Analysis of the influence of epitope flanking regions on MHC class I restricted antigen presentation [Elektronische Ressource] / Alejandra Nacarino Martínez

Martinea

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2007
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

U T O M N E S U N U M S I N T

The
SCHOOL OF MEDICINE and the DEPARTMENT OF BIOLOGY
of the
JOHANNES GUTENBERG-UNIVERSITY OF MAINZ
confer on
Alejandra Nacarino Martínez
born on 22.10.1976 in Cádiz (Spain)
in recognition of her dissertation

“Analysis of the influence of epitope flanking regions on MHC class I
restricted antigen presentation“

and the successful examinations
the academic degree
„Doktor der Naturwissenschaften"

Mainz, 12.09.2007
Dean Department of Biology




























Day of the disputation: 07.12.2007







Index
INDEX
1 INTRODUCTION 1
1.1 THE IMMUNE SYSTEM 1
1.2 INNATE IMMUNE RESPONSE 1
1.3 THE ADAPTIVE 2
1.3.1 Humoral immunity 2
1.3.2 Cell-mediated immunity 2
1.4 THE MHC COMPLEX 3
1.4.1 Structure of MHC molecules 4
1.4.2 Peptide binding on MHC molecules 4
1.5 MHC CLASS I ANTIGEN PROCESSING PATHWAY 5
1.5.1 The source of MHC class I peptides 5
1.5.2 ubiquitin-proteasome pathway 6
1.5.3 Peptide trimming by cytosolic aminopeptidases 6
1.5.3.1 Leucine aminopeptidase 7
1.5.3.2 Puromycin sensitive aminopeptidase & bleomycin hydrolase 7
1.5.3.3 Thimet oligopeptidase 8
1.5.3.4 Tripeptidyl peptidase II
1.5.3.5 Insulin degrading enzyme 9
1.5.4 Transport into the ER 10
1.5.4.1 Transporter associated with antigen processing (TAP) 10
1.5.4.2 Transport into the ER by signal sequences 11
1.5.5 Peptide trimming in the ER 11
1.5.6 loading complex 12
1.6 THE PROTEASOME 13
1.6.1 Structure and composition of the eukaryotic proteasome 14
1.6.2 Proteasomal activity 14
1.6.3 Conjugates of the 20S proteasome 16
1.6.4 The immunoproteasome 16
1.7 MHC CLASS II ANTIGEN PROCESSING PATHWAY 17
1.7.1 The source of MHC class II peptides 17
1.7.2 Antigen generation for MHC class II binding 18
1.7.3 Antigen loading onto MHC class II molecules 19
1.8 PREDICTION OF CTL EPITOPES 19
i Index
1.8.1 Prediction methods based on proteasomal cleavage 20
1.8.2 Prediction methods based on TAP transport 20
1.8.3 Prediction methods based on MHC class I binding 20
1.8.4 methods merging all three events 21
1.9 AIM OF THE THESIS PROJECT 21

2 MATERIALS AND METHODS 23
2.1 MATERIALS 23
2.1.1 Bacterial strains 23
2.1.2 Media for bacterial cells 23
2.1.3 Eukaryotic cell lines
2.1.4 Cell culture media
2.1.5 Plasmids 24
2.1.6 Constructs 25
2.1.7 Restriction enzymes 25
2.1.8 Primer 25
2.1.9 siRNA oligonucleotides 29
2.1.10 Antibodies 30
2.1.11 Proteins
2.1.12 Inhibitors
2.1.13 Chemicals & buffers 31
2.1.14 Plastic materials and equipment 31
2.2 METHODS 32
2.2.1 Molecular cloning methods 32
2.2.1.1 Plasmid preparation (Mini) 32
2.2.1.2 (Maxi) 33
2.2.1.3 DNA sequencing 33
2.2.1.4 Nucleic acid quantification
2.2.1.5 Agarose gel electrophoresis
2.2.1.6 Purification of DNA fragments from agarose gels 34
2.2.1.7 DNA ligation 34
2.2.1.8 Transformation of competent bacteria 34
2.2.1.9 Sitespecific mutagenesis by quickchange PCR 34
2.2.1.10 Mutagenesis by annealing of oligonucleotides 35
ii Index
2.2.2 Cell culture methods 36
2.2.2.1 Long-term storage 36
2.2.2.2 Thawing and repropagating cells 36
2.2.2.3 Trypan blue exclusion 36
2.2.2.4 Transfection with Fugene 37
2.2.3 Acid wash analysis 37
2.2.3.1 Inhibition assays for acid wash analysis 37
2.2.4 Flow cytometry analysis 38
b 2.2.5 siRNA mediated gene silencing in Flp-In 293K transfectants 38
2.2.5.1 Electroporation
2.2.5.2 mRNA isolation 39
2.2.5.3 Reverse transcription
2.2.5.4 Real time PCR
2.2.6 Methods for protein analysis 40
2.2.6.1 Determination of protein concentration 40
2.2.6.2 Fluorogenic activity assays in vitro 41
2.2.7 LCL721 cells cytosol purification 41
2.2.7.1 Cytosol extraction 41
2.2.7.2 Lysate purification of LCL721 cells by HPLC 42
2.2.7.3 Reducing SDS-PAGE 45
2.2.7.4 Coomassie staining
2.2.7.5 Analysis by mass spectrometry 46

