| Popis: |
Background: The importance of N-glycosylation for the IgG effector functions is well established. Studies describing associations of total and Fc IgG N-glycosylation with various physiological and pathological conditions in human populations are plentiful, while the knowledge on the importance of Fab glycans is lagging behind. Mouse is the main model animal in biomedical research, and is often used for in vivo studies on IgG glycosylation. Its total IgG N-glycosylation is routinely analyzed by ultraperformance liquid chromatography or capillary gel electrophoresis of released glycans and its Fc IgG N-glycosylation by liquid chromatography mass spectrometry of Fc glycopeptides. Its Fab glycosylation, however, remains underexplored. With the importance of Fab IgG glycans in the pathogenesis of autoimmune and other conditions entering the research spotlight in recent years, there is a growing need for a method enabling in vivo studies on Fab IgG glycosylation in mice. Aim: The aim of this study was to optimize the analytical method for the analysis of mouse Fab and Fc IgG N-glycosylation based on the method used for human IgG. Materials and methods: IgG was isolated from the serum of CBAT6T6 mice by affinity chromatography on Protein G monolithic plate. After binding to the IgG-Fc affinity matrix, IgG was digested with SpeB protease into Fab and Fc fractions. The efficiency of protease digestion was confirmed by SDS PAGE. After the Fab and Fc separation, N- glycans were released from each fraction by peptide N glycosidase F, the efficiency of deglycosylation was determined by SDS PAGE, and the released glycans were labeled with a fluorescent dye, 8-aminopyrene-1, 3, 6-trisulfonic acid. Labeled glycans were analyzed by capillary gel electrophoresis laser-induced fluorescence on an ABI3130 DNA sequencer. Results: IgG was successfully cleaved into Fab and Fc fragments. The Fc fragment was only partially deglycosylated, while the level of deglycosylation for the Fab fragment could not be confirmed. N glycans from both fractions were successfully analyzed. The N-glycosylation pattern of mouse Fab vs Fc roughly corresponds to the glycopattern of human Fab vs Fc fraction, with the Fab fraction bearing the most of highly processed, sialylated N-glycans, and the Fc fraction bearing a bigger proportion of neutral: a-, mono- and digalactosylated N-glycans. Conclusion: While additional optimization is required to completely deglycosylate both fractions, the initially implemented method for the analysis of mouse Fab and Fc IgG N-glycans is functional. The first analysis shows a distribution of mouse Fab and Fc N-glycans similar to the one observed in human IgG. |
