| Glycobiology | Pages |
Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites
Introduction
Results
Discussion
Materials and methods
Acknowledgments
References
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, 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. Key words: IL-2/cytokine/heparin/monoclonal antibodies/IL-2 receptor
Introduction
Interleukin 2 (IL-2) is a cytokine synthesized and secreted by antigen- or mitogen-activated T-cells (Gillis et al., 1978). This glycoprotein (polypeptide Mr 15.5 kDa) promotes the proliferation of IL-2 dependent T-cells, and has an immunomodulatory effect on cytotoxic T-cells, natural killer cells, activated B-cells, and lymphokine activated cells (Taniguchi et al., 1986). The high resolution model structure of human IL-2 is a bundle of four [alpha]-helices, with a short section of anti-parallel ß-sheet protein involving polypeptide loops A-B and C-D (Bazan and McKay, 1992; Mott et al., 1995). Together with this model, the use of truncation and single amino acid replacement mutations, as well as anti-IL-2 monoclonal antibodies, there is a good understanding of the interaction of IL-2 with its receptor complex (Ju et al., 1987; Sauve et al., 1991; Zurawski et al., 1992; Moreau et al., 1995). The high affinity IL-2 receptor complex (Kd = 10 pM) consists of three receptor subunits, one low-affinity p55 [alpha]-chain subunit (Kd = 10 nM) and two other p75 ß- and [gamma]-chain subunits, with intermediate affinity, Kd = 1 nM (Minami et al., 1993). The ß and [gamma] chain subunits, but not [alpha], are required for IL-2 signal transduction (reviewed in Theze et al., 1996). Current work implicates residues Lys35, Lys38, Thr42, and Lys43 as being critical in the binding site for the p55 [alpha]-chain (Sauve et al., 1991). These are located in the loop region between the A and B helices. Residues Leu17 and Asp20 of the region 1-30 (helix A) are critical for p75 ß-chain subunit binding (Eckenberg et al., 1997). The third receptor chain, [gamma]c, is thought to contact residue Gln126 which lies close to the carboxy terminus of the D helix (Buchli and Ciardelli, 1993; Theze et al., 1996).
An increasing number of cytokines are found to bind not only to their high affinity cell surface receptors, but also to glycosaminoglycans, particularly the heparin/heparan sulfate family. These polysaccharides are long, linear, highly sulfated chains. Although based on an N-acetylglucosamine-uronic acid disaccharide repeat, they are extremely heterogenous in structure (Gallagher et al., 1986). Heparan sulfate is widely synthesized by many cell types (Kolset and Gallagher, 1990), and is mainly present on cell surfaces and in the extracellular matrix borne on proteoglycans (Yanagashita and Hascall, 1992). However, heparin, a particularly highly sulfated variant with a high ratio of iduronic acid to glucuronic acid, is only produced and secreted by mast cells.
The binding of a cytokine to glycosaminoglycans may have a number of functions. One important role is the localization of the cytokine close to its site of secretion. In this way the biological activity of the cytokine will be focused at local tissue sites. This appears to be particularly the case for FGF-2 and related cytokines (reviewed by Schlessinger et al., 1995). Moreover, interaction with glycosaminoglycan may well protect the cytokine from proteolytic degradation thus enhancing its biological activity in the tissues. This has been established for FGF-2 (Saksela et al., 1988) and also for interferon-[gamma] (Lortat-Jacob et al., 1996). In the latter case this protection from proteolysis results in a considerably prolonged circulatory half-life. Binding to glycosaminoglycan may also be a mechanism for presenting the cytokine to its high affinity receptor. This is certainly the case for FGF-2, which in the absence of heparin or heparan sulfate cannot engage its receptor (Rapraeger et al., 1991; Yayon et al., 1991).
