Roles of Disulfide Bonds in Recombinant Human Interleukin 6 Conformation

Abstract

Human IL-6 has two disulfide bonds linking Cys45 to Cys51 and Cys74 to Cys84, respectively. Previous site-directed mutagenesis studies have demonstrated that the Cys74-Cys84 bond is essential for full biological and receptor binding activities. To address the structural importance of these disulfide bonds in the formation and stabilization of IL-6 secondary and tertiary structures, we have generated a panel of disulfide bond-deficient rIL-6 analogs both by chemical reduction and alkylation as well as by site-directed mutagenesis. Conformational changes affecting these rIL-6 analogs were probed by circular dichroism spectroscopy, as well as reactivity with monoclonal antibodies, and correlated with changes in biological activities. We have shown that the first disulfide bridge (Cys45-Cys51) is highly sensitive to reduction and, therefore, more solvent-exposed or less thermodynamically stable. Contrary to previous reports, this bridge contributes, although minimally, to the full biological activity of the cytokine. However, no significant changes in secondary or tertiary structures were observed upon removal of this bond. In marked contrast, analogs lacking the disulfide bridge between Cys74 and Cys84 exhibited as little as 0.5% and 0.05% wild-type biological and receptor binding activities, respectively. These dramatic changes correlated with a slight reduction in alpha-helical content and a decreased reactivity with the neutralizing monoclonal antibody mAb8 which recognizes a conformational epitope associated with the active site. Our results suggest that the second disulfide bridge plays a critical role in maintaining the spatial relationship between the putative IL-6 A and D helices.

Interleukin‐6: Structure‐function relationships

“…The first disulfide bond (Cys 44-Cys 50) can be selectively reduced (Rock et al, 1994) and plays only a minor role in hIL-6 activity (Snouwaert et al, 1991b;Rock et al, 1994). The second disulfide bond, however, plays a much larger role in hL-6 activity (Snouwaert et ai., 1991b;Rock et al, 1994).…”

“…The first disulfide bond (Cys 44-Cys 50) can be selectively reduced (Rock et al, 1994) and plays only a minor role in hIL-6 activity (Snouwaert et al, 1991b;Rock et al, 1994). The second disulfide bond, however, plays a much larger role in hL-6 activity (Snouwaert et ai., 1991b;Rock et al, 1994). The disulfide bonds are apparently responsible for maintaining structural integrity of receptor binding sites rather than conformational stability (Rock et al, 1994).…”

“…The second disulfide bond, however, plays a much larger role in hL-6 activity (Snouwaert et ai., 1991b;Rock et al, 1994). The disulfide bonds are apparently responsible for maintaining structural integrity of receptor binding sites rather than conformational stability (Rock et al, 1994). Substitution of the four cysteine residues in IL-6 by other amino acids by recombinant means results in biological activities of less than 0.1% when tested on human cell lines (Snouwaert et al, 1991b;Rock et al, 1994).…”

“…The disulfide bonds are apparently responsible for maintaining structural integrity of receptor binding sites rather than conformational stability (Rock et al, 1994). Substitution of the four cysteine residues in IL-6 by other amino acids by recombinant means results in biological activities of less than 0.1% when tested on human cell lines (Snouwaert et al, 1991b;Rock et al, 1994). However, when tested on rat and The numbering is based on the mature human sequence (Hirano et al, 1986) and the consensus sequence is shown below.…”

Structure, Stability and Biological Properties of a N‐terminally Truncated form of Recombinant Human Interleukin‐6 Containing a Single Disulfide Bond

“…It has been found that the first disulfide bridge Cys45 Cys51 does not play a functional role, whereas the activity of the mutant lacking the second disulfide bridge, Cys74 Cys84, was significantly reduced (Snouwaert et al, 1991b;Jean et al, 1993). In a recent study, Rock et al (1994) reexamined the structural and functional properties of several disulfide-bond-deficient analogs of human interleukin-6 prepared both by chemical reduction and S-alkylation of the protein, as well as by site-directed mutagenesis employing Cys-+.4la replacements. Unlike previous reports (Snouwaert et al, 1991b;Jean et al, 1993), these authors coneluded that the Cys45 Cys51 disulfide bridge slightly contributes to the full biological activity of the cytokine.…”