Post-translational modifications alter intact IgG1 conformation and influence Fc(gamma)RIIIa binding

摘要

Complete analytical & biophysical characterization was performed on IgG1 isoforms. Differences were detected by DSC, Fc(gamma)RIIIa binding and H/DX-MS. Oxidation did not impact Fc(gamma)RIIIa binding whereas elevated galactosylation increased Fc(gamma)RIIIa binding over 3-fold (removal of galactose resulted in lower binding). DSC and H/DX-MS revealed conformational differences in IgG1 isoforms. Differences were localized to CH2 domain, heavy chain residues 242-254. (1) Oxidation and deglycosylation both exposed residues to surrounding solvent and or weaken backbone H-bonding. (2) Higher levels of galactosylation provided more protection from solvent and or strengthened H-bonding. (3) Galactose likely IgG1 Fc region to occupy an overall conformation more sensitive to Fc(gamma)RIIIa binding. (4) Residues 242-254 are likely not important for Fc(gamma)RIIIa binding. H/DX-MS analysis of IgG1-Fc(gamma)RIIIa interaction indicated IgG1 heavy chain residues 157-164, 244-253 and 320-344 were protected upon binding. IgG1 residues 320-344 are known to interact with Fc(gamma)RIIIa but residues 157-164 and 240-255 were not previously known to be involved.