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Az FHR-1, FHR-3 És FHR-5 H-Faktorral Rokon Fehérjék Szerepe a Komplementrendszer Szabályozásában Doktori Értekezés

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Az FHR-1, FHR-3 És FHR-5 H-Faktorral Rokon Fehérjék Szerepe a Komplementrendszer Szabályozásában Doktori Értekezés Az FHR-1, FHR-3 és FHR-5 H-faktorral rokon fehérjék szerepe a komplementrendszer szabályozásában Doktori értekezés Csincsi Ádám István Témavezető: Dr. Józsi Mihály, DSc, egyetemi docens Biológia Doktori Iskola Immunológia Program Doktori Iskola és Program vezető: Prof. Erdei Anna, DSc, egyetemi tanár, az MTA rendes tagja ELTE TTK Biológiai Intézet, Immunológiai Tanszék Eötvös Loránd Tudományegyetem, Természettudományi Kar Budapest 2018 A disszertációban ismertetett kutatásokat a Magyar Tudományos Akadémia (LP2012-43 sz. pályázat) és a Nemzeti Kutatási, Fejlesztési és Innovációs Hivatal (K 109055 sz. pályázat) támogatta. Dr. Tóth Adriánnak Tartalomjegyzék Rövidítésjegyzék ........................................................................................................ 6 1. Bevezetés ............................................................................................................... 8 2. Irodalmi összefoglalás .......................................................................................... 9 2.1. Az emberi komplementrendszer .......................................................................................... 9 2.2. A komplementrendszer szabályozása ................................................................................ 12 2.2.1. A klasszikus, a lektin és a terminális utak szabályozása .........................................................12 2.2.2. Az alternatív komplement út szabályozása ..............................................................................13 2.3. A H-faktor molekulacsalád ................................................................................................. 14 2.3.1. Az FHR fehérjéket kódoló gének .............................................................................................16 2.3.2. Az FHR fehérjék szerkezete és tulajdonságaik .......................................................................17 2.3.3. Az FHR fehérjék és betegségek kapcsolata .............................................................................20 2.4. A tárgyalt betegségekkel kapcsolatos FH/FHR ligandumok ........................................ 27 2.4.1. A C3b .............................................................................................................................................27 2.4.2. A pentraxinok ...............................................................................................................................28 2.4.3. Az extracelluláris mátrix (ECM) ................................................................................................30 2.4.4. A malondialdehid (MDA) ...........................................................................................................31 3. Célkitűzések ......................................................................................................... 32 4. Felhasznált anyagok és módszerek ................................................................... 33 4.1. A felhasznált anyagok .......................................................................................................... 33 4.1.1. Fehérjék és ellenanyagok ............................................................................................................33 4.1.2. Szérumok .......................................................................................................................................34 4.1.3. Pufferek és egyéb reagensek .......................................................................................................35 4.1.4. Az mCRP előállítása CRP-ből denaturációval .........................................................................37 4.2. Az alkalmazott módszerek .................................................................................................. 37 4.2.1. Fehérje-fehérje kölcsönhatások kimutatása .............................................................................37 4.2.2. Kompetíciós kísérletek ................................................................................................................38 4.2.3. Kofaktor esszék ............................................................................................................................38 4.2.4. In vitro alternatív C3-konvertáz esszék ......................................................................................40 4.2.5. Komplementaktivációs kísérletek..............................................................................................41 4.2.6. Statisztikai analízis ........................................................................................................................42 5. Eredmények ........................................................................................................ 