Razvoj metode za analizu glikanskog profila IgG-a izoliranog iz sinovijalne tekućine

master's thesis

• Author: Bernard Krešić
• Mentor: Gordan Lauc (mentor)
• Committee member: Gordan Lauc (predsjednik povjerenstva)
• Committee member: Sanja Dabelić (član povjerenstva)
• Committee member: Ana Mornar Turk (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2017-09-04, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Protutijela imaju središnju ulogu u molekulskim mehanizmima imunosnog odgovora. Glavno i najzastupljenije protutijelo u krvnoj plazmi je imunoglobulin G (IgG). IgG se sastoji od dvije vrste polipeptidnih lanaca: teškog (veličine oko 50 kDa) i lakog lanca (veličine oko 25 kDa). Svaka IgG molekula se sastoji od dva teška i dva laka lanca (ukupne veličine oko 150 kDa). Glikozilacija je od velike važnosti za pravilno funkcioniranje IgG-a. Svaki teški lanac nosi kovalentnu vezani bi-antenarni N-glikan na asparaginskom ostatku 297. Ovi oligosaharidi imaju strukturnu i funkcionalnu važnost za imunoglobulin G i njegove efektorske funkcije. Normalni sinovijalni zglobovi sadrže šupljinu ispunjenu sinovijalnom tekućinom koju izlučuje sinovijalna membrana. Sinovijalna tekućina je viskozna tekućina koja ima podmazujuću i metaboličku funkciju unutar zgloba. Održavanje normalnog sastava sinovijalne tekućine je nužno za homeostazu zgloba. Reumatoidni artritis (RA), koji zahvaća otprilike 1% populacije, predstavlja autoimunosni napad primarno usmjeren na sinovijalne membrane pokretnih zglobova. Svrha ovog rada je razvoj metode za analizu N-glikana IgG-a iz sinovijalne tekućine kao važnog biološkog uzorka u proučavanju reumatoidnog artritisa. IgG je izoliran iz sinovijalne tekućine imunoprecipitacijom uz korištenje protein G zrna, te su optimirani uvjeti pročišćavanja. Nakon izolacije IgG-a provedena je deglikozilacija, N-glikani su obilježeni i analizirani UPLC-om. Razvijena metoda će biti korištena u daljnjem istraživanju povezanosti reumatoidnog artritisa i N-glikana IgG-a, poželjno u obliku koji omogućuje visokoprotočnu analizu velikog broja uzoraka.

Abstract (english)

Antibodies play a central role in molecular mechanisms of immune response. The main and most common antibody in blood plasma is immunoglobulin G (IgG). IgG consists of two types of polypeptide chains: heavy (approximately 50 kDa) and light chain (approximately 25 kDa). Each IgG molecule consists of two heavy and two light chains (total molecular weight of approximately 150 kDa). Glycosylation is of great importance for proper functioning of IgG. Each heavy chain carries a covalently bonded bi-antenary N-glycan on the asparagine residue 297. These oligosaccharides have structural and functional importance for immunoglobulin G and its effector functions. Normal synovial joints contain a cavity filled with synovial fluid secreted by the synovial membrane. Synovial fluid is a viscous liquid that has a lubricating and metabolic function within a joint. Maintaining normal composition of synovial fluid is necessary for homeostasis of a joint. Rheumatoid arthritis (RA), which affects approximately 1% of the population, represents an autoimmune attack primarily directed to synovial membranes of synovial joints. The purpose of this paper is to develop a method for IgG N-glycan analysis from synovial fluid as an important biological sample in the study of rheumatoid arthritis. IgG was isolated from synovial fluid by immunoprecipitation using protein G beads, and purification conditions were optimized. N-glycans were released from IgG, labeled and analyzed by UPLC. The developed method will be used in further research of the association of rheumatoid arthritis and IgG N-glycans, preferably in a form that allows high-throughput analysis of a large number of samples.

• Keywords: imunoglobulin G; sinovijalna tekućina; imuoprecipitacija; analiza N-glikana; UPLC
• Keywords (english): immunoglobulin G; synovial fluid; immunoprecipitation; N-glycan analysis; UPLC
• Language: croatian
• URN:NBN: urn:nbn:hr:163:436700
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-11-28 16:21:33