Određivanje epitopa autoantigena tetraspanina 7 za vezanje antitijela u dijabetesu tipa I

master's thesis

• Title (english): Mapping of autoantibody epitopes of the novel tetraspanin 7 autoantigen relevant in type I diabetes
• Author: Gloria Kraus
• Mentor: Anne Eugster (mentor)
• Mentor: Petra Korać (mentor)
• Committee member: Petra Korać (član povjerenstva)
• Committee member: Tomislav Ivanković (član povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2016-12-13, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Dijabetes mellitus tipa 1 (T1DM) je autoimuna bolest koja je specifična za gušteraču, a karakterizira ju nedostatak proizvodnje inzulina. Najčešći biomarkeri koji se koriste u dijagnozi i predviđanju nastanka bolesti su autoantitijela iz seruma za četiri najznačajnija antigena beta stanica: inzulin, GAD65, IA2 i ZnT8. Međutim, primijećeno je da još jedan protein koprecipitira s četiri spomenuta autoantigena. Istraživanja su pokazala da se radilo o glikoziliranom membranskom proteinu molekularne težine 38 kDa koji je nazvan Glima 38. Glima 38 je kasnije identificiran kao tetraspanin 7 (TSPAN7). TSPAN7 pripada proteinskoj obitelji tetraspanina te se sastoji od sedam domena: tri citoplazmatske (C1 do C3), četiri transmembranske (TM1 do TM4) i dvije ekstracelularne (E1 i E2), a u gušterači se nalazi specifično u stanicama Langerhansovih otoka. Hipoteza ovog istraživanja glasi da je imunoprecipitacijskim testom luciferaze moguće kvantificirati vezanje autoantitijela na proteinske fragmente TSPAN7. Cilj ovog istraživanje je bio utvrditi na koje domene se vežu autoantitijela kako bi se detaljnije mogli opisati autoimuni procesi u osobama koje boluju od dijabetesa tipa I. Glavna metoda korištena u ovom istraživanju je bila imunoprecipitacijski test luciferazom (LIPS), gdje su se proteinski fragmenti TSPAN7 obilježeni nanoluciferazom testirali na 41 uzorku seruma pacijenata koji su sadržavali autoantitijela na cjeloviti TSPAN7 i 20 uzoraka seruma koji nisu sadržavali autoantitijela na cjeloviti TSPAN7. Rezultati sugeriraju da se najvjerojatnije u domenama TSPAN7 E2 i C3 nalaze epitopi za vezanje autoantitijela te da domena TM4 također ima ulogu ili tako što i sama sadržava epitope ili tako što smješta domene E2 i C3 u pravilnu konformaciju.

Abstract (english)

Type 1 diabetes mellitus (T1DM) is an organ-specific autoimmune disease, which is characterized by a deficiency in endogenous pancreatic insulin production. The typical biomarkers that predict type I diabetes are serum autoantibodies against beta-cell antigens: insulin, GAD65, IA2, and ZnT8. Along with the four known autoantigens, autoantibodies were also detected to another potential autoantigen, a 38 kDa glycosylated membrane protein known as Glima 38. Glima 38 was later identified as tetraspanin 7 (TSPAN7). TSPAN7 is a member of the tetraspanin protein family, it has seven domains: three cytoplasmic (C1 to C3), four transmembrane (TM1 to TM4), and two extracellular (E1 and E2), and in pancreas it is found specifically in the islets of Langerhans. Hypothesis of this study is that by using luciferase immunoprecipitation systems (LIPS) assay, binding of autoantibodies to TSPAN7 protein fragments can be observed and quantified. The aim of this study was to map autoepitopes of the TSPAN7 in order to provide a more detailed description of autoimmune responses in T1DM individuals. The main method was the luciferase immunoprecipitation systems (LIPS) assay, where nanoluciferase-tagged truncated TSPAN7 forms were tested against 41 serum samples that had autoantibodies against full-length TSPAN7 and 20 samples that did not have autoantibodies against full-length TSPAN7. The results suggest that E2 and C3 TSPAN7 domains are the most likely candidates that carry autoepitopes, and that TM4 could play a role in the binding as well, either by carrying autoepitopes, or by positioning E2 and C3 in the proper conformation.

• Keywords: dijabetes mellitus tipa 1; T1DM; tetraspanin 7; TSPAN7; autoantitijela; imunoprecipitacijski test luciferazom; LIPS; epitopi
• Keywords (english): type I diabetes mellitus; T1DM; tetraspanin 7; TSPAN7; autoantibodies; luciferase immunoprecipitation systems assay; LIPS; autoepitopes
• Language: croatian
• URN:NBN: urn:nbn:hr:217:845666
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-05-04 08:53:16