FURTHER CHARACTERIZATION OF THE BINDING OF HUMAN RECOMBINANT INTERLEUKIN-2 TO HEPARIN AND IDENTIFICATION OF PUTATIVE BINDING-SITES

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, h ighly sulfated heparan sulfate and fucoidan, Here we show that IL-2 bi nding is dependent on heparin chain length, but with fragments as smal l as 15-mers retaining binding activity. The addition of exogenous hep arin has no effect on the in vitro biological activity of IL-2, In add ition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2, IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2-8, w hose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin, Human IL-2 analogs with s ingle amino acid substitutions at positions Lys43, Thr51, and Gln126 a nalogs no longer bind to heparin, By contrast the Arg38Ala analog reta ins heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48 , Lys49, Lys54, and His55, and the other being a discontinuous site co mprising Lys43, Lys64, Arg81, and Arg83, Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not int erfere with IL-2/IL-2 receptor interactions. Therefore, binding to gly cosaminoglycan may be a mechanism for retaining the cytokine in an act ive form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.