Solution structure of deglycosylated human IgG1 shows the role of CH2 glycans in its conformation

DOI: 10.1016/j.bpj.2021.02.038

Authors: Valentina A. Spiteri (University College London (UCL)) , James Doutch (ISIS Facility) , Robert P. Rambo (Diamond Light Source) , Jayesh Gor (University College London) , Paul A. Dalby (University College London) , Stephen J. Perkins (University College London (UCL))
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biophysical Journal

State: Published (Approved)
Published: March 2021
Diamond Proposal Number(s): 18022

Abstract: The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is comprised of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are conserved in the Fc region in IgG, however their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. Following deglycosylation using PNGase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s020,w of IgG1 decreased from 6.45 S by 0.16-0.27 S. This change was attributed to the reduction in mass following glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. While the radius of gyration RG was unchanged, the cross-sectional radius of gyration, RXS-1, increased by 0.1 nm and the commonly occurring distance peak M2 of the distance distribution curve P(r) increased by 0.4 nm. These changes revealed that the Fab-Fc separation in IgG1 was perturbed following deglycosylation. To explain these changes, atomistic scattering modelling based on Monte Carlo simulations resulted in 123,284 and 119,191 trial structures for glycosylated and deglycosylated IgG1 respectively. From these, 100 X-ray and neutron best-fit models were determined. For these, principal component analyses identified five groups of structural conformations that were different for glycosylated and deglycosylated IgG1. The Fc region in glycosylated IgG1 showed a restricted range of conformations relative to the Fab regions, while the Fc region in deglycosylated IgG1 showed a broader conformational spectrum. These more variable Fc conformations account for the loss of binding to the FcγR receptor in deglycosylated IgG1.

Journal Keywords: Analytical ultracentrifugation; antibody modelling; small-angle neutron scattering; human IgG subclasses; small angle X-ray scattering

Subject Areas: Biology and Bio-materials


Instruments: B21-High Throughput SAXS

Other Facilities: SANS2D at ISIS

Added On: 08/03/2021 11:29

Discipline Tags:

Structural biology Biophysics Life Sciences & Biotech

Technical Tags:

Scattering Small Angle X-ray Scattering (SAXS)