To overcome problems with the
To overcome problems with the biochemical reconstitution of these heterodimeric cytokines fusion strategies linking both subunits together have been proposed inspired by the “hyper-IL-6” . Much like fusing a protein to a solubility enhancing protein such as MBP or SUMO, fusing two subunits of a potential heterodimer together with a flexible linker could lead to the maturation and secretion of the heterodimer. As such one might however inadvertently create an artificially bioactive molecule displaying potentially novel properties . The validation of the existence of these heterodimers at the protein level in primary animal tissue is therefore crucial. Not only could the cytokine subunits reciprocally stabilize each other, but interactions between cytokines and their receptors might further stabilize the cytokines which could play a role in case of an autocrine system . Throughout our experiments we used the IL27 gene variant encoding for the common L119P variant (rs181206) of the protein which is found in about 30% of the human population . This variant is common in research as it was the first reported sequence of human IL27  and is part of the fused IL-27 variant sold by R&D systems (cat n° 2526-IL), which has been reported to be bioactive. Aside from its high prevalence in humans, a proline instead of leucine can be found at this position in some marsupial IL-27 proteins but not in other orthologues. Based on homology models, position 119 likely maps to the B-helix of the IL-27 helical bundle and a proline at this position may locally affect the alpha helix. This part of the helical bundle is however not part of any known epitope engaged by a receptor(-like) molecule in the IL-12 or IL-6 family. The variant might however affect protein stability or maturation and has been identified in a recent meta-analysis as a risk factor for several human diseases in Asian populations . EBI3 was shown to associate with Calnexin-1 (referred to as p95), Sequestosome-1 (referred to as p60) and an uncharacterized protein (referred to as p78) in BJAB Azithromycin Dihydrate synthesis overexpressing EBI3 . Recently p28 was also shown to interact with Endoplasmatic reticulum chaperone BiP . These identified proteins play a role in protein folding and quality control. It is not known how important this association with chaperones is for proper folding and maturation of the cytokine subunits and if it contributes to their ability to form heterodimeric cytokines. It nevertheless highlights a role for chaperones in the folding and maturation of the proteins under investigation. It is worth noting that the role of chaperones and ER-stress is being increasingly recognized in tuning immunity and inflammation , . A common view is that the covalent character of IL-12 and IL-23, whereby a disulfide bond is formed between p40 Cys199 and p35 Cys130 or p19 Cys73, is the driving force between the observed stability of these cytokines. It must however be noted that this disulfide is not required ,  for the formation of the heterodimer and that the disulfide is not fully formed when analyzing these recombinantly produced cytokines. Furthermore, the complex between and IL-6 and IL-6R is also not covalent. While it is indeed unlikely that EBI3 containing cytokines are disulfide linked as there are no predicted free cysteine residues in human EBI3, and a free cysteine residue in mouse EBI3 (Cys198) maps to a different location on the protein , the lack of a disulfide is unlikely the whole reason why IL-27 and IL-35 are more challenging to produce. Throughout the experiments reported herein there is a clear discrepancy between protein expression, as can be observed in the cellular fraction, and protein secretion and by extrapolation potential activity. This is an important distinction yet one that would be hard to make when relying on transcriptional data or intracellular antibody stains. IL-6 appears to be the only human alpha helical bundle cytokine (subunit) we tested which is inherently secreted independent of the presence of a receptor (or a receptor-like) subunit. However, the receptor (-like) subunits p40, EBI3, CRLF1 and IL-6R can all be secreted independently. In the case of IL-6 this enables the well documented cis- and trans-signaling whereby in the latter case IL-6 is pre-associated with soluble IL-6R . p40 and its homodimer IL-12B (p80) are both recognized as weak IL-12 and IL-23 antagonists and reports have attributed agonistic properties to p80 and a role as chemoattractant in humans and mice . Furthermore, differences in protein secretion have also been reported for certain murine orthologues such as p28 or p19 ,  which in contrast to their human counterparts can be secreted as stand-alone subunits. Thus, the secretion of these cytokine subunits on their own might constitute a layer of possible regulation of signaling by heterodimeric IL-12 family cytokines. However, the biogenesis of the human heterodimeric IL-12-family cytokines upon immune stimulus is strictly dependent on the expression of the receptor-like subunit .