In this laboratory we have previously shown that IL-2 is among the growing list of cytokines which bind to glycosaminoglycans of the heparin/heparan sulfate family. Affinity chromatography on immobilized glycosaminoglycans showed that recombinant human IL-2 (rhIL-2) was bound by heparin and fucoidan, whereas chondroitin sulfate gave poor binding (Ramsden and Rider, 1992). More recently we have developed a quantitative ELISA technique to investigate the binding of proteins to an immobilized complex of heparin covalently coupled to BSA. Using this approach, in addition to confirming that rhIL-2 is a heparin-binding cytokine, we showed that only soluble heparin, fucoidan, and a highly sulfated preparation of heparan sulfate are able to compete with the heparin complex for the binding of rhIL-2. In the case of heparin, an IC50 value of 0.5 µM was obtained (Najjam et al., 1997). In the present article, we further characterize the interaction between heparin and rhIL-2. Moreover, using monoclonal antibodies, recombinant soluble IL-2 receptor polypeptides, and mutant rhIL-2s with single amino acid replacements, we localize the heparin binding on the surface of IL-2, identifying two putative binding sites.
Results
Recognition of heparin-bound IL-2 by mAbs
We have previously described an ELISA method demonstrating that rhIL-2 binds to a heparin-BSA complex in a dose-dependent manner. In this assay binding of IL-2 to heparin is detected by subsequent incubation with either goat polyclonal or a commercial murine mAb specific for rhIL-2 (Najjam et al., 1997). In order to determine whether particular epitopes on the surface of IL-2 remain accessible to antibody or not after heparin binding, we employed three murine anti-rhIL-2 mAbs, 3H9, H2-8 and 16F11. These mAbs, all IgG, have been fully characterized elsewhere (Rebollo et al., 1992; Moreau et al., 1995; Eckenberg et al., 1997). In Figure 1A, we show that with both 3H9 and H2-8, dose-dependent binding of IL-2 to the heparin complex is readily detectable, indicating that their respective epitopes are accessible and unaltered by the previous binding of rhIL-2 to heparin. By contrast, with up to 50 ng of rhIL-2, no absorbance above background is detectable in presence of 16F11 (data not shown). However, this mAb is known to recognize a conformational epitope, and only binds to rhIL-2 in solution (Rebello et al., 1992). With mAbs 3H9 and H2-8, the binding of rhIL-2 to BSA-coupled heparin is detected with high sensitivity, the lower limit of detection being less than 5 ng IL-2, although the curve obtained with H2-8 is lower than that obtained with 3H9 mAb (Figure 1A). The background binding of IL-2 to mock treated-BSA (t-BSA) is virtually undetectable with all mAbs.
Figure 1. Recognition of heparin-bound rhIL-2 by mAbs. (A) Dose-dependent binding of rhIL-2 to immobilized heparin complex, detected using mAbs 3H9 ([squf]) and H2-8 ([squ]). (B) Competitive binding to rhIL-2, 10 ng/well, of UFH, HS6, and fucoidan, 50 µg/ml. The absorbances obtained in the absence of competitor (control, 100%) were 0.088 for H2-8, and 0.271 for 3H9. Error bars represent standard deviation (n = 3). For both (A) and (B), 3H9 and H2-8 mAbs were used for detection at 140 and 80 nM, respectively. These concentrations were found to give maximal detection of IL-2 binding directly to the ELISA well surface.
Structural specificity of the interaction between heparin and rhIL-2
We previously showed that the binding of rhIL-2 to the heparin-BSA complex was competed out by free heparin with an IC50 of 5 µg/ml (Najjam et al., 1997). Moreover, we were able to demonstrate that fucoidan and a highly sulfated heparan sulfate, HS6, were also good competitors whereas other glycosaminoglycans (chondroitin sulfate, dermatan sulfate, and several other heparan sulfates) showed little or no competition. In Figure 1B we show using mAbs H2-8 and 3H9, that the competition for IL-2 binding between coupled-heparin and soluble GAGs is fully demonstrable, as it was in our previous work with polyclonal anti-IL-2. Thus under the conditions employed, with both mAbs fucoidan gives complete inhibition of binding, heparin gives around 80% inhibition. However, HS6 is only a partial inhibitor, giving around 60% inhibition with mAb 3H9 and 25% using H2-8. With both mAbs four other heparan sulfates (HS2, HS4, HSA, and HSB), were inactive as competitors (results not shown), a finding in agreement with that we previously obtained using polyclonal anti-IL-2.
