Structural and biological stability of the human interleukin 10 homodimer

Human interleukin 10 (huIL-10) is a cytokine that regulates the synthesis o f type 1 helper T cell derived cytokines such as gamma-interferon, interleu kin 2, and tumor necrosis factor alpha. The potential immunosuppressive act ivities of huIL-10 suggest that this protein may be clinically useful for t reating autoimmune diseases. Due to the potential clinical value of this cy tokine, physicochemical studies have been performed regarding its associati on state and biological/structural stability. These studies include pet-for ming size-exclusion chromatography, chemical cross-linking, equilibrium ult racentrifugation, and circular dichroism spectroscopy. The results indicate huIL-10 is predominantly a noncovalent homodimer at neutral pH and 4 degre es C for concentrations greater than 0.003 mg/mL (0.08 mu M dimer). An appa rent pK(a) value of similar to 4.8 was calculated for both the pH-dependent subunit dissociation and pH-induced loss in MC/9 biological activity. A te mperature analysis revealed a linear relationship between the percent dimer and relative MC/9 activity, thus, these results and the pH-dependent activ ity results suggest that the huIL-10 dimer is the active species. The GndHC l-induced unfolding of rhuIL-10, monitored by far-UV circular dichroism, re vealed a unique biphasic unfolding process which contained both a subunit d issociation process (<1.6 M GndHCl) as well as the unfolding of a highly a- helical monomer intermediate ([GndHCl](1/2) = 3.5 M). The monomer intermedi ates generated with 1.6 M GndHCl or pH 2.5 retained similar to 80% and 89% of the alpha-helical content of the native protein, respectively. Although a soluble and highly helical monomer state can be generated, the observed c orrelation between unfolding studies and biological activity suggests the d imer is the active species. These results are consistent with both the rece nt observation that the three-dimensional structure of rhuIL-10 is a 2-fold symmetric homodimer and that a complex between the extracellular domain of the recombinant human IL-10 receptor and IL-10 is consistent with two IL-1 0 homodimers and four receptors.