Definition of receptor binding sites on human interleukin-11 by molecular modeling-guided mutagenesis

Eur J Biochem. 1999 Oct;265(2):645-55. doi: 10.1046/j.1432-1327.1999.00755.x.

Abstract

Interleukin-11 (IL-11) belongs to the interleukin-6 (IL-6)-type subfamily of long-chain helical cytokines including IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, and cardiotrophin-1, which all share the glycoprotein gp130 as a signal transducing receptor component. IL-11 acts on cells expressing gp130 and the IL-11 receptor (IL-11R) alpha-subunit (IL-11Ralpha). The structural epitopes of IL-11 required for the recruitment of the individual receptor subunits have not yet been defined. Based on the structure of CNTF, a three-dimensional model of human IL-11 was built. Using this model, 10 surface exposed amino acid residues of IL-11 were selected for mutagenesis using analogies to the well-characterized receptor recruitment sites of IL-6, CNTF, and LIF. The respective mutants of human IL-11 were expressed as soluble fusion proteins in bacteria. Their biological activities were determined on HepG2 and Ba/F3-130-11alpha cells. Several mutants with substantially decreased bioactivity and one hyperagonistic mutant were identified and further analyzed with regard to recruitment of IL-11Ralpha and gp130. The low-activity mutant I171D still binds IL-11Ralpha but fails to recruit gp130, whereas the hyperagonistic variant R135E more efficiently engages the IL-11R subunits. The low-activity mutants R190E and L194D failed to bind to IL-11Ralpha. These findings reveal a common mechanism of receptor recruitment in the family of IL-6-type cytokines and offer considerable perspectives for the rational design of IL-11 antagonists and hyperagonists.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antigens, CD / metabolism*
  • Binding Sites
  • Cell Division
  • Cytokine Receptor gp130
  • Cytokines / agonists
  • Cytokines / antagonists & inhibitors
  • Growth Inhibitors*
  • Humans
  • Interleukin-11 / chemistry
  • Interleukin-11 / genetics*
  • Interleukin-6 / metabolism
  • Leukemia Inhibitory Factor
  • Leukemia Inhibitory Factor Receptor alpha Subunit
  • Lymphokines*
  • Membrane Glycoproteins / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Receptors, Cytokine / metabolism
  • Receptors, OSM-LIF
  • Recombinant Fusion Proteins / genetics
  • Thioredoxins / genetics
  • Tumor Cells, Cultured

Substances

  • Antigens, CD
  • Cytokines
  • Growth Inhibitors
  • IL6ST protein, human
  • Interleukin-11
  • Interleukin-6
  • LIF protein, human
  • LIFR protein, human
  • Leukemia Inhibitory Factor
  • Leukemia Inhibitory Factor Receptor alpha Subunit
  • Lymphokines
  • Membrane Glycoproteins
  • Receptors, Cytokine
  • Receptors, OSM-LIF
  • Recombinant Fusion Proteins
  • Cytokine Receptor gp130
  • Thioredoxins