Structural consequences of aglycosylated IgG Fc variants evolved for FcγRI binding

Mol Immunol. 2015 Oct;67(2 Pt B):350-6. doi: 10.1016/j.molimm.2015.06.020. Epub 2015 Jul 4.

Abstract

In contrast to the glycosylated IgG antibodies secreted by human plasma cells, the aglycosylated IgG antibodies produced by bacteria are unable to bind FcγRs expressed on the surface of immune effector cells and cannot trigger immune effector functions. To avoid glycan heterogeneity problems, elicit novel effector functions, and produce therapeutic antibodies with effector function using a simple bacterial expression system, FcγRI-specific Fc-engineered aglycosylated antibodies, Fc11 (E382V) and Fc (E382V/M428I), containing mutations in the CH3 region, were isolated in a previous study. To elucidate the relationship between FcγRI binding affinity and the structural dynamics of the upper CH2 region of Fc induced by the CH3 mutations, the conformational variation of Fc variants was observed by single-molecule Förster resonance energy transfer (FRET) analysis using alternating-laser excitation (ALEX). In sharp contrast to wild-type Fc, which exhibits a highly dynamic upper CH2 region, the mutations in the CH3 region significantly stabilized the upper CH2 region. The results indicate that conformational plasticity, as well as the openness of the upper CH2 region, is critical for FcγR binding and therapeutic effector functions of IgG antibodies.

Keywords: Aglycosylated antibody; Alternative laser excitation; Antibody engineering; Effector function; Förster resonance energy transfer; Single-molecule analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Crystallography, X-Ray
  • Electrophoresis, Polyacrylamide Gel
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Immunoglobulin Fc Fragments / chemistry*
  • Immunoglobulin Fc Fragments / isolation & purification
  • Immunoglobulin Fc Fragments / metabolism
  • Immunoglobulin G / chemistry*
  • Immunoglobulin G / genetics
  • Mice
  • Models, Molecular
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Point Mutation / genetics
  • Protein Binding
  • Protein Conformation
  • Protein Engineering
  • Protein Stability
  • Receptors, IgG / metabolism*

Substances

  • Immunoglobulin Fc Fragments
  • Immunoglobulin G
  • Mutant Proteins
  • Receptors, IgG
  • glycosylated IgG