Functional validation of genes associated with glycosylation of immunoglobulin G

doctoral thesis

• Title (croatian): Funkcionalna analiza gena povezanih s glikozilacijom imunoglobulina G
• Author: Karlo Miškec
• Mentor: Aleksandar Vojta (mentor)
• Committee member: Vlatka Zoldoš (predsjednik povjerenstva)
• Committee member: Dunja Leljak-Levanić (član povjerenstva)
• Committee member: Gordan Lauc (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2024-01-18, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 577 - Biochemistry. Molecular biology. Biophysics

Abstract

Immunoglobulin G (IgG) is a key player in the human immune system. Pletohora of its roles is determined by its alternative N-glycosylation of Fc region, which has a role in defining its pro- or antiinflamatory character. Except Fc region, 20-25% of IgG molecules are glycosylated on the Fab region. Genome-wide association studies (GWAS) showed that IgG glycosylation is regulated by a complex gene network including genes with roles in immune cell development and immune response. However, role of these genes in IgG glycosylation is unknown. Therefore, twenty-three of GWAS hits were selected for functional validation in a model system FreeStyle™ 293-F with VPR-dSaCas9 and KRAB-dSpCas9 stably integrated in the genome. This system was modified for simultaneous analysis of Fab and Fc glycosylation followed by gene manipulation. Ten genes were functionally proven to be relevant for Fab and/or Fc glycosylation: KIF3C, MANBA, TNFRSF13B, EEF1A1, HIVEP2, TBX21, SPPL3, DERL2, RRBP1, and KDELR2.

Abstract (croatian)

Imunoglobulin G (IgG) jedan je od ključnih igrača u imunom sustavu čovjeka s velikim brojem različih uloga koje su određene altrernativnom N-glikozilacijom njegove Fc regije, uključujući njegovo proupalno ili protuupalno djelovanje. Izuzev regije Fc, 20-25% molekula IgG je glikozilirano na regiji Fab. Cjelogenomske studije povezanosti (engl. genome wide association studies, GWAS) su otkrile da je glikozilacija IgG-a regulirana kompleksnom mrežom gena koja uključuje gene s ulogom u razvoju imunoloških stanica i imunološkim odgovorima, čija uloga u IgG glikozilaciji nije poznata. Dvadesettri gena otkrivenih studijama GWAS odabrano je za funkcionalnu validaciju u staničnom modelu FreeStyle™ 293-F sa stabilno ugrađenim fuzijama VPR-dSaCas9 i KRAB-dSpCas9 za manipulaciju gena, a koji je modificiran u svrhu istovremene analize glikozilacije regija Fab i Fc. Rezultat ovog istraživanja otkriva deset gena (identificiranih studijama GWAS) s ulogom u Fab i/ili Fc glikozilacijskom putu: KIF3C, MANBA, TNFRSF13B, EEF1A1, HIVEP2, TBX21, SPPL3, DERL2, RRBP1 i KDELR2.

• Keywords: Fab and Fc glycosylation; IgG; GWAS; CRISPR/dCas9; FreeStyle™ 293-F cells; epigenetic editing; gene regulation
• Keywords (english): Fab i Fc glikozilacija; imunoglobulin G; cjelogenomske studije povezanosti; CRISPR/dCas9; stanice FreeStyle™ 293-F; epigenetičko uređivanje; regulacija genske ekspresije
• Language: english
• URN:NBN: urn:nbn:hr:217:829523
• Promotion: 2024
• Project: Number: KK.01.1.1.04.0085; Title: Genomsko inženjerstvo i genska regulacija u staničnim linijama i modelnim organizacijama tehnologija CRISPR / Cas9 CasMouse; Title: Genomic engineering and gene regulation in the cell lines model organisms CRISPR / Cas9 CasMouse technology; Acronym: CRISPR/Cas9-CasMouse; Leader: Vlatka Zoldoš; Jurisdiction: Croatia; Funder: EK; Funding stream: EFRR
• Study programme: Title: Doctoral study; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti u području prirodnih znanosti (doktor/doktorica znanosti u području prirodnih znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2026-01-18
• Created on: 2024-02-23 12:36:09