Characterization of human recombinant interleukin 2 binding to heparin and heparan sulfate using an ELISA approach.
We have developed an enzyme-linked immunosorbent assay (ELISA) approach for the study of interactions between cytokines and glycosaminoglycans. This involves, as solid phase, a synthetic heparin-bovine serum albumin (BSA) complex in which the heparin is coupled via its reducing terminus to the protein using sodium cyanoborohydride. We have investigated the sensitivity and specificity of this experimental technique, employing antithrombin (AT III) and fibroblast growth factor 2 (FGF-2) as well-characterized heparin binding proteins. Using this ELISA method, we have established that human recombinant interleukin (IL-2) binds to heparin in a concentration-dependent manner. Soluble heparin competes for the binding of IL-2 to the complex with 50% inhibition at 5 microg/ml. This IC50 value provides an estimate of the binding constant of around 0.5 microM. This value is at least two orders of magnitude larger than that for the binding of IL-2 to its dimeric and trimeric cell surface receptors, but similar to that for binding to the IL-2 receptor beta polypeptide acting alone. Our ELISA shows that in addition to soluble heparin, fuciodan also competes for IL-2 binding, but chondroitin sulfate and dermatan sulfate are inactive. Of six heparan sulfates tested, only one highly sulfated preparation competed for IL-2. The interaction between IL-2 and heparin-like glycosaminoglycans is likely to be an important mechanism for retaining IL-2 close to its sites of secretion, thus giving rise to localized concentration gradients in the tissues.
Najjam
S
S
Division of Biochemistry, Royal Holloway University of London, Egham, Surrey, UK.
Gibbs
R V
RV
Gordon
M Y
MY
Rider
C C
CC
D000990:Antithrombin III / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007376:Interleukin-2 / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
D000990:Antithrombin III; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D006497:Heparan Sulfate; D007376:Interleukin-2; D011994:Recombinant Proteins