Effect of chain length on heparin binding
In our ELISA assay a range of four different clinical low molecular weight heparins (Enoxaparin, Fragmin, Tinzaparin, and Fraxiparine) failed to compete with coupled-heparin for IL-2 binding (data not shown). These preparations typically have mean number average molecular weights (Mn) 3-5.5 kDa, corresponding to around 10-17 hexose residues in length. In order to investigate further the size requirement for IL-2 binding we separated uncleaved heparin chains into four size fractions by gel-filtration. The characteristics of the four fractions, F1-F4, are shown in Table 01. As may be seen in Figure 3, unfractionated heparin under the conditions employed gives 80-85% competition of IL-2 binding. The F1 pool is a more effective competitor, whereas the F2 pool gives the same level of competition found with the parent, unfractionated material. With the F3 pool only 35-40% competition is observed making it the weakest competitor of the four size fractions, since F4 gives approximately 50% inhibition. Essentially the same results are obtained irrespective of whether H2-8 or 3H9 is used as the detecting antibody. Taken overall these results indicate that heparin binding activity has a marked size dependency, although chains as small as 5 kDa Mn retain the ability to bind IL-2.
Figure 2. Competitive binding of heparin size fractions. Fractions F1-F4 are described fully in the text. All were tested at 50 µg/ml giving concentrations of 3.1, 1.4, 2.5, 4.2, and 7.1 nM for UFH and fractions F1-F4, respectively. RhIL-2 was used at 10 ng/well, and detection was with either mAb H2-8, 80 nM, or 3H9, 140 nM. The control values of 100% correspond to absorbances of 0.088 and 0.27 for H2-8 and 3H9, respectively. Error bars represent standard deviation.
Figure 3. Binding of rhIL-2 in the presence and absence of soluble rIL-2 subunits. Hatched bars, rhIL-2, 200 ng/ml (13.3 nM), in the absence of soluble receptor subunits. Open bars, rhIL-2 mixed with IL-2R[alpha], 3.7 nM, or IL-2Rß, 4.0 nM prior to addition to the complex. Stippled bars, rhIL-2 mixed with 37 nM IL-2R[alpha], or 40 nM IL-2Rß. The mAbs 3H9 and H2-8 were used for detection in the presence of IL-2R[alpha] and IL-2Rß, respectively. Control absorbances in the absence of receptor subunit were 0.313 for 3H9 and 0.225 for H2-8.
Table 1
Fraction
Mean number average Mr (kDa)
Polydispersitya
F1
32
1.1
F2
17
1.15
F3
9.2
1.3
F4
5.2
1.37
Unfractionated heparin
12
1.3
Effect of IL-2 receptor polypeptides [alpha] and ß on heparin binding
In order to examine whether or not heparin might bind at the same sites on IL-2 as its receptors subunits, we preincubated rhIL-2, 200 ng/ml equivalent to 13.3 nM, with increasing concentrations of soluble recombinant receptor polypeptides, prior to plating out aliquots (100 µl) in heparin-BSA complex coated wells. For sIL-2R[alpha], up to 37 nM was used, this maximal concentration corresponding to the dissociation constant for the binding of this receptor to IL-2 (Myszka et al., 1996). As shown in Figure 3, there was no apparent competition for subsequent heparin binding. Indeed at the highest concentration of sIL-2R[alpha] employed, there is a statistically significant increase (p < 0.005, by Student's t test) in heparin binding. In the case of the lower affinity polypeptide, sIL-2Rß, Kd 400 nM (Myszka et al., 1996), the use of concentrations equal to the Kd is impractical. However, similarly preincubation with up to 40 nM sIL-2Rß, showed no evidence of competition, again a modest but statistically significant (p < 0.005) increase in binding is observed. Therefore, these data provide no evidence of competition between these receptor subunits and heparin for the binding of IL-2.
The effect of heparin on the binding of IL-2 to its receptor was also examined in a bioassay involving the IL-2-dependent cell line, CTLL-2. As shown in Figure 4A, the presence of 100µg/ml heparin neither inhibited nor enhanced the proliferative response of these cells to rhIL-2 in vitro. Moreover, pretreatment of the cells with heparinase II also had no detectable effect (Figure 4B). Taken overall, our data strongly suggests that the binding of heparin to IL-2 does not affect receptor interactions.
