title: 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.
authors.affiliation: Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.
authors.forename: Samadhi
authors.initials: S
authors.lastname: Aparicio-Siegmund
authors.affiliation: Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.
authors.forename: Jens M
authors.initials: JM
authors.lastname: Moll
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Lokau
authors.affiliation: Department of Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Grusdat
authors.affiliation: Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Schröder
authors.affiliation: Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.
authors.forename: Svenja
authors.initials: S
authors.lastname: Plöhn
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Rose-John
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Grötzinger
authors.affiliation: Department of Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany; Department of Molecular Medicine II, Heinrich-Heine University, Düsseldorf, Germany.
authors.forename: Philipp A
authors.initials: PA
authors.lastname: Lang
authors.affiliation: Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Scheller
authors.affiliation: Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Garbers
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D016207:Cytokines / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D018664:Interleukin-12 / Q000737:chemistry / Q000378:metabolism; D053765:Interleukin-12 Subunit p35 / Q000737:chemistry / Q000378:metabolism*; D053762:Interleukin-12 Subunit p40 / Q000737:chemistry / Q000378:metabolism; D007378:Interleukins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016207:Cytokines; D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40; D007378:Interleukins; D021122:Protein Subunits; D011994:Recombinant Proteins; C569375:interleukin-35, mouse; D018664:Interleukin-12