title: Distinct subunit pairing criteria within the heterodimeric IL-12 cytokine family.
abstract: The heterodimeric IL-12 cytokine family is characterized by the sharing of three α (p19, p28, p35) and two β (p40 and Ebi3) subunits, and includes IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Ebi3) and IL-35 (p35/Ebi3). In this study, the dimerization interfaces of IL-12 family members were characterized, with emphasis on IL-35. Ebi3 and p35 subunits from human and mouse paired effectively with each other, indicating there is no species barrier to IL-35 dimerization and suggesting a conserved dimerization interface. Specific p35 residues that contribute to formation of the IL-12 interface were assessed for their contribution to the IL-35 interface, and candidate Ebi3 residues were screened for their contribution to both IL-27 and IL-35 interfaces. Several residues were identified as critical to the IL-12 or IL-27 interfaces. Conversely, no single mutation was identified that completely disrupts p35/Ebi3 pairing. Linear alanine scanning mutagenesis on both p35 and Ebi3 subunits was performed, focusing on residues that are conserved between the mouse and human proteins. Additionally, a structure-based alanine-scanning approach in which mutations were clustered based on proximitiy was performed on the p35 subunit. Both approaches suggest that IL-35 has distinct criteria for subunit pairing and is remarkabley less sensitive to structural perturbation than IL-12 and IL-27. Additionally, studies using a panel of anti-p35 and anti-Ebi3 antibodies indicate differential availability of epitopes within IL-12 family members that share these subunits, suggesting that IL-35 has distinct structural features, relative to IL-12 and IL-27. These results may be useful in future directed therapeutic targeting of IL-12 family members.
authors.affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Lindsay L
authors.initials: LL
authors.lastname: Jones
authors.forename: Vandana
authors.initials: V
authors.lastname: Chaturvedi
authors.forename: Catherine
authors.initials: C
authors.lastname: Uyttenhove
authors.forename: Jacques
authors.initials: J
authors.lastname: Van Snick
authors.forename: Dario A A
authors.initials: DA
authors.lastname: Vignali
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D018664:Interleukin-12 / Q000737:chemistry* / Q000378:metabolism*; D053765:Interleukin-12 Subunit p35 / Q000737:chemistry / Q000378:metabolism; D007378:Interleukins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D018121:Receptors, Cytokine / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013045:Species Specificity
chemical_list: C097732:EBI3 protein, human; C499601:Ebi3 protein, mouse; D053765:Interleukin-12 Subunit p35; D007378:Interleukins; D015778:Minor Histocompatibility Antigens; D021122:Protein Subunits; D018121:Receptors, Cytokine; D018664:Interleukin-12