Foot-and-mouth disease viruses (FMDVs) target epithelial cells via integrin receptors, but can acquire the capacity to bind cell-surface heparan sulphate (or alternative receptors) on passage in cell culture. Vaccine viruses must be propagated in cell culture and, hence, some rationale for the selection of variants in this process is important. Crystal structures are available for type O, A and C viruses and also for a complex of type O strain O 1 BFS with heparin. The structure of FMDV A10 61 (a cell culture-adapted strain) complexed with heparin has now been determined. This virus has an RGSD motif in place of the otherwise conserved RGD integrin-binding motif and the potential to bind heparan sulphate (suggested by sequence analyses). FMDV A10 61 was closely similar in structure to other serotypes, deviating most in antigenic sites. The VP1 GH loop comprising the integrin-binding motif was disordered. Heparin bound at a similar site and in a similar conformation to that seen in the analogous complex with O 1 BFS, although the binding had a lower affinity and was more ionic.

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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.

Using an ELISA approach, we demonstrate that recombinant human IL-12 (rhIL-12) binds strongly to an immobilized heparin-BSA complex. This binding is completely displaceable with soluble heparin, IC50∼ 0.1 μg/ml, corresponding to ∼ 10 nM. By interpolation with our previous findings, this indicates an affinity for heparin greater than that of antithrombin III and comparable with that of FGF-2, two high-affinity heparin-binding proteins. Recombinant murine IL-12 also binds strongly to heparin. The binding of rhIL-12 to heparin shows specificity because chondroitin sulfates A and C fail to compete, whereas chondroitin B inhibits weakly. A highly sulfated heparan sulfate is a strong competitor, whereas other heparan sulfates show weak or no activity. Small heparin fragments inhibit binding, although activity decreases with size. An octasaccharide pool derived by cleavage of heparin with nitrous acid is a significantly stronger inhibitor than its heparinase I-derived counterpart, further indicating structural specificity in the interaction between rhIL-12 and heparin. The binding of recombinant p40 to heparin appears indistinguishable from that of the IL-12 heterodimer, implying that the heparin binding site is largely if not solely located in this subunit. These results show for the first time that IL-12 is a heparin-binding cytokine, a property common to the other Th1-response-inducing cytokines, IFN-γ and IL-2. Our findings strongly suggest that IL-12 will tend to be retained close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine role for IL-12.

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An increasing number of polypeptide growth factors and cytokines are now known to bind to heparin and heparan sulphate glycosaminoglycans. Well studied examples are members of the chemokine and fibroblast growth factor families. However other growth factors including proinflammatory and haematopoietic cytokmes also bind to heparin [ 1-41, Such interaction with glycosaminoglycans of the extracellular matrix and cell surface may be important in protecting cytokines against degradation, and in localising them close to their sites of secretion.We have developed a novel ELISA approach for the investigation of heparin-cytokine interactions. For this heparin is covalently bound via its reducing terminus to a protein carrier, bovine serum albumin (BSA), using sodium cyanoborohydnde. This coupling method is intended to ensure that the heparin chains remain accessible, for subsequent engagement in protein-binding interactions.Antithrombin 111 (AT 111) and fibroblast growth factor-2 (basic-FGF), both proteins with well characterised heparinbindmg properties, bind to the heparin-BSA complex in our ELISA. In both cases, bindmg is a dose dependent and saturable over the 0-3Ong range. Free heparin competes with binding of both proteins to the complex, with an EC,, value of around 50Ong/ml. With AT III, we have found that an AT 111-binding heparin fiaction separated by aflimty chromatography on immobilised AT 111, is a strong competitor in our ELISA. Conversely the low affinity heparin fraction is a poor competitor. Thus our ELISA shows the anticipated specificity in heparin-protein interactions.We have now shown that recombinant human i n t e r l e h 2, rIL-2, binds to the heparin complex (see Fig 1). The binding is dose dependent, but over a range of 0-100ng does not reach saturation. As with AT 111 and FGF-2, free heparin displaces rIL-2 fiom the complex, but with rIL-2 the EC,, for mhibition of binding is an order of magnitude higher, at around 7pg/ml. Thus it would appear that the afhuty of IL-2 for heparin is lower than that of AT 111 and By using various polysaccharides to compete with the immobilised heparin-BSA complex for binding to rIL-2, we are able to examine the specificity of the interaction. Thus far we have found that chondroitin sulphate fails to compete, whereas fucoidin competes strongly. These results therefore confirm and extend previous affinity chromatography studies which showed that rIL-2 binds fucoidin and heparin but not chondroitin sulphate [3].T h s binding of IL-2 to heparin-like glycosaminoglycans is llkely to maintain high concentrations of the cytolune at local tissue sites of release, thus enabling a paracrine mode FGF-2. 0.5 0.4 E m 0 * 0.3 0 c -0 9 : f < 0.1 0 Fig. 1. The ELISA. 0 10 20 30 40 50 IL-2 ngbinding of recombinant IL-2 to heparin in Wells were coated with either heparin-BSA complex ( 0 ) or mock treated BSA (0). After blocking and washing, wells were incubated with human recombinant IL-2. Bindmg was then disclosed with goat anti-human IL-2 and peroxidase-labelled donkey anti-goat I...

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