Masking of the Fc region in human IgG4 by constrained X-ray scattering modelling: implications for antibody function and therapy.
Of the four human IgG antibody subclasses IgG1-IgG4, IgG4 is of interest in that it does not activate complement and exhibits atypical self-association, including the formation of bispecific antibodies. The solution structures of antibodies are critical to understand function and therapeutic applications. Thus IgG4 was studied by synchrotron X-ray scattering. The Guinier X-ray radius of gyration R(G) increased from 5.0 nm to 5.1 nm with an increase of concentration. The distance distribution function P(r) revealed a single peak at 0.3 mg/ml, which resolved into two peaks that shifted to smaller r values at 1.3 mg/ml, even though the maximum dimension of IgG4 was unchanged at 17 nm. This indicated a small concentration dependence of the IgG4 solution structure. By analytical ultracentrifugation, no concentration dependence in the sedimentation coefficient of 6.4 S was observed. Constrained scattering modelling resulted in solution structural determinations that showed that IgG4 has an asymmetric solution structure in which one Fab-Fc pair is closer together than the other pair, and the accessibility of one side of the Fc region is masked by the Fab regions. The averaged distances between the two Fab-Fc pairs change by 1-2 nm with the change in IgG4 concentration. The averaged conformation of the Fab regions appear able to hinder complement C1q binding to the Fc region and the self-association of IgG4 through the Fc region. The present results clarify IgG4 function and provide a starting point to investigate antibody stability.
Abe
Yuki
Y
Department of Biochemical Engineering, University College London, UK.
Gor
Jayesh
J
Bracewell
Daniel G
DG
Perkins
Stephen J
SJ
Dalby
Paul A
PA
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000627:therapeutic use; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000627:therapeutic use; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use; D012542:Scattering, Radiation; D012996:Solutions; D017356:Synchrotrons; D014461:Ultracentrifugation; D014965:X-Rays
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012996:Solutions