Figure 4. Effect of heparin on proliferation of CTLL-2 cells. (A) Increasing concentrations of rhIL-2 were added to 104 cells/well in the absence (dashed line) and presence (solid line) of 100 µg/ml unfractionated heparin. (B) As for (A) except cells were pretreated for 3 h with heparinase II, (1.25 U/ml). After 2 days, cell proliferation was monitored by MTS colorimetric assay. All points are means of triplicates, shown ± SD. The results shown are for a single experiment on the same batch of cells. A repeat experiment reproduced these results.
Heparin binding properties of rhIL-2 mutants
We sought to establish the effect of single amino acid substitutions on the binding of rhIL-2 to heparin. Four different purified rhIL-2 muteins (R38A, K43E, T51P, and Q126D) (Sauve et al., 1991; Buchli and Ciardelli, 1993; Chang et al., 1995) were investigated. We first performed direct ELISA by adding increasing concentrations, 0-200 ng/ml, of each analog or wild type rhIL-2 to uncoated plate wells, with binding detected by either 3H9 or H2-8. With both mAbs, analogs R38A, T51P, and Q126D gave higher absorbances than wild type. However, compared to wild type, K43E analog was either less well, or similarly recognized by 3H9 or H2-8, respectively. Representative values obtained for 100 ng/ml of each protein are shown in Figure 5A for 3H9, and in Figure 5B for H2-8.
Figure 5. Binding of rhIL-2 analogs to heparin complex. Hatched bars, direct binding of rhIL-2, 10 ng/well, to ELISA plate. Solid bars, binding of rhIL-2, 30 ng/well, to immobilized heparin complex. (A) Detection with mAb 3H9. (B) Detection with mAb H2-8.
We then performed similar titration curves, 0-500 ng/ml protein, to examine the binding of these muteins to wells coated with heparin complex. As may be seen in Figure 5, the analogs K43E and T51P completely failed to bind to the complex. With mutant Q126D trace amounts of binding to the complex was detectable. However R38A gives much higher absorbances than wild type. This is also the case albeit to a lesser extent with direct binding to the plate. The reasons for this are unclear. Nonetheless, it appears that possession of a basic residue at position 38 is not important for heparin binding. By contrast the three residues Lys43, Thr51, and Gln126 are essential in the binding of IL-2 to heparin.
Discussion
In the present study we have examined the size requirements for the binding of heparin to rhIL-2. We clearly show that small size fractions of heparin chains retain binding of the cytokine. Even the smallest size fraction studied, [sim]5 kDa, competes well for rhIL-2 binding. However, clinical low Mr heparins, which are of a similar size to F4 (data not shown), failed to compete. This lack of binding with these preparations may arise because particular cleavage methods which have been employed in their preparation preferentially cut the heparin chains within sequences which would have bound IL-2. Taken overall, our data suggest that the minimal size for binding is around 15 hexose residues. The well studied, high affinity interaction between heparin and antithrombin III, requires a pentasaccharide sequence (Lindahl et al., 1984). However the minimal sequence required for binding to the growth factor FGF-2 has been variously described as a decasaccharide (Turnbull et al., 1992) or a pentasaccharide (Ishihara et al., 1993; Maccarana et al., 1993). Further studies using monodisperse oligosaccharides are required to fully define the size of the IL-2 binding sequence.
For a heparin binding cytokine, a key question is whether such binding results in sequestration, i.e., the cytokine is no longer available for interaction with its high affinity cell surface receptor, or presentation, in which case the heparin-bound cytokine would be fully active and able to engage its receptors. In the case of FGF-2, binding to heparin in fact appears to be an essential prerequisite or subsequent receptor engagement (Rapraeger et al., 1991; Yayon et al., 1991). Here we found that neither the addition of soluble heparin, nor exposure to heparinase II, reduced or potentiated the proliferative activity of rhIL-2 on the dependent cell line CTLL-2. Moreover in our binding ELISA we found that neither IL-2R[alpha] (p55, CD25) nor IL-2Rß (p75, CD122) compete with heparin. In fact in both cases the receptor subunits appeared to give a modest potentiation of binding. Overall, we therefore conclude that heparin-bound IL-2 is fully able to bind to the [alpha] and ß chains of its cell surface receptor complex and retains complete bioactivity.
