The binding of human betacellulin to heparin, heparan sulfate and related polysaccharides.
Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.
Mummery
Rosemary S
RS
School of Biological Sciences, Royal Holloway University of London, Egham Hill, Egham, Surrey TW20 OEX, UK.
Mulloy
Barbara
B
Rider
Christopher C
CC
D000107:Acetylation; D000818:Animals; D016667:Bacterial Capsules; D066259:Betacellulin; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002417:Cattle; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D013552:Swine
D066259:Betacellulin; D004815:Epidermal Growth Factor; D006493:Heparin; D006497:Heparan Sulfate; D036341:Intercellular Signaling Peptides and Proteins; D011135:Polysaccharides, Bacterial; C587851:BTC protein, human; C050673:capsular polysaccharide K5