Abstract
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.
Highlights
Glycosylation of proteins is one of the most common cotranslational and post-translational modifications that consists of the covalent attachment of carbohydrate moieties to polypeptide chains
Since carcinoma development is accompanied by inflammation, we previously analyzed the N-glycosylation profile of acute-phase proteins isolated from serum samples of epithelial ovarian cancer (EOC) patients [14]
We investigated the EOC-related glycosylation changes in the three most abundant immunoglobulin G (IgG) subclasses, namely, IgG1, IgG2 and IgG3
Summary
Glycosylation of proteins is one of the most common cotranslational and post-translational modifications that consists of the covalent attachment of carbohydrate moieties to polypeptide chains. Apart from increasing protein diversity, glycans are known to play a broad range of roles; that is, they govern the folding of nascent polypeptides in the ER, provide protein physicochemical stability and modify their functions They are involved in various biological processes including cell signaling, extracellular interactions and immune responses [1, 2]. In the absence of biomarkers able to reliably detect OC at an early, still asymptomatic stage, altered glycosylation of proteins attracted attention as a potential source of complementary screening markers. Since carcinoma development is accompanied by inflammation, we previously analyzed the N-glycosylation profile of acute-phase proteins isolated from serum samples of epithelial ovarian cancer (EOC) patients [14]. Triantennary N-glycans containing a [1,2,3,4,5,6] branch and a LewisX epitope or tetraantennary N-glycans containing three LewisX epitopes were detected only in EOC patients, which demonstrates their diagnostic potential [14]
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