Preparation of affinity-fractionated, heparin-derived oligosaccharides and their effects on selected biological activities mediated by basic fibroblast growth factor.
Homogeneously sized, heparin-derived oligosaccharides were prepared from heparin following partial depolymerization with nitrous acid, reduction with sodium borohydride, and fractionation by gel permeation chromatography. The resulting pools of di-, tetra-, hexa-, octa-, and decasaccharides were sequentially applied to an affinity column of human recombinant basic fibroblast growth factor (bFGF) covalently attached to Sepharose 4B and further fractionated into subpools based on their elution from this column in response to gradients of sodium chloride. In general, pools of smaller heparin-derived oligosaccharides required relatively lower salt concentration for complete elution, and pools of larger oligosaccharides required higher salt concentration. The homogeneously sized pools and affinity-fractionated subpools of heparin-derived oligosaccharides were quantitatively assessed as inhibitors or enhancers of specific bFGF-mediated biological activities in five separate assay systems as follows: assay 1, to compete with human lymphoblastoid cells expressing syndecan (RO-12 UC cells) for binding to bFGF-coated wells (Ishihara, M., Tyrrell, D.J., Kiefer, M.C., Barr, P.J., and Swiedler, S.J. (1992) Anal. Biochem. 202, 310-315); assay 2, to inhibit 125I-bFGF binding to "low affinity sites" of adrenocortical endothelial (ACE) cells; assay 3, to inhibit bFGF-induced proliferation of ACE cells; assay 4, to support mitogenic activity of bFGF in a growth stimulation assay of chlorate-treated ACE cells; and assay 5, to enhance the in vitro interaction between 125I-bFGF and the recombinant extra-cellular domain of FGF high affinity receptor. The data derived from the five assay systems demonstrated that heparin-derived hexa- and octasaccharides inhibited the interaction between cell surface heparan sulfate proteoglycan and bFGF (assays 1 and 2) and bFGF-induced proliferation of ACE cells (assay 3) but were unable to enhance the binding of bFGF to its high affinity receptor in vitro (assay 5) or to support bFGF-induced mitogenesis in ACE cells (assay 4). These two activities required at least a decasaccharide with high affinity for bFGF.
Ishihara
M
M
Glycomed Incorporated, Alameda, California 94501.
Tyrrell
D J
DJ
Stauber
G B
GB
Brown
S
S
Cousens
L S
LS
Stack
R J
RJ
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002704:Chlorates / Q000494:pharmacology; D002846:Chromatography, Affinity; D004727:Endothelium / Q000166:cytology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D006493:Heparin / Q000737:chemistry*; D006497:Heparan Sulfate / Q000096:biosynthesis; D006801:Humans; D015394:Molecular Structure; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000494:pharmacology; D013552:Swine
D002704:Chlorates; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D006497:Heparan Sulfate; D009844:Oligosaccharides