Analysis of affinity and structural selectivity in the binding of proteins to glycosaminoglycans: development of a sensitive electrophoretic approach.
Members of several families of cell surface and secreted proteins bind glycosaminoglycans (GAGs), the structurally heterogeneous polysaccharides found on proteoglycans. To understand the physiological significance of the interactions of proteins with GAGs, it is critical that relationships between GAG structure and binding be analyzed. It is particularly important that interactions depending on common structural features of GAGs (e.g., size, charge density, and disaccharide repeat unit) be distinguished from those mediated by specific sequences of carbohydrate modification. Gathering the information needed to make such distinctions has so far been difficult, however, partly because structurally homogeneous samples of GAGs are lacking but also because of technical difficulties associated with performing and interpreting assays of protein-GAG binding. We describe an electrophoretic method useful for both measuring affinity and evaluating structural selectivity in protein-GAG binding. Data are presented on the binding of the GAG heparin to the protease inhibitor antithrombin III, the acidic and basic fibroblast growth factors, and the extracellular matrix protein fibronectin. Results obtained with fibronectin are consistent with a model in which high-affinity binding (Kd approximately 34 nM) is mediated through the recognition of specific carbohydrate sequences.
Lee
M K
MK
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Lander
A D
AD
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D009414:Nerve Growth Factors / Q000378:metabolism; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013329:Structure-Activity Relationship
D005353:Fibronectins; D006025:Glycosaminoglycans; D009414:Nerve Growth Factors; D016222:Fibroblast Growth Factor 2; D012710:Serum Albumin, Bovine; D000990:Antithrombin III; D006493:Heparin; D002809:Chondroitin Sulfates