Abstract
We have previously shown that the heterodimeric cytokine interleukin-12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin-12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin-12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C′D′ loop within D3 is important in heparin binding. However since the human and murine C′D′ loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL-12 via its p40 subunit to heparin-related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence.
Highlights
Interleukin 12 (IL-12) plays a key role in establishing cell-mediated, TH1-type, immune responses by stimulating natural killer (NK) and T lymphocytes to secrete γ-IFN
We sought to characterise the interaction between rIL-12 and heparin/heparan sulfate (HS) using a series of chemically modified heparin preparations in which specific sulfate moieties had been removed
Preincubation for 15 min of rhIL-12 with soluble heparin and HS reduces binding to the complex to the low background levels which occur in control wells coated with mock-conjugated BSA
Summary
Interleukin 12 (IL-12) plays a key role in establishing cell-mediated, TH1-type, immune responses by stimulating NK and T lymphocytes to secrete γ-IFN (for reviews see [1,2]). Γ-IFN stimulates further production of IL-12, giving rise to a positive feedback loop at the initiation of TH1 responses. IL-12 is a relatively large cytokine, Mr 70kD, being a heterodimer of two disulphide-bridged subunits, p40 and p35. IL-23 is a pro-inflammatory and immunostimulatory cytokine expressed by antigen presenting cells upon activation. IL-23 stimulates the TH17 lymphocyte subset, thereby promoting both early responses to microbial infection and chronic autoimmune states. The high affinity cell surface signalling receptor for IL-12 is a heterodimer of the IL-12Rβ1 polypeptide, which is a component of the IL-23 receptor, and IL-12Rβ2, which is shared with the IL-35 receptor [3]. Free p40 is found in serum in a number of disease states and in efforts to identify the biological role of this polypeptide a number of binding proteins have been identified, including the CD5 glycoprotein [4]
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