Crystallographic modeling of the heparin binding proteins FGF-2 and lactoferrin shows that clusters of between four and seven basic residues are the primary determinants of heparin binding sites (Mann et al., 1994; Thompson et al., 1994; Faham et al., 1996). Moreover, several studies of heparin binding proteins and peptide have shown that binding sites frequently consist of basic residues forming loci about 20 Å apart on the protein surface (Spillman and Lindahl, 1994). Our studies reveal that IL-2 will only recognize the highly sulfated polysaccharides heparin and fucoidan, together with a highly sulfated preparation of heparan sulfate. This strongly implies that the binding site for these acidic polysaccharides on the surface of IL-2 is rich in basic residues. On this basis, the high resolution models of human IL-2, obtained by both x-ray crystallographic and NMR studies (Bazan et al., 1992; Mott et al., 1995) were examined and several plausible clusters and alignments of basic residues were identified. Prominent among these are four which we designate Clusters A, B, C, and D comprising, respectively: A, Lys32, Lys35, Arg38, Lys76, His79, and Arg81; B, Lys48, Lys49, Lys54, and His55; C, Lys32, Lys76, Arg81, and Arg83; and D, Lys43, Lys64, Arg81, and Arg83. Interestingly, none of these sites is fully conserved in murine IL-2, which we have found not to be a heparin binding protein (data not shown).
Residues 35 and 38 of Cluster A are involved in defining the IL-2R[alpha] (p50) receptor binding site (Sauve et al., 1991). The absence of competition between this polypeptide and heparin in our binding studies, together with the inability of heparin to influence the biological activity of IL-2 argue against Cluster A being important in heparin binding. Moreover, the full retention of heparin binding in a mutein in which Arg38 is replaced by Ala, also tends to rule out this cluster. Similarly, Cluster D can also be dismissed as it contains Lys43 which is also a component of the p50 receptor binding site. Our only evidence implicating both Clusters A and D is that replacement of Lys43 with glutamate abolishes heparin binding. However, since this residue is located at one end of a short section of ß-sheet (Mott et al., 1995), its nonconservative replacement is very likely to disrupt the folding patterns in this region of the molecule which lies outside the more stable four [alpha]-helical bundle region of IL-2. Any such disruption would be likely to affect cluster B as the four basic residues it comprises lie at the end of this [beta]-sheet and in the loop connecting it to the B helix. The nonconservative replacement of Thr51 in this region with Pro completely abolishes binding to heparin, an observation which suggests that residues adjacent to Thr51, such as those defining Cluster B, are involved in binding. Similarly, the point substitution of Gln126 with Asp, which also abolishes heparin binding, is located near the carboxy terminal end of the D helix and is adjacent to Cluster B. This evidence lends considerable support to Cluster B being the heparin binding site. A counterargument is that mAb 3H9 is reported to recognize an epitope on IL-2 within residues 30-54, and in particular residues 44-54 (Moreau et al., 1995) in which Cluster B lies, and our studies show that this mAb is still able to bind to its epitope in the presence of bound heparin. However, mAb 3H9 can also recognize the IL-2:IL-2R[alpha] complex (Moreau et al., 1995) and components of the IL-2R[alpha] (p50) site, Lys 35, Arg38, Phe42, and Lys43 (Sauve et al., 1991), are also located within this region. Thus until the exact residues defining the 3H9 epitope are known, the binding of this mAb cannot be used to discount Cluster B.
