Mouse Fc and Fab IgG N-glycosylation

master's thesis

• Author: Nikolina Vuković
• Mentor: Marija Pezer (mentor)
• Mentor: Sofia Ana Blažević (sumentor)
• Committee member: Sofia Ana Blažević (predsjednik povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Committee member: Ana Galov (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-02-24, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

N-glikani su kompleksni oligosaharidi vezani na dušikov atom asparagine. Imunoglobulin G (IgG) sadrži dva N-glikana vezana na kristalizirajućem fragmentu (Fc). Manji udio molekula IgG ima vezane N-glikane na antitijelo vezujućem fragment (Fab). Fab i Fc N-glikani utječu na funkciju molekule IgG u imunološkom odgovoru, što se često istražuje na mišjim modelima. Cilj ovog diplomskog rada je bio uspostaviti analitičku metodu za usporednu analizu N-glikozilacije Fc i Fab fragmenta mišjeg imunoglobulina G na temelju metode koja se koristi za analizu glikoma ljudskog IgG-a. IgG je izoliran afinitetnom kromatografijom iz seruma miševa soja CBAT6T6. Optimirani protokol uključuje dvadesetsatnu razgradnju na kuglicama specifičnim za Fc fragment u reducirajućim uvjetima. Nakon razgradnje na Fab i Fc fragment, uzorci su deglikozilirani. Oslobođeni N-glikani su fluorescentno obilježeni i analizirani kapilarnom gel-elektroforezom s laserom induciranom fluorescencijom u DNA sekvenceru. Prikupljeni podaci su obrađeni u programu Empower. Ukupno je određeno 35 N-glikanskih pikova, od kojih je za njih 16 otprije poznat N-glikanski sastav. Inicijalno upotrijebljena metoda za analizu N-glikana Fab i Fc fragmenta mišjeg IgG-a procijenjena je učinkovitom. Usporedba postotaka ukupne visine (PHP%) pojedinih pikova pokazala je razlike u glikanskom profilu Fc i Fab fragmenata nalik onima uočenim kod ljudskog IgG-a.

Abstract (english)

N-glycans are complex oligosaccharides bound to the nitrogen atom of asparagine. Immunoglobulin G (IgG) molecule contains two N-glycans attached to the fragment crystallizable (Fc) region, but only a minority of IgG molecules contain antigen binding fragment (Fab) N-glycans. Both Fab and Fc N-glycans influence the molecule's function in the immune response, a phenomenon often explored through mouse models. The aim of this study was to establish an analytical method for comparative N-glycosylation analysis of mouse Fc and Fab IgG fragments based on the method used for human IgG. IgG was isolated by affinity chromatography from serum of CBAT6T6 mice. The optimized protocol included a 20h-digestion on Fc beads in reducing conditions and fragments separation. Each IgG portion was deglycosylated and the released N-glycans were fluorescently labelled and analysed by capillary gel-electrophoresis with laser-induced fluorescence on an DNA sequencer. Collected data were processed in the Empower program. A total of 35 N-glycan peaks were defined and 16 of them annotated. The initially implemented method for the analysis of mouse Fab and Fc IgG N-glycans is judged functional. The comparison of proportion height percentages (PHP%) showed differences in the Fab and Fc glycoprofile similar to those observed for human IgG.

• Keywords: imunuglobulin G; kristalizirajući fragment; antitijelo vezujući fragment; N-glikani; SDS-PAGE; kapilarna gel-elektroforeza s laserom induciranom fluorescencijom
• Keywords (english): fragment crystallizable; antigen binding fragment; N-glycans; SDS-PAGE; CGE-LIF
• Language: english
• URN:NBN: urn:nbn:hr:217:427228
• Study programme: Title: Experimental Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-02-24
• Created on: 2021-03-08 14:12:12