Henry Frederick Earps
Structural characterisation of human immunoglobulin G Fc fragment and its interaction with heparin
Earps, Henry Frederick
Human immunoglobulin (Ig) G is the most commonly found class of antibody in the human body and is involved in pathogen recognition as a part of the adaptive and innate immune response. Recognition occurs through the fragment antibody binding (FAB) region and interaction with the innate immune system occurs via interactions between the fragment crystallisable (Fc) region and cell surface Fc receptors as well as select proteins of the complement system. It is this interaction that allows IgG to activate the immune system. The aim of this work was to characterise, at the atomic level, the recognition of heparin analogues using IgG-Fc fragments.
One native crystal structure of purified IgG-Fc and two crystal structures of heparin soaked IgG were determined, one with purified IgG-Fc soaked in heparin of size degree of polymerisation (dp) 12 and a recombinant IgG-Fc soaked in a rough heparin fraction. These structures have been successfully solved in P212121 using rigid body refinement. The native purified IgG-Fc was refined to 2.30 A?, with refinement statistics: Rwork 0.296 and Rfree 0.356. And the dp12-soaked purified IgG-Fc was refined to 2.15 A? with refinement statistics: Rwork 0.250 and Rfree 0.301. The rough heparin-soaked recombinant IgG-Fc was refined to 3.55 A? with refinement statistics: Rwork 0.325 and Rfree 0.297. Whilst no ligand binding was observed in the heparin soaked structures, the N-linked glycan of the Fc region is clearly defined.
The proportions of IgG subtypes in the human sera sample were also characterised through the use of matrix assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) mass spectrometry. Using these techniques, the sequence coverage of IgG-Fc subtypes in the sample of purified human sera used in this work were found to be: 84.5% IgG1-Fc, 28.2% IgG2-Fc, 12.1% IgG3-Fc and 11.3% IgG4-Fc respectively.
|Publicly Available Date||May 26, 2023|