43 5.1. Az FHR-1 komplementaktivációban játszott szerepének vizsgálata ............................ 43 5.1.1. Az FHR-1 kötődése CRP-hez ...................................................................................................43 5.1.2. Az mCRP kötőhely azonosítása az FHR-1-ben ......................................................................48 5.1.3. Az FHR-1 és a FH közötti kompetíció az mCRP kötésért ...................................................49 5.1.4. Az FHR-1 szerepének vizsgálata a C3-konvertáz és a terminális út szintjén in vitro .........51 5.1.5. Az alternatív útvonal C3-konvertázának (C3bBb) felépülése FHR-1-en ............................53 4 5.2. Az FHR-3 komplementaktivációban játszott szerepének vizsgálata ............................ 56 5.2.1. Az FHR-3 kofaktor aktivitásának vizsgálata ............................................................................56 5.2.2. Pentraxinok kötődése FHR-3-hoz ............................................................................................57 5.2.3. Az alternatív útvonal C3-konvertázának (C3bBb) felépülése FHR-3 fehérjén ..................58 5.2.4. Az FHR-3 komplement működésre gyakorolt hatása pentraxin ligandumokon ...............62 5.2.5. Az FHR-3 kompetíciója FH-ral malondialdehiden ................................................................62 5.3. Az FHR-5 komplementaktivációban játszott szerepének vizsgálata ............................ 65 5.3.1. Az FHR-5 kötődése PTX3-hoz .................................................................................................65 5.3.2. Az FHR-5 – PTX3 kölcsönhatás jellemzése ...........................................................................67 5.3.3. A C3b és a C1q hatása az FHR-5 – PTX3 kölcsönhatásra ...................................................70 5.3.4. Az FHR-5 gátolja a FH – PTX3 kölcsönhatást ......................................................................71 5.3.5. Az FHR-5 kötődése a CRP módosult formájához és ennek következménye ....................72 5.3.6. Az FHR-5 kölcsönhatása az extracelluláris mátrixszal ...........................................................74 5.3.7. Az FHR-5 és FH kompetíciója humán szérumban ................................................................76 5.3.8. Az alternatív útvonal C3-konvertázának (C3bBb) felépülése FHR-5-ön ...........................77 6. Diszkusszió .......................................................................................................... 80 6.1. Az FHR-1, az FHR-3 és az FHR-5 komplementaktivációt gátló szerepének vizsgálata in vitro ........................................................................................................................... 81 6.2. Az FHR-1, az FHR-3 és az FHR-5 közvetlen szerepe az alternatív út C3- konvertázának képződésében in vitro ........................................................................................ 83 6.3. Az FHR-1 kölcsönhatása CRP-vel .................................................................................... 83 6.4. Az FHR-3 kölcsönhatása pentraxinokkal és malondialdehiddel ................................... 86 6.5. Az FHR-5 kölcsönhatása pentraxinokkal és az extracelluláris mátrixszal ................... 87 6.6. Összegzés, kitekintés ........................................................................................................... 90 7. Összefoglalás ....................................................................................................... 91 Irodalomjegyzék ...................................................................................................... 93 Köszönetnyilvánítás .............................................................................................. 103 5 Rövidítésjegyzék A dolgozatban bemutatott H-faktor molekulacsalád, valamint a többi komplement komponens elnevezése során figyelembe vettem a legutóbbi, nevezéktanra vonatkozó ajánlást [Kemper 2014]. Ennek alapján a H-faktor fehérjecsalád tagjainak elnevezése: H-faktor FH H-faktor szerű fehérje FHL-1 H-faktorral rokon fehérje 1 FHR-1 H-faktorral rokon fehérje 2 FHR-2 H-faktorral rokon fehérje 3 FHR-3 H-faktorral rokon fehérje 4 FHR-4 H-faktorral rokon fehérje 5 FHR-5 A disszertációban előforduló egyéb rövidítések: aHUS atípusos hemolítikus urémiás szindróma AMD időskori makuladegeneráció (age-related macular degeneration) ANOVA varianciaanalízis (analysis of variance) BSA szarvasmarha szérum albumin (bovine serum albumin) CARPA komplementaktivációval kapcsolatos pszeudoallergiás reakció (complement activation-related pseudoallergy) C3G C3 glomerulopátia C3GN C3 glomerulonefritisz C4BP C4b-kötő fehérje (C4b-binding protein) CCP komplement-szabályozó fehérje [domén] (complement control protein [domain]) CD59 MAC-inhibitor fehérje (cluster
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