3 RESULTS 47
3.1 SCREENING SYSTEM 47
3.1.1 The Flp-In system 48
3.1.2 Generation of the SIINFEKL constructs 49
b 3.1.3 Detection of the H-2K/SIINFEKL complex 52
3.1.4 Steady state SIINFEKL presentation level of the transfectants 53
3.1.5 Determination of SIINFEKL re-presentation rate on the cell surface 55
3.1.6 In vitro inhibitor assays 58
3.1.7 MHC class I analysis after the acid treatment 59
3.1.8 Determsentation rate on the cell surface of
all tranfectants and the influence of the proteasome on its presentation 60
iii Index
3.1.9 Proteasomal inhibition with epoxomicin 64
3.1.10 Role of TPPII in antigen processing and presentation of SIINFEKL 65
3.2 SILENCING OF CYTOSOLIC AMINOPEPTIDASES 68
b 3.2.1 Gene silencing of TPPII, THOP1 and IDE in Flp-In 293K cells 68
3.2.2 Activity analysis in the electroporated cells for the silenced cytosolic
peptidases (ThOP1, TPPII, IDE) 69
b 3.2.3 Presentation of H-2K /SIINFEKL after gene silencing of the cytosolic
b peptidases ThOP1, TPPII and IDE in Flp-In 293K _P’IR cells 72
b 3.2.4 /SIINFEKL after simultaneous gene silencing of
b two peptidases ThOP1 and TPPII in Flp-In 293K _P’IR cells 73
3.3 IDENTIFICATION OF AN ENDOPEPTIDASE ACTIVITY IN THE
CYTOSOL INVOLVED IN ANTIGEN PROCESSING 75
3.3.1 Purification strategy of the LCL 721 cytosol 75
3.3.2 Desalting step 76
3.3.3 Anion exchange chromatography (DEAE column) 76
3.3.4 Gelfiltration (Superdex S200 column) 80
3.3.5 atography (Mini Q™ column) 84
3.3.6 SDS-PAGE and coomassie staining 85
3.3.7 Mass spectrometry analysis of the SDS-PAGE bands 86

4 DISCUSION 88
4.1 S8L EXPRESSING CELLS WITH CONSTRUCTS CARRYING
DIFFERENT C-TERMINAL REGIONS 88
4.2 SILENCING OF CYTOSOLIC PEPTIDASES 92
4.3 IDENTIFICATION OF ENDOPEPTIDASES FROM THE LCL721
CYTOSOL 94
4.4 FURTHER ANALYSIS OF THE S8L GENERATION 98
4.5 THE LINK BETWEEN VACCINE DESIGN AND ANTIGEN
PROCESSING/PRESENTATION 98
4.6 FURTHER POSSIBLE MODEL INVOLVED IN ANTIGEN
PROCESSING/PRESENTATION: HEAT SHOCK PROTEINS 101

5 SUMARY 104

6 REFRENCES 105
iv Index

7 ABREVIATIONS 121







v 1 Introduction
1 Introduction

Immunology was born with the discovery of Edward Jenner (1796), when he discovered that
cowpox (or vaccinia) induced protection against human smallpox. He injected the material
from a pustule into the arm of an 8-year-old boy. When this boy was later intentionally
inoculated with smallpox, the disease did not develop. This procedure was called vaccination
(Latin: vaccinus, or from cows), which still remains to be the most effective method for
preventing infections (Abul K.Abbas and Andrew H.Lichtman, 2003). Although it took
almost two centuries for smallpox vaccination to become universal, the interest in this new
science increased and with the help of the discoveries of further great scientists we have
gained a remarkable knowledge in the understanding of the immune system and its functions.

1.1 The immune system
The immune system has evolved to protect against the continuous attack of viruses, bacteria,
fungi and parasites. It is able to recognize and eliminate the pathogens. This defense against
microbes is mediated by the early reactions of innate immunity and the later responses of
adaptive (or specific) immunity (Abul K.Abbas and Andrew H.Lichtman, 2003).

1.2 The innate immune response
Innate immunity provides the early lines of defense against microbes. The principal
components are physical and chemical barriers (e.g.: skin and antimicrobial substances
produced at epithelial surfaces), phagocytic cells (neutrophils, macrophages), natural killer
cells and proteins, like members of the complement system and cytokines that regulate and
coordinate many activities of the cells of the innate immune response. Molecules produced
during innate immune responses stimulate adaptive immunity and influence the nature of
adaptive immune responses. For example, macrophages activated by microbes and interferon-
gamma (IFN- γ) produce costimulators that enhance T cell activation.

The innate immune system uses pattern recognition receptors to recognize struct