Visokoprotočna analiza N-glikozilacije alfa-1-kiseloga glikoproteina u identifikaciji biljega šećerne bolesti

doctoral thesis

• Title (english): High-throughput N-glycosylation analysis of alpha-1-acid glycoprotein in the identification of diabetes mellitus biomarkers
• Author: Marko Tijardović
• Mentor: Olga Gornik Kljaić (mentor)
• Committee member: Roberta Petlevski (predsjednik povjerenstva)
• Committee member: Gordan Lauc (član povjerenstva)
• Committee member: Diana Muačević-Katanec (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2022-01-10, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry
• Universal decimal classification (UDC): 615 - Pharmacology. Therapeutics. Toxicology

Abstract

N-glikozilacija, proces enzimski posredovanog vezanja šećernih struktura na asparaginske ostatke proteina,
istraživana je u brojnim bolestima, u mnogima od kojih su promjene i potvrđene. Pronalazak specifičnih promjena
N-glikozilacije nudi mogućnost razvoja predikcijskih, dijagnostičkih i prognostičkih alata potrebnih u sprječavanju
i liječenju različitih bolesti. Studije ukupne N-glikozilacije proteina plazme provedene u istraživanju predispozicije
za šećernu bolest tipa 2, kao i istraživanju šećerne bolesti zrele dobi kod mladih osoba (MODY), identificirale su
promjene u glikozilaciji za koje se vjeruje da potječu s alfa-1-kiseloga glikoproteina (AGP). S ciljem detaljnijeg
istraživanja ovih promjena, razvijena je metoda za visokoprotočnu analizu N-glikozilacije AGP-a koja uključuje
obogaćenje ovog proteina te analizu njegovih glikopeptida metodom tekućinske kromatografije spregnute sa
spektometrijom masa (LC-MS). Inicijalnom analizom 14 zdravih ispitanika utvrđena je temporalna stabilnost Nglikoma
AGP-a, kao općeniti preduvjet za dijagnostičku upotrebu. Novorazvijenom metodom zatim je analizirana
glikozilacija AGP-a 108 ispitanika koji se razlikuju po svojoj predispoziciji za razvoj šećerne bolesti tipa 2 te je
utvrđena povezanost porasta grananja i smanjenja sijalinizacije glikana AGP-a s povećanim rizikom od razvoja
šećerne bolesti tipa 2. Nadalje, kod 466 pacijenata oboljelih od različitih tipova šećerne bolesti (HNF1A-, HNF4AMODY,
šećerna bolesti tipa 1 i tipa 2) analizom fukozilacije AGP-a ustanovljeno je njezino smanjenje koje je
pokazano specifično za HNF1A-MODY. Nadalje, analizom uzoraka 98 pacijenata oboljelih od šećerne bolesti ranog
nastupa bez autoimunosne podloge, pokazano je da je smanjena fukozilacija prisutna samo kod pacijenata s
oštećujućim HNF1A mutacijama – sržnim uzrokom bolesti. Također, pokazana je visoka uspješnost identifikacije
HNF1A-MODY pacijenata na temelju individualnih glikopeptida AGP-a. Prikupljene spoznaje upućuju na
mogućnost upotrebe glikozilacije AGP-a kao predikcijskog biljega za šećernu bolest tipa 2, kao i biljega
identifikacije pacijenata oboljelih od HNF1A-MODY-a što bi dalo velik doprinos pravovremenom prepoznavanju
rizika kao i diferencijalnoj dijagnostici šećerne bolesti.

Abstract (english)

N-glycosylation, the process of enzymatically mediated binding of sugar structures to proteins' asparagine residues,
has been investigated in a number of diseases, in many of which the changes have been confirmed. The discovery
of specific N-glycosylation changes offers a possibility for development of prediction, diagnostic and prognostic
tools needed in the prevention and treatment of various diseases. Studies of total plasma protein N-glycosylation
performed in type 2 diabetes predisposition research, as well as in the research of maturity-onset diabetes of the
young (MODY), identified changes in glycosylation believed to originate from alpha-1-acid glycoprotein (AGP).
In order to investigate these changes in more detail, a method for high-throughput AGP N-glycosylation analysis
was developed. The method is based on enrichment of AGP and analysis of its glycopeptides by liquid
chromatography coupled to mass spectrometry (LC-MS). Initial analysis performed on 14 healthy subjects
confirmed the temporal stability of AGP N-glycome, as a general prerequisite for diagnostic use. The newly
developed method was then used to analyze AGP glycosylation in 108 subjects differing in their predisposition for
type 2 diabetes development. The results confirmed an association of increased branching and decreased sialylation
of AGP glycans with an increased risk of type 2 diabetes development. Analysis of samples from 466 patients
diagnosed with different types of diabetes (HNF1A-, HNF4A-MODY, type 1 and type 2 diabetes) revealed a
reduction in AGP fucosylation, which was shown to be specific for HNF1A-MODY. Furthermore, analysis of
samples from 98 patients with early-onset non-autoimmune diabetes showed that decreased fucosylation was
present only in patients carrying damaging HNF1A mutations - the core cause of the disease. Also, high success
rate of identification of HNF1A-MODY patients based on individual AGP glycopeptides was demonstrated. The
findings suggest the possibility of utilizing AGP glycosylation as a predictor of type 2 diabetes, as well as a marker
for identification of HNF1A-MODY patients. This would present a valuable contribution to timely risk
identification and differential diagnosis of diabetes.

• Keywords: N-glikozilacija; alfa-1-kiseli glikoprotein; šećerna bolest; tekućinska kromatografija; spektrometrija masa
• Keywords (english): N-glycosylation; alpha-1-acid glycoprotein; diabetes; liquid chromatography; mass spectrometry
• Language: croatian
• URN:NBN: urn:nbn:hr:163:500265
• Promotion: 2022
• Study programme: Title: Pharmacy and biochemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor znanosti (doktor znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-11-08 16:37:10