Recombinant p35 from Bacteria Can Form Interleukin (IL-)12, but Not IL-35

Abstract

The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.

The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition

“…and more rigorous studies are needed to fully understand the different receptor complexes and the signal transduction of the cytokine IL-35. Biochemical analysis are further complicated by the fact that p35 protein produced in bacteria can be used to assemble IL-12, but not IL-35 [45].…”

Expanding Diversity in Molecular Structures and Functions of the IL-6/IL-12 Heterodimeric Cytokine Family

“…So far, only the anti-inflammatory property is known for IL-35; therefore, this cytokine could be one of the most promising candidates for clinical application against allergic and autoimmune diseases. However, among the IL-6/IL-12 family cytokines, IL-35 is considered to differ from the other cytokines (75). Although bacterially produced and purified recombinant proteins of EBI3 and p35 were correctly folded and biologically active in combination with p28 and p40, respectively, no biologically active IL-35 was reported to be formed when the p35 and EBI3 were combined (75).…”

“…However, among the IL-6/IL-12 family cytokines, IL-35 is considered to differ from the other cytokines (75). Although bacterially produced and purified recombinant proteins of EBI3 and p35 were correctly folded and biologically active in combination with p28 and p40, respectively, no biologically active IL-35 was reported to be formed when the p35 and EBI3 were combined (75). There are currently no reasons to explain this, but IL-35 might need additional, yet unidentified, molecules for efficient secretion and exertion of biological activity (75).…”

“…Although bacterially produced and purified recombinant proteins of EBI3 and p35 were correctly folded and biologically active in combination with p28 and p40, respectively, no biologically active IL-35 was reported to be formed when the p35 and EBI3 were combined (75). There are currently no reasons to explain this, but IL-35 might need additional, yet unidentified, molecules for efficient secretion and exertion of biological activity (75). One of the best criteria to prove the relationship between a bioactive cytokine and its receptor should be to examine whether the recombinant cytokine can proliferate a factor-dependent cell line such as Ba/F3 cells expressing its receptor subunits.…”

The biology of interleukin-27 reveals unique pro- and anti-inflammatory functions in immunity

“…Through different subunit pairing especially in the IL-12 family EBI3 as well as p28 are used by different cytokines. EBI3 can form the cytokine IL-35 by binding the IL-12 subunit p35 [15,16], although the interaction site seems to be very unconventional in the context of this cytokine family [17]. For p28 it was shown that the site II binding to the cytokine binding module (CBM) of different cytokine b-receptors is depending on the bound a-receptor, in this case EBI3 or the IL-6R, which results in the activation of a gp130/WSX-1 heterodimer by IL-27 and the activation of a gp130 homodimer by p28 through the IL-6R [10].…”