Distinct subunit pairing criteria within the heterodimeric IL-12 cytokine family

Mol Immunol. 2012 Jun;51(2):234-44. doi: 10.1016/j.molimm.2012.03.025. Epub 2012 Apr 7.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Conserved Sequence
  • Dimerization
  • Enzyme-Linked Immunosorbent Assay
  • HEK293 Cells
  • Humans
  • Immunoblotting
  • Immunoprecipitation
  • Interleukin-12 / chemistry*
  • Interleukin-12 / metabolism*
  • Interleukin-12 Subunit p35 / chemistry
  • Interleukin-12 Subunit p35 / metabolism
  • Interleukins / chemistry
  • Interleukins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Minor Histocompatibility Antigens
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Subunits / chemistry*
  • Protein Subunits / metabolism*
  • Receptors, Cytokine / chemistry
  • Receptors, Cytokine / metabolism
  • Sequence Alignment
  • Species Specificity


  • EBI3 protein, human
  • Ebi3 protein, mouse
  • Interleukin-12 Subunit p35
  • Interleukins
  • Minor Histocompatibility Antigens
  • Protein Subunits
  • Receptors, Cytokine
  • Interleukin-12