The fourth basic residue cluster, C, also forms a plausible heparin binding region. This cluster produces a so-called discontinuous site (Spillman and Lindahl, 1994), as the four constituent residues are from different regions of the primary and secondary structure of IL-2. Lys32 is located on the AA[prime] loop whereas Lys76, Arg81, and Arg83 are all located on the loop joining the B and C helices. Such sites are highly susceptible to disruption through conformational changes. Thus although these residues are not in the immediate vicinity of Thr51 and Gln126, nonconservative mutations of these residues could induce conformational changes that disrupt the alignment of the basic residues forming Cluster C. In fact the replacement of Thr51 with Pro induces some changes in the tertiary structure since the binding of this mutein to the [alpha][beta] receptor dimer shows reduced affinity compared to wild type IL-2 (Chang et al., 1995). Additional evidence supporting Cluster C comes from our mAb studies since the epitopes of H2-8 and 3H9, namely, residues 1-30 and 30-54, respectively (Eckenberg et al. 1997), lie well outside this cluster.
Figure 6. Molecular models of human IL-2 bound to heparin and IL-2 receptor polypeptides. The molecular model of a dodecamer of the main repeating unit of heparin (Mulloy et al., 1993) was taken from the Brookhaven Protein Database, PDB (1hpn), and is shown as a white ball and stick structure with enlarged yellow sulfur atoms. A theoretical model of IL-2 attached to its three receptor subunits (Bamborough et al., 1994) also came from the PDB (1iln). IL-2 is shown in green; the receptor subunits are red ([alpha]), orange ([beta]), and turquoise ([gamma]). The heparin is simply shown adjacent to the binding sites; no docking calculations have been performed. (A) Cluster C is picked out in pink; K32 and K76, with R81 and R83, form two pairs of basic residues about 18 Å apart. This corresponds approximately to the distance between two adjacent sulfate clusters down one side of the heparin chain. (B) As (A) rotated through 90° to show that Cluster C lies in a shallow, groove formed by the three receptor subunits, sufficiently wide to accommodate the heparin and allow extension of the polysaccharide chain at each end. On the opposite face of IL-2, residues forming the putative binding site B, also highlighted in pink, can be seen. (C) Cluster B, in which K48 and K49, with K54 and H55 form a similar pattern to Cluster C. Cluster B lies on the opposite face of IL-2 to Cluster C. The receptor subunit [gamma] would interfere with heparin extending in its direction from this potential binding site. Again, the spacing between the two pairs of basic residues matches the spacing of sulfate clusters on heparin.
Since the binding of IL-2 to heparin does not influence the biological activity of IL-2, it is important to establish that the putative heparin-binding sites formed from the basic residues in Clusters B and C do not interfere with the [alpha], [beta] and [gamma] receptor binding sites. In order to evaluate this, these clusters were highlighted (Figure 6a-c) on one of two theoretical models of wild type IL-2 complexed with all three receptor polypeptides (Bamborough et al., 1994). (The coordinates of IL-2 in this theoretical model were compared with those of the experimentally determined solution structure for IL-2 (Mott et al., 1995), and the backbone atoms of the two structures were found to have a R.M.
S. deviation of 3.3 Å). Also shown in Figure 6 is a dodecamer of the most common repeat unit of heparin (Mulloy et al., 1993) positioned so as to demonstrate the correspondence between the spacing of basic residues and the distance between two adjacent clusters of sulfate groups, 17 Å, on the same side of the heparin chain. When oriented so as to fit approximately the putative binding site formed from Cluster C, the heparin polysaccharide sits neatly between the receptor subunits (Figure 6a). Figure 6b shows the same model but rotated through 90° to emphasize the position of heparin in a shallow channel in the complex. In murine IL-2, two of the residues defining Cluster C are replaced by nonbasic alternatives, R81E and R83A, and this would be expected to remove affinity for heparin, as observed in our studies.
Cluster B occupies a location on the opposite face of the IL-2 molecule, nearer in space to residues 51 and 126 (Figure 6c). The four residues form a suitable site for heparin binding, with approximately 17 Å separating the two pairs of basic residues. The major disadvantage of this site, however, is that heparin, positioned so as to bind with the site, meets an obstacle in a protrusion of the [gamma] receptor subunit. Again, in murine IL-2, two of these four residues are nonconservatively replaced, namely K49Q and H55D, which would result in a loss of affinity for heparin.
