Optimizacija simuliranih fizioloških pogojev vezave protiteles proti β2 – glikoproteinu I na kristalizirani aneksin A5 na fosfolipidnem dvosloju

Tahirović, Aneja (author), Božič, Borut (mentor), Škarabot, Miha (comentor)

Antifosfolipidni sindrom je sistemska avtoimunska bolezen, ki se kaže v obliki tromboz in zapletov v nosečnosti, in pri kateri so prisotna antifosfolipidna protitelesa v plazmi bolnikov. Katastrofni antifosfolipidni sindrom je redka, življenjsko ogrožajoča oblika bolezni, pri kateri se krvni strdki oblikujejo hkrati po vsem telesu in povzročijo večkratno odpoved organov. Za nastanek bolezni so pomembne interakcije med protitelesi in membrane vezočimi proteini, vendar mehanizmi še niso povsem pojasnjeni. Cilj magistrskega dela je bil določiti fiziološke pogoje vezave protiteles proti β2-glikoproteinu I na način, da to lahko opazujemo pod mikroskopom na atomsko silo. Iz seruma bolnika s katastrofnim antifosfolipidnim sindromom smo v več korakih izolirali protitelesa proti β2-glikoproteinu I po principu afinitetne kromatografije in specifičnost njihove vezave na antigen potrdili z ELISA. Na mikroskopu na atomsko silo smo na sljudo zaporedno nanesli fosfolipidne vezikle, aneksin A5 ter kompleks antigen – protitelo (β2-glikoprotein I, protitelesa proti β2-glikoproteinu I). Sprva smo opazovali nastanek planarnega fosfolipidnega dvosloja. Ko smo dobili več kot 50 % prekritost površine z dvoslojem, smo nanj nanesli aneksin A5 ter opazovali in analizirali tvorbo kristalnega plašča. V zadnji fazi smo opazovali vezavo protiteles proti β2-glikoproteinu I na aneksinski kristalni plašč in posledice morebitnih medmolekulskih interakcij. Na osnovi tega smo ocenili njihov morebitni patološki potencial. Ob popolni kristalizaciji aneksina A5 na fosfolipidnem dvosloju ni prišlo do vezave β2-glikoproteina I, protiteles proti β2-glikoproteinu I ali kompleksa antigen – protitelo. Afiniteta vezave aneksina A5 do fosfatilidilserina in posledično tvorbe kristalne plasti je namreč večja od afinitete vezave kompleksa. Kompleks antigen – protitelo se pod eksperimentalnimi pogoji preferenčno veže na sljudo, ki je bolj negativno nabita kot fosfolipidni dvosloj. Ob prisotnosti popolnoma kristaliziranega aneksina A5 ne pride do izrivanja aneksinske kristalne plasti in širitve kompleksa po fosfolipidnem dvosloju. Aneksinski kristalni plašč tako nudi zaščito pred vezavo protiteles, kar lahko razumemo tudi kot zaviranje napredovanja bolezni. Potrdili smo domnevo, da zgolj s prisotnostjo protiteles proti β2-glikoproteinu I ob aneksinskem plašču na fosfolipidni membrani ne moremo razložiti razvoja in/ali napredovanja bolezni.

• Language: Slovenian
• Keywords: β2-glikoprotein I, protitelesa proti β2-glikoproteinu I, katastrofni antifosfolipidni sindrom, mikroskop na atomsko silo, optimizacija fizioloških pogojev
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-115974
• Publication date in RUL: 05.05.2020

Secondary language

• Language: English
• Title: Optimization of simulated physiological binding conditions of antibodies against β2-glycoprotein I to crystallized annexin A5 on a phospholipid bilayer

Abstract

Antiphospholipid syndrome (APS) is an autoimmune disease in which antiphospholipid antibodies are present in the plasma of patients. Catastrophic antiphospholipid syndrome (cAPS) is a rare, life-threatening form of antiphospholipid syndrome that occurs as a result of a trigger in patients positive for antiphospholipid antibodies. Interactions between antibodies and membrane-binding proteins are important for the development of the disease, but the mechanisms are not yet fully understood. The aim of the master's thesis was to optimize the physiological conditions of binding of antibodies to anti-β2GPI in such a way that it can be observed under an atomic force microscope. In the serum of a patient with catastrophic antiphospholipid syndrome, antibodies to β2-glycoprotein I were isolated in several steps according to the principle of affinity chromatography. The antibodies obtained were evaluated with anti-β2GPI IgG ELISA. At the atomic force microscope, phospholipid vesicles, annexin A5, and the antigen-antibody complex (β2-glycoprotein I, antibodies against β2-glycoprotein I) were sequentially bound to mica. Initially, we observed the formation of a planar phospholipid bilayer. If more than 50% of the surface was obtained, annexin A5 was bonded to it and the formation of the crystal coat was observed and analyzed. In the final phase, binding of antibodies to β2-glycoprotein I to the annexin crystal coat was observed and their possible pathological potential was assessed. Upon complete crystallization of annexin A5 on the phospholipid bilayer, no binding of β2-glycoprotein I, antibodies against β2-glycoprotein I, or the antigen-antibody complex were observed. The binding affinity of annexin A5 to phosphatylidylserine and, consequently, the formation of the crystalline layer, is greater than the binding affinity of the antigen-antibody complex. The antigen-antibody complex, under experimental conditions, preferentially binds to the mica, which is more negatively charged than the phospholipid bilayer. In the presence of fully crystallized annexin A5 the complex is unable to expand along the phospholipid bilayer. The annexin crystal mantle thus offers protection against antibody binding, which can also be understood as inhibiting disease progression. We confirmed the assumption that the mere presence of antibodies against β2-glycoprotein I at the annexin mantle on the phospholipid membrane could not explain the development and / or progression of the disease.

• Keywords: β2-glycoprotein I, antibodies against β2-glycoprotein I, catastrophic antiphospholipid syndrome, atomic force microscopy, optimization of physiological conditions