A cell-based assay for evaluating the interaction of heparin-like molecules and basic fibroblast growth factor.
A simple panning procedure that allows for the evaluation of interactions between various heparin-like molecules and basic FGF has been developed. This assay measures the ability of compounds to inhibit the interaction of transfected human lymphoblastoid cells, UC 729-6 (UC cells), expressing hamster syndecan and basic FGF-coated plastic plates. The transfected cells bind rapidly to basic FGF-coated plates while the control cells do not bind well. Binding of the transfected cells to basic FGF was inhibited by heparin and heparin sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. There was little inhibition of binding by chemically modified heparin such as completely desulfated, N-acetylated heparin, completely desulfated, N-sulfated heparin, and N-desulfated, N-acetylated heparin. These results suggested that both the N-sulfate and O-sulfate groups of heparin are required for binding to basic FGF. In addition, inhibition by oligosaccharides derived from depolymerized heparin increased with fragment size; partial inhibition was observed with oligosaccharides as small as hexamers. The biochemical basis for the binding of transfected cells to basic FGF was established by showing a significant increase of 35SO4 incorporation into HS. In particular, the level of 35SO4-HS in the trypsin-releasable (cell surface) pool increased fivefold. This increase was accounted for by demonstration of the presence of HS on immunoprecipitated syndecan from the transfected cells.
Ishihara
M
M
Glycomed Inc., Alameda, California 94501.
Tyrrell
D J
DJ
Kiefer
M C
MC
Barr
P J
PJ
Swiedler
S J
SJ
D000818:Animals; D001667:Binding, Competitive; D006224:Cricetinae; D005109:Extracellular Matrix; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D006493:Heparin / Q000031:analogs & derivatives / Q000494:pharmacology*; D006801:Humans; D011509:Proteoglycans; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured
D011509:Proteoglycans; D013462:Sulfur Radioisotopes; C036090:heparin proteoglycan; D016222:Fibroblast Growth Factor 2; D006493:Heparin