Overall, we have shown that the binding of human IL-2 to heparin does not affect its biological activity and there appears to be no competition between binding to heparin, and binding to the [alpha] and [beta] IL-2 receptor chains. Moreover our modeling studies reveal that our putative binding sites for heparin on IL-2 are well separated from the receptor binding sites. The only possible mutual interference may be between occupancy of Cluster B and binding of the [gamma] receptor chain to its site. It would therefore appear that on secretion, IL-2 associates with heparin and highly sulfated heparan sulfate chains present on cell surfaces or in the neighboring extracellular surface, thereby reducing its rate of diffusion away from the source of its release. In this retained form, IL-2 would be able to associate with the [alpha],[beta] receptor dimer without any requirement for release from the glycosaminoglycan. This proposal amounts to a mechanism favoring the delivery of active cytokine to neighboring receptor-bearing target cells. Thus, a paracrine role for IL-2, focusing its activity local tissue sites, would be supported.
Materials and methods
Reagents and antibodies
Porcine intestinal mucosal heparin (unfractionated heparin, UFH), fucoidan (polymer of fucose of algal origin), and heparan sulfate (from bovine intestinal mucosa) were all purchased from Sigma Chemical Co., St. Louis, MO. Two porcine intestinal mucosal heparan sulfates (HSA and HSE) were obtained as described previously (Johnson, 1984). Three bovine lung heparan sulfate preparations (HS2, HS4, and HS6) were kindly provided by Dr. A. Malstrom (University of Lund, Sweden). Four size fractions of heparin were obtained by gel filtration of 216 mg of UFH on a column of Sephadex 75-G, 1 × 85 cm, eluted in 10 mM Tris-HCl, pH 7.5, containing 15 mM NaCl (Jordan et al., 1982). Elution of heparin was monitored by dimethylmethylene blue colorimetric assay (Rider, 1998) and size profile was visualized on 20% polyacrylamide gel electrophoresis (Hampson and Gallagher, 1984). Four pools were prepared and their Mr distributions determined by high performance gel permeation chromatography, using as calibrant the 1st International Reference Preparation Low Molecular Weight Heparin for Molecular Weight Calibration (NIBSC 90/686) (Mulloy et al., 1997). The clinical low molecular weight heparins, Enoxaprin (Clexane, Rhone-Poulenc, Dagenham, Essex, UK), Fragmin (KabiVitrum, Stockholm, Sweden), Tinzaparin (Logiparin, Novo Nordisk, Gentofte, Denmark), and Fraxiparine (Sanofi Chimie, Notre Dame de Bondeville, France) were obtained from their respective producers.
Recombinant human IL-2 (rhIL-2) was purchased from Amersham International (Amersham, Bucks, UK). RhIL-2 analogs, Arg38His and Lys43Glu (Sauve et al., 1991), were kindly provided by Dr. G. Ju (Hoffman-La Roche, New Jersey); and Thr51Pro (Chang et al., 1995) and Gln126Asp (Buchli and Ciardelli, 1993) were kindly provided by Dr. T. L. Ciardelli (Dartmouth Medical School, Hanover, NH). All four muteins have been previously characterized as well folded proteins.
Heparinase II (165 U/mg) from Flavobacterium heparinum was purchased from Sigma Chemical Co. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) was obtained from Promega (Madison, WI). All the constituents for complete RPMI-1640 cell culture medium containing 10% fetal calf serum were obtained from Life Technologies Ltd. (Paisley, Scotland). Murine cytotoxic cell line CTLL-2 was a kind gift of Dr. A. Saoudi (INSERM, Toulouse, France).
The three murine anti-rhIL-2 monoclonal antibodies (Mabs), 3H9, H2-8, and 16F11, were as previously characterized (Rebollo et al., 1992; Moreau et al., 1995; Eckenberg et al., 1997). Peroxidase-coupled sheep anti-mouse IgG was purchased from Amersham International . Soluble rhIL-2 receptor polypeptides [alpha] and ß were from R&D Systems Europe Ltd. (Abingdon, Oxon, UK).
