TY - JOUR
T1 - Recombinant p35 from bacteria can form interleukin (IL-)12, but not IL-35
AU - Aparicio-Siegmund, Samadhi
AU - Moll, Jens M.
AU - Lokau, Juliane
AU - Grusdat, Melanie
AU - Schröder, Jutta
AU - Plöhn, Svenja
AU - Rose-John, Stefan
AU - Grötzinger, Joachim
AU - Lang, Philipp A.
AU - Scheller, Jürgen
AU - Garbers, Christoph
N1 - Publisher Copyright:
© 2014 Aparicio-Siegmund et al.
PY - 2014/9/26
Y1 - 2014/9/26
N2 - 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 p40C197Amutation 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.
AB - 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 p40C197Amutation 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.
UR - http://www.scopus.com/inward/record.url?scp=84907909046&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0107990
DO - 10.1371/journal.pone.0107990
M3 - Article
C2 - 25259790
AN - SCOPUS:84907909046
SN - 1932-6203
VL - 9
JO - PLoS ONE
JF - PLoS ONE
IS - 9
M1 - e107990
ER -