| Abstract | Glikan je kompleksan spoj sastavljen od velikog broja monosaharida povezanih glikozidnim vezama, a koji biomolekule
dobivaju procesom glikozilacije. Termin se glikan često koristi za opisivanje ugljikohidratnog dijela glikokonjugata. Najčešći
monosaharidi u glikokonjugatima su Glc, Gal, GlcNAc, GalNAc, Man, Fuc, Xyl i Sia. Glikom se odnosi na cjelokupan repertoar
glikanskih struktura unutar organizma, stanice ili tkiva u određenom vremenu i uvjetima. Stoga, N-glikom IgG podrazumijeva
cijeloukupni spektar glikana koji se mogu pronaći na IgG. Glikani su specifično vezani za pojedinca te nisu zapisani u genomu i
podliježu utjecaju epigenetike, prehrane i trenutnog stanja pojedinca. Analiza glikoma može pružiti uvid u trenutno zdravstveno
stanje i personalizirati terapiju zbog čega su glikani vrijedni prediktivni biomarkeri. Kako bi mogli poslužiti za takvo što moramo
biti sigurni da je N-glikom IgG stabilan u određenom vremenskom periodu, a pošto su strukture glikana jako osjetljive na male
promjene korisno je i praktično stvoriti automatizirani pristup prilikom izolacije glikana, što su i ciljevi ovog istraživačkog rada.
U ispitivanju su prisustvovali dvojica zdravih muškaraca kojima su uzimani uzorci krvi u periodu od pet, odnosno, deset
godina. Uzorci u razdoblju od pet godina uzimani su svaka tri mjeseca, dok su uzroci u razdoblju od deset godina uzimani svaka
tri tjedna. Krv je uzeta na antikoagulans (EDTA) i centrifugirana čime je odvojena plazma iz koje je, uz pomoć pločice monolita s
proteinom G automatiziranom metodom koristeći Tecan Freedom EVO sustav i robotiku, izoliran IgG. N-glikani oslobođeni su
PNGaza F i fluorescentno obilježeni APTS nakon čega su analizirani xCGE-LIF metodom. Izračunata su četiri derivirana svojstva
(S, G1, G2 i G0) jer je pokazano kako upravo ona igraju ključnu ulogu u starenju. Također, izračunata su i dva derivirana svojstva
F i B koja doprinose uvidu u stabilnost glikana tijekom godina.
Analizom je podataka uočeno kako N-glikom IgG pokazuje vrlo dobru stabilnost u vremenskom periodu od deset godina što
znači da se uzorci plazme stari do deset godina pouzdano mogu koristiti za daljnja biološka istraživanja. Trend starenja prati
smanjenje sijalinizacije (S) i povećanje degalaktozilacije (G0), uz nepromijenjenu sržnu fukozilaciju (F) i monogalaktozilaciju
(G1), a odstupanja ukazuju na trenutna stanja i promjene u organizmu i oko njega. |
| Abstract (english) | A glycan is a complex compound consisting of a large number of monosaccharides linked by glycosidic bonds that biomolecules obtain through the process of glycosylation. The term "glycan" is often used to describe the carbohydrate portion of glycoconjugates. The most common monosaccharides in glycoconjugates include Glc, Gal, GlcNAc, GalNAc, Man, Fuc, Xyl, and Sia. Glycome refers to the total repertoire of glycan structures in an organism, cell, or tissue at a particular time and under particular conditions. Therefore, the N-glycome of IgG refers to the entire spectrum of glycans that can be found in IgG. Glycans are individually specific, not encoded in the genome, and are subject to the influence of
epigenetics, diet, and the current state of the individual. Analysis of glycans can provide information about current health status and personalize therapy, making glycans valuable predictive biomarkers. To fulfill this purpose, it is essential to ensure that the N-glycome of IgG remains stable over a specific time period and understand how it changes with aging. Due to the sensitivity of glycan structures to minor changes, it is useful and practical to develop an automated approach for glycan isolation, which is the objective of this research study. The study involved two healthy male participants from whom blood samples were collected over a period of five and ten years. During the fiveyear period, samples were collected every three months, while during the ten-year period, samples were collected every three weeks. Blood was
collected using an anticoagulant (EDTA) and centrifuged to separate plasma. IgG was then isolated from the plasma using an automated method with a protein G monolith plate, utilizing the Tecan Freedom EVO system and robotics. N-glycans were released by PNGase F and fluorescently labeled with APTS before being analyzed using the xCGE-LIF method. Four derived traits (S, G1, G2, and G0) were calculated as they have been shown to play a crucial role in aging. Additionally, two derived traits, F and B, were calculated to provide insights into the stability of glycans over the years. Data analysis revealed that the N-glycome of IgG exhibits excellent stability over a ten-year period, indicating that plasma samples aged up to ten years can reliably be used for further biological research. The aging trend is characterized by decreased sialylation (S) and increa sed degalactosylation (G0), with unchanged core fucosylation (F) and monogalactosylation (G1). At the same time, deviations indicate current states and changes in the organism and its environment. |