Heparin binding ELISA
The binding of rhIL-2 to heparin was measured by a solid phase ELISA method as described previously (Najjam et al., 1997). Briefly, heparin chains were covalently coupled to BSA molecules via their reducing ends, using Na-cyanoborohydride. The heparin-BSA complexes thus obtained are employed as a solid phase with BSA binding to the ELISA well surfaces, while the heparin chains remain free to interact with potential ligands. For each assay, BSA molecules treated with Na-cyanoborohydride (t-BSA) in the absence of heparin was used as control. ELISA plate wells were coated with 100 µl of t-BSA or heparin/BSA complex, 2.5 µg/ml protein, in 50 mM Tris-HCl buffer, pH 7.4, containing 12.7 mM EDTA. After washing the plate with phosphate-buffered saline, pH 7.4 (PBS), then blocking nonspecific sites with 2% Marvel milk in PBS, rhIL-2 diluted in PBS was added to interact for 2 h with the heparin complex. Bound IL-2 molecules were detected by incubating with mAbs 3H9, H2-8, or 16F11 (at 140, 80, and 120 nM, respectively, in blocking buffer) for a further 2 h. Following incubation for 30 min with peroxidase-coupled sheep anti-IgG (at 1 µg/ml in blocking buffer), peroxidase activity was detected by ABTS in 50 mM Na-Citrate buffer, pH 4. Between each ELISA steps the plates were washed three times with PBS-0.05% (vol/vol) Tween 20. Plates were read at 405 nm after incubating for 1 h in the dark at room temperature. For each IL-2 concentration, absorbance value was calculated as follows: OD = (A1[prime] - A1) - (A2[prime] - A2), where A1[prime] and A1 represent absorbances on heparin/BSA complex in the presence and absence of IL-2, respectively; and A2[prime] and A2 indicate the equivalent absorbances on t-BSA.
Each ELISA experiment was carried out on at least two occasions, with each absorbance value being the mean of triplicate wells.
Competitive binding assays
Using the same ELISA approach, rhIL-2 was added, alone or premixed with glycosaminoglycans (GAGs), to coupled-heparin for 2 h of incubation. The detection of heparin-bound IL-2 was performed as described above, using 3H9 or H2-8 mAbs. Binding of rhIL-2 to coupled-heparin in presence of its soluble receptor p55 [alpha]-chain and p75 ß-chain were similarly tested using mAbs 3H9 and H2-8, respectively. In the various competitive binding experiments, A1 and A2 were measured in presence of the relevant competitor.
Bioassay of rhIL-2
CTLL-2 cells were maintained in complete RPMI 1640 medium, containing 10% (vol/vol) fetal calf serum, 213 nM l-glutamine, 106 nM pyruvate, 0.1% (vol/vol) PEN-STREP, 0.1% (vol/vol) nonessential amino acids, and 50 µM 2 mercaptoethanol at 37°C with 5% CO2. Cells were refed every 2 days with rhIL-2, 5 IU/ml final concentration. For bioassay, 2 days after the last addition of IL-2, cells were washed twice in incomplete RPMI 1640 medium. Cells at a density of 105 cells/ml were incubated for 3 h at 37°C with 5% CO2 in the presence and absence of heparinase II, 1.25 U/ml, and washed twice again in incomplete medium. Cells were then resuspended in complete medium and plated out in microtiter wells at a density of 105 cells/ml with 100 µl added per well. A range of rhIL-2 concentrations diluted in 100 µl complete medium was then added. Finally, PBS, 10 µl/well, containing either no heparin, or heparin to give a final concentration of 100µg/ml, was added. After 2 days MTS solution, 10 µl/well, was added and absorbance at 525 nm was read 4 h later.
Protein modeling studies
Molecular modeling was carried out using InsightII software (MSI, San Diego, CA) running on a Silicon Graphics Indigo 2 computer.
Acknowledgments
This work was supported by Leukaemia Research Fund. We thank Dr. Anders Malmstrom (University of Lund, Sweden) for providing heparan sulfates. We thank Dr. Grace Ju (Hoffman-La Roche Inc., New Jersey) and Dr. Thomas L. Ciardelli (Dartmouth Medical School, Hanover) for providing the rhIL-2 analogs.
References
6To whom correspondence should be addressed
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