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Dissertation Zur Erlangung Des Grades ‚Doktor Der Naturwissenschaften‘

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Dissertation Zur Erlangung Des Grades ‚Doktor Der Naturwissenschaften‘ FUNKTIONELLE UND STRUKTURELLE CHARAKTERISIERUNG DES OVASTACINS IN VIVO UND IN VITRO Dissertation zur Erlangung des Grades ‚Doktor der Naturwissenschaften‘ Am Fachbereich Biologie der Johannes Gutenberg-Universität Mainz Vorgelegt von André Hildebrand geboren am 16.01.1979 in München Mainz, März 2013 Gutachter: Dekan: 1. Berichterstatter: 2. Berichterstatter: Tag der mündlichen Prüfung: 24.07.2013 Für meine Familie. INHALTSVERZEICHNIS Inhaltsverzeichnis 1. Zusammenfassung ............................................................................................ 1 2. Einleitung ........................................................................................................... 2 2.1 Proteasen ............................................................................................................ 2 2.2 Serinproteasen .................................................................................................... 4 2.2.1 Elastase .............................................................................................................. 5 2.2.2 Trypsin ................................................................................................................ 6 2.3 Metalloproteasen ................................................................................................. 7 2.3.1 MMPs .................................................................................................................. 7 2.3.2 Astacine .............................................................................................................. 9 2.3.3 Ovastacin ...........................................................................................................10 2.4 Inhibitoren ..........................................................................................................13 2.4.1 Fetuine ...............................................................................................................13 2.5 Oogenese und Reifung der Eizellen ...................................................................14 2.5.1 Aufbau der Oocyten und Spermien ....................................................................15 2.5.2 Fertilisation von Oocyten ....................................................................................16 2.5.3 Embryonalentwicklung von Mus musculus .........................................................17 2.6 Ziele der Arbeit ...................................................................................................18 3. Material und Methoden ....................................................................................19 3.1 Verwendete Geräte und Chemikalien .................................................................19 3.2 Polymerase-Kettenreaktion (PCR) .....................................................................20 3.3 Agarose-Gelelektrophorese ................................................................................22 3.4 Restriktion ..........................................................................................................23 3.5 Ligation ..............................................................................................................24 3.6 Transformation - Einschleusung fremder DNA in Bakterien ................................25 3.7 Eukaryotische Zellkultur .....................................................................................27 3.7.1 Baculovirus-Expressionssystem .........................................................................27 3.7.2 Sf9-Zellen ...........................................................................................................30 3.7.3 HighFive-Zelllen .................................................................................................30 3.7.4 CHO-Zellen zur Expression von Pro-Ovastacin-Strep ........................................31 3.7.5 HEK-293-Zellen zur Expression von Pro-Ovastacin-Strep ..................................32 3.7.6 Transfektion .......................................................................................................32 3.8 Antikörperherstellung .........................................................................................33 3.9 Ermittlung der Proteinkonzentration ...................................................................34 3.10 SDS-Polyacrylamid-Gelelektrophorese ..............................................................35 3.11 Western-Blot (Semi-dry Verfahren) ....................................................................36 I INHALTSVERZEICHNIS 3.12 Immunologischer Nachweis von Proteinen mittels Chemilumineszenz ...............37 3.13 Native Polyacrylamid-Gelelektrophorese ............................................................41 3.14 Zymographie-Gelelektrophorese ........................................................................42 3.15 Native Zymographie-Gelelektrophorese .............................................................42 3.16 Coomassie Brillant Blue-Färbung .......................................................................42 3.17 Reinigung ...........................................................................................................44 3.17.1 Ammoniumsulfat-Fällung ....................................................................................44 3.17.2 Dialyse ...............................................................................................................44 3.17.3 Affinitätschromatographie über Strep-Tactin ......................................................44 3.17.4 Gelchromatographie über Hema Bio 1000 .........................................................45 3.18 Herstellen von Zelllysaten ..................................................................................46 3.19 Herstellung von Organlysaten ............................................................................47 3.20 Limitierte Proteolyse ...........................................................................................48 3.21 FRET-Assay .......................................................................................................49 3.22 pH Abhängigkeit der Aktivierung von Pro-Ovastacin-Strep .................................51 3.23 Sequenz-Alignments und Modellierung von Pro-Ovastacin ................................52 3.24 Superovulation von Mäuseeizellen .....................................................................52 3.25 Präparation der Eileiter aus FVB Mäusen ...........................................................52 3.26 Entnahme der Zygoten .......................................................................................53 3.27 Vorbereitung der Zygoten für die Kultivierung ....................................................54 3.28 In vitro-Kultivierung der Zygoten .........................................................................55 3.29 Immunhistochemischer Antikörpertest mit Sf9-Zellen .........................................55 3.30 Antikörperbehandlung und Fixierung von Embryonen ........................................56 3.31 Transmissions‐Elektronenmikroskopie (TEM) ....................................................58 4. Ergebnisse ........................................................................................................59 4.1 Herstellung der DNA-Konstrukte für die Expression ...........................................59 4.1.1 Ovastacin-Strep .................................................................................................59 4.1.2 Ovastacin-Kat-Strep ...........................................................................................62 4.1.3 Zusammenfassung der Klonierung der 3 Ovastacin-Varianten ...........................65 4.2 Nachweis der Ovastacin mRNA in HUVEC Zellen ..............................................65 4.3 Herstellung eines polyklonalen Anti-Pro-Ovastacin Antiserums ..........................67 4.4 Expression und Reinigung von Ovastacin-Strep .................................................67 4.5 Expression und Reinigung von Ovastacin-Kat-Strep ..........................................69 4.6 Expression und Reinigung von Pro-Ovastacin-Strep ..........................................71 4.7 Massenspektrometrische Analyse und N-terminale Sequenzierung ...................73 4.8 Untersuchung der Oligomerisierung von Pro-Ovastacin .....................................74 4.9 Stabilität des Pro-Ovastacins in Abhängigkeit von pH-Wert und Temperatur .....77 II INHALTSVERZEICHNIS 4.10 Aktivierung von Pro-Ovastacin-Strep ..................................................................81 4.10.1 Sequenzalignment und Homologiemodellierung zur Identifizierung der potenziellen Aktivierungsschnittstelle des Pro-Ovastacins..................................81 4.10.2 Aktivierung von Pro-Ovastacin mit MMP-2 .........................................................86 4.10.3 Aktivierung von Pro-Ovastacin-Strep mit Trypsin ...............................................87 4.10.4 Aktivierung von Pro-Ovastacin mit neutrophiler Elastase ...................................89 4.10.5 Interaktion von Pro-Ovastacin mit Proteaseninhibitoren .....................................94 4.10.6 Massenspektrometrische Analyse und N-terminale Sequenzierung des aktivierten Ovastacins..................................................................................95 4.11 Nachweis von Ovastacin in Geweben ................................................................96 4.12 Expression von Pro-Ovastacin in Cho-K1-Zellen ................................................97
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