Abstract
It has been hypothesized that interleukin-6 (IL-6) and granulocyte-colony-stimulating factor (G-CSF) may fold as four-alpha-helix bundle proteins. To probe the functional role of the putative fourth helical segment of IL-6 (D-helix), a chimeric IL-6/G-CSF analog containing the predicted D-helix of G-CSF as well as a panel of IL-6 D-helix point mutants were analyzed for their respective secondary structure, antigenicity, and receptor binding and biological activities. The putative D-helix of IL-6 could not be replaced by its G-CSF counterpart in spite of their high degree of similarity and thus is indispensable for the antigenic and functional integrity of the IL-6 receptor binding site. Conversely, the grafting of the G-CSF D-helix did not confer any G-CSF activity to IL-6. A synthetic helical peptide containing the IL-6 D-helix was inactive, even when mixed with or linked to a peptide from the A-helix known to be involved in the active site. However, the conserved residues F173, R179, and R182 found in the D-helices of both IL-6 and G-CSF critically contribute to the architecture of the IL-6 active site. Indeed, mutation of F173 or R179 markedly affected IL-6 receptor binding and biological activities, but not the conformation of a major neutralization epitope. Furthermore, substitution of R182 resulted in a significant unfolding of the D-helix accompanied by a drastic loss in IL-6 antigenicity and functional activities. Nevertheless, residues other than F173, R179, and R182 also contribute to IL-6 specificity.
Highlights
It has been hypothesizedthat interleukin-6 (IL-6)and the pathogenesisof certain malignancies (Bataille et al, 1989; granulocyte-colony-stimulatingfactor (G-CSF) mayfold Blankenstein et al, 19901, autoimmune disorders
IL-6 first binds to the point mutants were analyzed for their respective sec- 80-kDa subunit of the IL-6 receptor expressed on target cells, ondary structure, antigenicity, and receptor binding this interactionpromotes the formation of a complex with and biological activities
The results reported bythese authors are diffi- extinction coefficient E'" of 5.2 for the fusion proteins and 4.6 for the cult to interpret unambiguously, since they introduceadnum- rIL-6 species, respectively(Rock et al, 1992).Since rIL-2/6 fusion prober of non-conservative and potentially helix-breaking residues without attempting topurify or fold the analogs.more recently, Fontaine et al (1993) demonstrated the functional importance of a positively charged residue at position teins containing the chimeric rIL-G/granulocyte-colony-stimulating factor (G-CSF) and the IL-6 point mutants F173A, R179A. and R182A were folded at low concentrations, protein concentrations were determined by duplicate amino acid analyses and bicinchoninic acidassays (Pierce Chemical Co.) using as standard purified wild-type rIL-2/6 whocseoncentrationhad been determined spectrophotometrically at 280 nm
Summary
It has been hypothesizedthat interleukin-6 (IL-6)and the pathogenesisof certain malignancies (Bataille et al, 1989; granulocyte-colony-stimulatingfactor (G-CSF) mayfold Blankenstein et al, 19901, autoimmune disorders In and IL-6Analogs-The recombinant fusion proteins were expressed as these assays, wild-type rIL-6 and rIL-2/6produced in our laboratory as inclusion bodies,denatured in 6 M Gu-HCl, folded,and oxidized in the well as human recombinant G-CSF (BoehringerMannheim)were used presence of a glutathione redox system as described byRocket al.
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