Utjecaj starenja stanične linije FreeStyleTM 293-F na glikozilaciju izlučenog imunoglobulina G

master's thesis

• Title (english): Influence of cell aging on glycosylation of immunoglobulin G secreted from cell line FreeStyleTM 293-F
• Author: Fran Lukšić
• Mentor: Vlatka Zoldoš (mentor)
• Committee member: Vlatka Zoldoš (predsjednik povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Mirjana Pavlica (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-09-27, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Alternativna glikozilacija fragmenta Fc imunoglobulina G (IgG) znatno mijenja ulogu ovog antitijela u imunom sustavu. Starenjem dolazi do promjena u glikozilaciji, prije svega galaktozilaciji, što dovodi do povećanja udjela IgG s proupalnom ulogom. Stanična linija FreeStyle™ 293-F koristi se za proučavanje regulacije glikozilacije IgG-a te za sintezu rekombinantnih imunoglobulina. Cilj ovog istraživanja bio je utvrditi utječe li starost stanične linije FreeStyle™ 293-F na glikom IgG-a te ukoliko utječe jesu li promjene uzrokovane starenjem nalik onima koje se dešavaju s glikomom IgG-a u serumu prilikom starenja u ljudi. Drugi dio istraživanja imao je za cilj utvrditi jesu li promjene glikoma izravna posljedica promjene ekspresije glikoziltransferaza koje sudjeluju u procesu glikozilacije IgG-a. Analizom glikana metodom tekućinske kromatografije ultravisoke djelotvornosti utvrđeno je da starenjem stanica modelne linije dolazi do promjena IgG glikoma. Najznačajnije promjene odnose se na porast udjela agalaktoziliranih glikana i smanjenje udjela fukoziliranih glikana. Promjene fukozilacije mogu se barem dijelom objasniti uočenim promjenama ekspresije fukoziltransferaze FUT8, dok promjene galaktozilacije nisu izravna posljedica promjene ekspresije galaktoziltransferaze B4GALT1. Promjene glikoma IgG-a zabilježene tijekom starenja modelne stanične linije uspoređene su s već poznatim promjenama koje nastaju u IgG glikomu u krvi čovjeka tijekom starenja.

Abstract (english)

Glycosylation of the Fc fragment of immunoglobulin G (IgG) affects the role of this antibody in the immune system. Aging is associated with changes in glycosylation, primarily galactosylation, which lead to an increase in proportion of proinflammatory IgG antibodies. The FreeStyle ™ 293-F is the preferred model cell line used for production of recombinant immunoglobulins, but also for studies of IgG glycosylation. The aim of this study was to determine whether the aging of the FreeStyle ™ 293-F cells affects IgG glycome and, if so, whether the changes caused by cell aging are similar to changes of IgG glycome found during aging of a human organism. The aim of the second part of this study was to determine whether changes in IgG glycome result directly from changes in the expression of glycosyltransferases involved in the process of IgG glycosylation. Analysis of glycans by ultra-high performance liquid chromatography revealed that cell aging leads to changes in IgG glycome. The most significant changes are an increase in the proportion of agalactosylated glycans and a decrease in the proportion of fucosylated glycans. Changes in fucosylation can be at least partially explained by the changes in FUT8 expression, while changes in galactosylation are not a direct consequence of changes in B4GALT1 expression. Changes of IgG glycome found during aging of model cells were compared to changes of IgG glycome which are known to occur during human ageing.

• Keywords: imunoglobulin G; N-glikozilacija; IgG glikom; HEK293 FreeStyle; in vitro stanično starenje
• Keywords (english): immunoglobulin G; N-glycosylation; IgG glycome; HEK293 FreeStyle; in vitro cell aging
• Language: croatian
• URN:NBN: urn:nbn:hr:217:541821
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-10-05 12:59:00