Određivanje stupnja glikozilacije alfa-1-kiselog glikoproteina na pet glikozilacijskih mjesta korištenjem LC-MS metode

master's thesis

• Title (english): AGP glycosylation site occupancy assessment at five glycosylation sites using the LC-MS method
• Author: Ema Rajković
• Mentor: Toma Keser (mentor)
• Committee member: Toma Keser (predsjednik povjerenstva)
• Committee member: Biljana Nigović (član povjerenstva)
• Committee member: Branimir Plavša (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2024-07-12, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Alfa-1-kiseli glikoprotein (AGP) je jedan od glavnih proteina akutne faze koji se ističe svojom izrazitom glikoziliranošću. Sadrži pet glikozilacijskih mjesta na kojima su povezani strukturno raznoliki N-glikani kompleksnog tipa. Biološka uloga mu nije u potpunosti poznata, ali uključuje različite protuupalne i imunomodulatorne učinke te prijenos raznolikog niza endogenih i egzogenih liganada. Koncentracija AGP se mijenja u različitim akutnim i kroničnim stanjima, a promjene u glikoformama... More AGP-a, uključujući varijacije u njihovim antenskim strukturama, razinama α1,3-fukozilacije ili obrascima sijalinizacije, opažene su u različitim patološkim i fiziološkim stanjima (npr. diabetes mellitus tipa II i rak).
Poznato je da u različitim bolestima dolazi do promjene u obrascima glikozilacije AGP-a te se postavlja pitanje dolazi li do promjene i u zauzetosti glikozilacijskih mjesta proteina. U ovom radu po prvi puta se ispituje stupanj glikozilacije AGP-a. AGP je obogaćen taloženjem "seromukoidne" frakcije iz uzoraka ljudske plazme, nakon čega je uslijedila redukcija i alkilacija. Zauzetost glikozilacije određena je uporabom PNGaze F, koja uklanja glikane s proteina pri čemu se glikozilirani asparagin pretvara u asparaginsku kiselinu. Uzorci su podijeljeni u četiri skupine: PNGaza F dodana prije tripsinizacije, PNGaza F dodana nakon tripsinizacije, voda dodana prije tripsinizacije (kontrola) i voda dodana nakon tripsinizacije (kontrola). Nakon dodatka enzima ili vode, glikopeptidi su obogaćeni ekstrakcijom čvrste faze i analizirani kromatografijom reverzne faze spregnutom s qTOF masenim spektrometrom. Postotak neglikoziliranih peptida dobio se iz omjera zbroja nedeamidiranih i deamidiranih peptida kontrole te zbroja nedeamidiranih i deamidiranih peptida uzorka s dodanom PNGazom F.
Dokazana je potpuna glikozilacija triju mjesta (I_ORM1*S/ORM2*A, IV_ORM1, V_ORM1 i V_ORM2), djelomična, ali visoka glikoziliranost I_ORM1*F1/ORM1*F i II_ORM1/ORM2, a za mjesto III_ORM1/ORM2 nije bilo moguće odrediti zbog premalog intenziteta. Ove spoznaje mogu poslužiti za daljnju usporedbu sa stupnjem glikozilacije kod osoba oboljelih od različitih bolesti te bi onda stupanj glikozilacije potencijalno mogao biti dijagnostički alat tih bolesti. Less

Abstract (english)

Alpha-1-acid glycoprotein (AGP) is one of the main acute-phase proteins, which is extremely glycosylated. It contains five glycosylation sites with structurally diverse N-linked complex glycans. Its biological role is not fully known, but it includes various anti-inflammatory and immunomodulatory effects, plus the transfer of a diverse range of endogenous and exogenous ligands. AGP concentration changes in various acute and chronic conditions, and changes in AGP glycoforms, including... More variations in their antenna structures, levels of α1,3-fucosylation or sialinization patterns, have been observed in various pathological and physiological conditions (e.g. diabetes mellitus type II and cancer).
It is known that in various diseases there is a change in the glycosylation patterns of AGP, and the question arises whether there is also a change in the occupancy of glycosylation sites of the protein. In this paper, AGP glycosylation site occupancy is examined for the first time. AGP was enriched by precipitation of the "seromucoid" fraction from human plasma samples, followed by reduction and alkylation. Glycosylation occupancy was determined using PNGase F, which removes glycans from proteins, converting glycosylated asparagine to aspartic acid. The samples were divided into four groups: PNGase F added before trypsinization, PNGase F added after trypsinization, water added before trypsinization (control group), and water added after trypsinization (control group). After addition of enzymes or water, glycopeptides were enriched by solid phase extraction and analyzed by reverse phase chromatography coupled to a qTOF mass spectrometer. The percentage of non-glycosylated peptides was obtained from the ratio of the sum of non-deamidated and deamidated control peptides and the sum of non-deamidated and deamidated peptides of the sample with added PNGase F.
Complete glycosylation of three sites (I_ORM1*S/ORM2*A, IV_ORM1, V_ORM1 and V_ORM2), partial glycosylation of I_ORM1*F1/ORM1*F and II_ORM1/ORM2 was demonstrated, and site III_ORM1/ORM2 gave no results. These findings can be used for further comparison with the glycosylation site occupancy in people suffering from various diseases, and then AGP glycosylation site occupancy could potentially be a diagnostic tool for these diseases. Less

• Keywords: alfa-1-kiseli glikoprotein; glikozilacija; LC-MS
• Keywords (english): alpha-1-acid glycoprotein; glycosylation; LC-MS
• Language: croatian
• URN:NBN: urn:nbn:hr:163:405050
• Study programme: Title: Pharmacy - integrated undergraduate and graduate - university study; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: sveučilišni magistar farmacije (sveučilišni magistar farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-09-26 11:25:05