Structural basis of adenovirus attachment to its receptor CD46 [Elektronische Ressource] = Strukturelle Basis der Bindung des Adenovirus an seinen Rezeptor CD46 / vorgelegt von David Persson

David Persson

Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2008
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 80 Mo

Extrait

Structural Basis of Adenovirus Attachment to
its Receptor CD46

Strukturelle Basis der Bindung des Adenovirus an seinen
Rezeptor CD46


Dissertation



der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen

zur Erlangung des Grades eines Doktors
der Naturwissenschaften


2008

vorgelegt von
David Persson

















Tag der mündlichen Prüfung: 04. Juli 2008
Dekan: Professor Dr. L. Wesemann
1. Berichterstatter: Professor Dr. T. Stehle
2. Berichterstatter: Dr. N. Arnberg

I The experimental part of this thesis was performed at the Interfaculty
Institute for Biochemistry at the University of Tuebingen between June 2005
and April 2008, under the supervision Prof. Dr. Thilo Stehle.




















"Millions saw the apple fall, but Newton
was the one who asked why."
-Bernard Baruch






II Abstract
Adenoviruses are common pathogens, causing a number of infectious diseases.
They have been found in virtually all human organs, producing symptoms that
range from the common cold to pneumonia (Hakim and Tleyjeh, 2008).
Additionally, they also cause a variety of clinical symptoms, for example
epidemic keratoconjunctivitis (EKC), pharyngoconjunctival fever (PCF), infantile
gastroenteritis, cystitis and urinary tract infections. Individuals with poorly
functioning immune systems are especially prone to severe and life-threatening
infections. Among these are persons infected with HIV, or organ transplant
patients (Hoffman, 2006). In 1977 Philip Sharp and Richard Roberts discovered the
ability of Adenoviruses to split genes (e.g. splicing), and they were in 1993
awarded the Nobel Prize in Physiology and Medicine for their discovery. Today,
Adenoviruses are mostly studied for the ability to be used as delivery vehicles in
gene therapy. About 25% of all clinical gene therapy trials are performed with
Adenovirus-based vectors, making Adenoviruses the largest group (Edelstein et
al., 2007). In this thesis we exclusively studied Adenoviruses binding to the cellular
receptor CD46, characterizing the interaction at an atomic level. We
experimentally connected structure and function, defining the Adenovirus
interaction with CD46. The structures presented in this thesis will have implications
for future drug design, focusing on reducing complications due to Adenoviruses
during organ transplantation. Furthermore, our findings will significantly improve
general knowledge of Adenovirus:receptor interactions, which will facilitate the
development of more efficient gene delivery vehicles.





III
1 Introduction..............................................................................................................1
1.1 History................1
1.2 Taxonomy.........2
1.3 The Adenovirus structure.................3
1.4 Adenovirus structural proteins........................................................................4
1.4.1 Hexon (polypeptide II)............4
1.4.2 Penton base (polypeptide III)6
1.4.3 Fiber (polypeptide IV).............7
1.5 Adenoviruses and the host................................................................8
1.6 The Adenovirus life cycle................9
1.7 The Adenovirus receptors.............10
1.7.1 The Coxsackie Adenovirus Receptor (CAR).......11
1.7.2 Sialic acid................................................................................................12
1.7.3 Membrane cofactor protein (MCP or CD46).....13
1.7.4 The role of integrins as entry receptors................................................15
1.8 Species B Adenoviruses ................................16
1.9 Clinical relevance.........................17
1.9.1 Gene therapy................................17
1.9.2 Vaccination and treatment.................................19
1.10 Aim ..................................................................................20
2 Materials.................21
2.1 Equipment......21
2.1.1 General purpose equipment ...............................................................21
2.1.2 Chromatography...................................................22
2.1.3 X-ray crystallography equipment........................22
2.1.4 Software..................................................................22
2.1.5 Filters and kits..........................................................23
2.2 Cloning and expression ................................................23
2.2.1 Bacterial strains......................23
2.2.2 Eukaryotic cell lines................................................23
2.2.3 Bacterial expression plasmid ................................................................23
2.2.4 CHO cell expression plasmid24
2.2.5 PCR-reactions.........................................................24
2.2.6 Primers.....24
2.2.7 Restriction enzymes...............................................25
2.2.8 Ligation of plasmids................................25
2.3 Cell culture .....................................26
2.3.1 Bacterial expression...............26
2.3.2 CHO cell expression................................26
2.4 Protein chemistry ...........................................................26
2.4.1 DNA electrophoresis..............26
2.4.2 SDS-PAGE................................................................26
2.4.3 Concentration determination of protein............27
2.4.4 Deglycosylation .....................................................27
2.5 SPR ...................................................................................27
2.5.1 SPR buffers..............................27
2.5.2 BiaCore chips.........................27
IV 2.6 Crystallization .................................................................................................28
2.6.1 Crystallization screens...........28
2.6.2 Crystallization reagents.........28
2.7 Buffers..............................................................................................................29
2.7.1 Generation of competent cells...........................29
2.7.2 Buffers protein purification....29
3 Methods..................31
3.1 Molecular biology..........................................................................................31
3.1.1 Polymerase Chain Reaction (PCR)......................31
3.1.2 Purification of plasmid DNA..................................32
3.2 Restriction enzyme digestion and plasmid ligation...32
3.2.1 Adenovirus knob ....................................................................................32
3.2.2 Human CD46..........................33
3.3 Protein chemistry...........................33
3.3.1 Protein expression..................33
3.4 Protein purification ........................................................................................34
3.4.1 Adenovirus knob purification...............................34
3.4.2 CD46 purification...................36
3.4.3 Formation of Adenovirus:CD46 SCR1-SCR2 complex........................38
3.4.4 Deglycosylation of CD46 ......................................................................39
3.4.5 Determination of protein concentration............39
3.5 Affinity measurements...................39
3.5.1 Isothermal Titration Calorimetry (ITC) ..................................................39
3.5.2 Surface Plasmon Resonance (SPR)......................40
3.6 Functional studies of binding and infectivity..............42
353.6.1 Propagation of S-labeled Adenoviruses...........42
3.6.2 Binding experiments ..............................................................................42
3.6.3 Fluorescent Focus Assay (FFA)..............................................................42
3.7 X-ray crystallography....................43
3.7.1 Crystallization of proteins......43
3.7.2 Crystals - the composition of the unit cell...........................................45
3.7.3 Crystal freezing.......................................................45
3.7.4 X-rays .......................................................................46
3.7.5 Diffraction of X-rays at a lattice...........................46
3.7.6 Data collection......................................................47
3.7.7 Data processing.....................................................48
3.7.8 Structure determination and truncation of data...............................51
3.7.9 The phase problem...............52
3.7.10 General - molecular replacement ......................................................53
3.7.11 General - structure refinement.............................55
3.7.12 The Matthews coefficient .....................................................................58
4 Results......................................................................................59
4.1 Protein purification........................59
4.1.1 Adenovirus knobs.................................