Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites

Abstract

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition 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, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains 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 comprising 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 interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.

Antibody–cytokine fusion proteins: applications in cancer therapy

“…This antibody-(IL-2) fusion protein retained the binding specificity of the antibody and cytokine biological activity similar to that of rhIL-2 [69]. We have also observed that anti-HER2/neu IgG3-(IL-2) retains the heparin-binding activity of IL-2 [70], a property that allows the binding of this cytokine to glycosaminoglycans present in the surface of cells [71] and that may play a role in the control of T-cell responses by IL-2 [72]. The authors explored the in vivo efficacy of anti-HER2 /neu IgG3-(IL-2) as a direct antitumor agent in a murine colon carcinoma cell line expressing human HER2/neu (CT26-HER2/neu), which was injected subcutaneously into immunocompetent syngeneic BALB/c mice.…”

Interleukin-2 Carbohydrate Recognition Modulates CTLL-2 Cell Proliferation

“…Sherblom et al (11) and Zanetta et al (12) reported that IL-2 recognizes high-mannose type glycans with five or six mannosyl residues as determined by the plate method. Later, Najjam et al (13) found that rhIL-2 binds to heparin specifically. However, since the addition of heparin did not show any inhibitory effect on IL-2-dependent cell proliferation, it was suggested that the interaction between IL-2 and heparin is not related to such activity.…”

Propagation and Control of T Cell Responses by Heparan Sulfate-Bound IL-2

“…However, our laboratory (9) and others (10,11) recently demonstrated that IL-2 binds to the extracellular matrix, suggesting the possibility that bound IL-2 might contribute to immune responsiveness. We tested this concept in model systems in which the functional effects of bound vs free IL-2 could be compared.…”

“…Because IL-2 has been reported to bind collagen and mannose as well as heparan sulfate (9 -11, 20), we asked whether IL-2 must bind heparan sulfate in vivo to manifest this function. To address this question, we compared T cell responses generated in the presence of WT IL-2, with responses generated using a mutant IL-2 that does not bind heparan sulfate (11). The mutant IL-2, bearing a proline for threonine substitution at aa 51, has equivalent bioactivity to that of WT IL-2 in vitro (21,22).…”