Photoinduced Trp and Tyr Side Chain Cleavage in IgG4-Fc: Impact on Physicochemical Stability and Receptor Binding and Effect of N- vs O-Glycosylation.

In previous studies, we discovered that the exposure of IgG4-Fc and IgG1 to UV light resulted in the side chain cleavage of specific Tyr and Trp residues, converting these amino acids into a series of products, including Gly [Haywood, J. Mol. Pharmaceutics 2013, 10(3), 1146-1150; Kang, H. Mol. Pharmaceutics 2019, 16, 258-272]. In order to evaluate the physicochemical consequences of such photochemical transformations, we prepared a series of IgG4-Fc mutants, in which Trp and Tyr residues were replaced by Gly, i.e., Y300G, Y373G, Y436G, and W381G, for biophysical studies. As the expression yields of W381G IgG4-Fc were low, we also prepared W381A to achieve higher yields required for some of the studies. Among these mutants, Y373G displayed significantly lower melting temperatures compared to wild-type IgG4-Fc, as analyzed by differential scanning calorimetry and fluorescence spectroscopy, indicating a decrease in thermal stability of both the CH2 and CH3 domains. In contrast, W381A showed no thermal transitions, indicating a significant loss of overall thermal stability. As for binding affinity to FcγRIIIA, Y300G and Y436G displayed ca. 10-fold reduction compared to wild-type IgG4-Fc. Interestingly, W381A and W381G IgG4-Fc did not only contain N-linked glycans but also high levels of O-mannose (>60%) at Ser375. Therefore, we prepared additional mutants, S375A and S375A/W381A IgG4-Fc, to evaluate the effect of N- vs O-glycosylation on thermal stability and the susceptibility of Tyr and Trp residues to undergo photodegradation. The removal of N-glycans specifically destabilized the CH2 domain of IgG4-Fc. The removal of O-glycans did not affect the overall thermal stability of S375A IgG4-Fc, and S371A/W381A shows no thermal transitions, suggesting that the W381A mutation alone is sufficient to perturb the protein structure irrespective of O-glycosylation. As for photostability, we observed a 2.6-13.5 fold reduction of yields of Tyr side chain fragmentation products for W381A IgG4-Fc, potentially due to a lower probability for electron transfer between Tyr and oxidized Trp, and photodegradation of alternative Trp residues in W381A IgG4-Fc.