High-throughput N-glycosylation analysis of complement component C3 as biomarker of type 1 diabetes

Autor: Šoić, Dinko
Přispěvatelé: Gornik Kljaić, Olga
Jazyk: chorvatština
Rok vydání: 2023
Předmět:
Popis: Ubrzani razvoj analitičkih tehnika u posljednjih nekoliko desetljeća omogućio je temeljito istraživanje procesa N-glikozilacije u brojnim patofioziološkim stanjima, a uočene promjene u mnogima od njih dovele su do strelovitog rasta interesa za glikobiologiju. Promjene u N-glikozilaciji danas se smatraju vrijednim biljezima raznih bolesti, te se njihova specifičnost pokušava iskoristiti u diferencijalne, dijagnostičke i prognostičke svrhe. Prethodne studije pokazale su da je N-glikanski profil plazmatskih proteina promijenjen u šećernoj bolesti tipa 1 (ŠBT1). Jedna od izraženije uočenih promjena bila je ona u zastupljenosti visoko-manoznih glikanskih struktura u plazmatskom N-glikomu. Ove strukture dominantno bi mogle potjecati s komponente komplementa C3, budući da je to glikoprotein s isključivo visoko-manoznim glikanima vezanima na proteinsku okosnicu za kojeg je poznato da doprinosi razvoju ŠBT1 pojačavanjem autoimunih upalnih procesa. Iz tog je razloga u ovom radu razvijena visokoprotočna metoda za analizu N-glikozilacije pojedinih glikozilacijskih mjesta ljudskog C3 proteina upotrebom tekućinske kromatografije spregnute sa spektrometrom masa (LC-MS) temeljena na njegovu obogaćivanju iz plazme pomoću lektinskog afinitetnog medija visokog afiniteta za manozu. Novorazvijenom metodom zatim je analizirana krvna plazma ispitanika novodijagnosticiranih šećernom bolesti tipa 1 i njihove zdrave braće i sestara te su uočene značajne promjene C3 N-glikoma. ŠBT1 povezana je s porastom manje procesuiranih struktura s više manoznih podjedinica na oba N-glikozilacijska mjesta. Nadalje, C3 N-glikozilacija je analizirana u odrasloj populaciji ispitanika s različitim stupnjem najčešćih komplikacija ŠBT1 – retinopatije i albuminurije. Pokazano je da se C3 N-glikom značajno mijenja u teškoj albuminuriji u ŠBT1, no da je neovisan o trajanju bolesti. Retinopatija je pak dovela do promjene samo jedne glikoforme, dok su svi osim jednog C3 glikopeptida značajno povezani s razinama hemoglobina A1c (HbA1c). Predstavljene spoznaje ukazuju na uključenost N-glikozilacije i C3 komponente komplementa u patofiziologiju šećerne bolesti tipa 1 te upućuju na značaj C3 N-glikoma kao potencijalnog dijagnostičkog i prognostičkog biljega ove bolesti i njoj pridruženih komplikacija. Rapid development of analytical techniques in the last few decades has enabled a thorough investigation of the N-glycosylation process in numerous pathophysiological conditions, and the observed changes in many of them have led to a rapid growth of interest in the field of glycobiology. Changes in N-glycosylation are now considered valuable biomarkers of various diseases, and their specificity is being exploited for differential, diagnostic and prognostic purposes. Previous studies have shown that the N-glycan profile of plasma proteins is altered in type 1 diabetes (T1D). One of the more pronounced changes observed was in he abundance of high-mannose glycans in the plasma N-glycome. These structures could predominantly originate from the complement component C3, a glycoprotein with exclusively high-mannose glycans attached to its protein backbone, which is known to contribute to the development of T1D by enhancing autoimmune inflammatory processes. For this reason, a high-throughput method for the site-specific N-glycosylation analysis of human C3 protein was developed in this study using liquid chromatography coupled to a mass spectrometer (LC-MS), based on its enrichment from plasma using lectin affinity matrix. The newly developed method was then used to analyze blood plasma of subjects newly diagnosed with type 1 diabetes and their healthy siblings, and significant changes in C3 N-glycome were observed. T1D was associated with an increase in less processed structures with more mannose subunits at both C3 N-glycosylation sites. Furthermore, C3 N-glycosylation was analyzed in the adult population of T1D subjects with different degrees of the most common complications of T1D – retinopathy and albuminuria. It was shown that C3 N-glycome significantly changes in severe albuminuria in T1D, but is independent of disease duration. Retinopathy led to a change in only one glycoform, while all but one C3 glycopeptide was significantly associated with levels of hemoglobin A1c (HbA1c). The findings presented indicate the involvement of N-glycosylation and the C3 complement component in the pathophysiology of type 1 diabetes and indicate the importance of C3 N-glycome as a potential diagnostic and prognostic biomarker of this disease and its associated complications.
Databáze: OpenAIRE
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