INSIGHTS FROM TRYPSIN DIGESTION STUDIES

In the present study we have attempted a characterization of the bioch emical bases of the interaction of human basic fibroblast growth facto r (bFGF) with glycosaminoglycans (GAGs) in solution. This interaction has been evidenced as the capacity of different GAGs and various sulfa ted compounds to protect bFGF and different bFGF mutants from tryptic cleavage. Heparin protects bFGF from trypsin digestion in a dose-depen dent fashion. Substitution by site-directed mutagenesis of two or more basic residues with neutral glutamine residues in the amino-terminal region bFGF(27 - 32) or in the carboxyl-terminal region bFGF(118 - 129 ) does not significantly affect the protective effect exerted by hepar in. In contrast, heparin protection is abolished when the full region bFGF(27 - 32) is deleted. The capacity of different GAGs to protect bF GF from proteolytic cleavage decreases in the following order: heparin > heparan sulfate > dermatan sulfate = chondroitin sulfates A and C > hyaluronic acid = K5 polysaccharide, indicating that both the degree of sulfation and the backbone structure of GAG modulate its interactio n with bFGF. This is confirmed by the different capacity of various su lfated compounds (including dextran sulfates, suramin, trypan blue, an d sulfate ion) to protect bFGF from tryptic digestion. Moreover, trypt ic digestion studies performed with various heparin molecules and dext ran sulfates of different size, ranging from 2.0 kDa to 500 kDa, indic ate that the number of bFGF molecules which interact with a single mol ecule of polysaccharide is related to the molecular mass of the GAG an d that six hexose residues are sufficient to protect 1-2 molecules bFG F. In conclusion, our findings indicate that the capacity of GAGs to p rotect bFGF from tryptic cleavage depends upon their size, sulfation, distribution of the anionic sites along the chain, and structural requ irements of the bFGF molecule. These studies will help to design synth etic oligosaccharides endowed with different bFGF agonist and/or antag onist activities.