Recombinant p35 from bacteria can form Interleukin (IL-)12, but Not IL-35

Samadhi Aparicio-Siegmund; Jens M Moll; Juliane Lokau; Melanie Grusdat; Jutta Schröder; Svenja Plöhn; Stefan Rose-John; Joachim Grötzinger; Philipp A Lang; Jürgen Scheller; Christoph Garbers

doi:10.1371/journal.pone.0107990

Recombinant p35 from bacteria can form Interleukin (IL-)12, but Not IL-35

PLoS One. 2014 Sep 26;9(9):e107990. doi: 10.1371/journal.pone.0107990. eCollection 2014.

Affiliations

¹ Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.
² Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany.
³ Department of Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany.
⁴ Department of Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany; Department of Molecular Medicine II, Heinrich-Heine University, Düsseldorf, Germany.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CD4-Positive T-Lymphocytes / immunology
CD4-Positive T-Lymphocytes / metabolism
Cell Line
Cytokines / chemistry
Cytokines / metabolism
Humans
Interleukin-12 / chemistry
Interleukin-12 / metabolism
Interleukin-12 Subunit p35 / chemistry
Interleukin-12 Subunit p35 / metabolism*
Interleukin-12 Subunit p40 / chemistry
Interleukin-12 Subunit p40 / metabolism
Interleukins / chemistry
Interleukins / metabolism
Mice
Models, Molecular
Protein Binding
Protein Conformation
Protein Multimerization*
Protein Subunits / chemistry
Protein Subunits / metabolism
Recombinant Proteins / chemistry
Recombinant Proteins / metabolism*

Substances

Cytokines
Interleukin-12 Subunit p35
Interleukin-12 Subunit p40
Interleukins
Protein Subunits
Recombinant Proteins
interleukin-35, mouse
Interleukin-12

Grants and funding

This work was funded by a grant from the Research Commission of the Medical Faculty of the Heinrich-Heine-University Düsseldorf (to C.G. and J.S.). S.R.-J. is funded by the Deutsche Forschungsgemeinschaft (SFB841, project C1) and the Cluster of Excellence “Inflammation at Interfaces”. J.G. is funded by the Deutsche Forschungsgemeinschaft (SFB877, project A6). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.