Immunoglobulin G Subclass-Specific Glycosylation Changes in Primary Epithelial Ovarian Cancer

Epithelial ovarian cancer (EOC) was previously shown to be associated with glycosylation changes of total serum and total IgG proteins. However, as a majority of previous studies analyzed released glycan profiles, still little is known about IgG subclass-specific alterations in ovarian cancer. Hence, in this study, we investigated EOC-related glycosylation changes of the three most abundant IgG subclasses, namely, IgG₁, IgG₂ and IgG₃ isolated from sera of 87 EOC patients and 74 age-matched healthy controls. In order to separate IgG₂ and IgG₃, we performed a two-step affinity purification employing Protein A and Protein G Sepharose. After tryptic digestion, IgG glycopeptides were enriched and measured by MALDI-TOF-MS. Finally, EOC-related glycosylation changes were monitored at the level of total agalactosylation, monogalactosylation, digalactosylation, sialylation, bisection and fucosylation, which were calculated separately for each IgG subclass. Interestingly, aside from an EOC-related increase in agalactosylation/decrease in monogalactosylation and digalactosylation observed in all IgG subclasses, some subclass-specific trends were detected. Glycosylation of IgG₁ was found to be most strongly affected in EOC, as it exhibited the highest number of significant differences between healthy controls and EOC patients. Specifically, IgG₁ was the only subclass that showed a significant decrease in sialylation and a significant increase in fucosylation in EOC patients. Interestingly, IgG₂ and IgG₃ that were often investigated collectively in previous studies, were found to have distinct glycosylation patterns. IgG₃ displayed stronger EOC-related increase in agalactosylation/decrease in digalactosylation and was characterized by notably higher sialylation, which consequently decreased in EOC patients. In conclusion, our study indicates that IgG subclasses exhibit subtly distinct glycosylation patterns of EOC-related alterations and that IgG₁ and IgG₃ agalactosylation show the strongest association with CA125, the routine diagnostic marker. Additionally, our results show that simultaneous analyses of IgG₂ and IgG₃ might lead to wrong conclusions as these two subclasses exhibit noticeably different glycosylation phenotypes.