Mutational analysis of the export targeting motif of fibroblast growth factor 2, a mediator of tumor-induced angiogenesis [Elektronische Ressource] / presented by Andre Engling

Andre Norton

Sujets

• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2005
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 26 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences


















presented by
Diplom-Biologe Andre Engling
born in Marl











Mutational Analysis of the Export Targeting Motif
of Fibroblast Growth Factor 2,
a Mediator of Tumor-Induced Angiogenesis














Referees:
Prof. Dr. Walter Nickel
Prof. Dr. Michael Brunner

Content


SUMMARY 1
1 INTRODUCTION 2
1.1 Classical Protein Secretion 2
1.2 Unconventional Protein Secretion 4
1.3 Unconventionally secreted proteins 7
1.3.1 Interleukin-1 8
1.3.1.1 Interleukin-1a 8
1.3.1.2 Interleukin-1b 8
1.3.2 Thioredoxin 9
1.3.3 Macrophage migration inhibitory factor (MIF) 10
1.3.4 Leishmania hydrophilic acylated surface protein B (HASPB) 10
1.3.5 Viral proteins: HIV Tat, FV Bet and HSV VP22 11
1.3.5.1 HIV Tat 11
1.3.5.2 HSV VP22 12
1.3.5.3 FV Bet 13
1.3.6 Homeodomain-containing transcription factors and HMG chromatin-binding proteins 13
1.3.7 Galectins 14
1.3.7.1 Galectin-1 15
1.3.7.2 Galectin-3 16
1.3.8 Fibroblast growth factors 16
1.3.8.1 Fibroblast growth factor 1 18
1.3.8.2 Fibroblast growth factor 2 19
Structural characteristics 19
Binding to heparin and heparan sulfate proteoglycans 21
Biological functions of FGF2 22
Unconventional secretion of FGF2 22
1.4 Aim of the present thesis 24
2 MATERIAL AND METHODS 26
2.1 Material 26
2.1.1 Chemicals 26
2.1.2 Enzymes 28
2.1.3 Antibodies 28
I Content


2.1.4 Equipment 28
2.1.5 Plasmids and Primers 29
2.1.6 Bacteria and Media 34
2.1.7 Eukaryotic Cells and Media 34
2.2 Molecular biological methods 36
2.2.1 Polymerase Chain Reaction (PCR) 36
2.2.1.1 Random Mutagenesis 36
2.2.1.2 Point mutations 37
2.2.1.3 Truncations 39
2.2.2 PCR Purification 39
2.2.3 Restriction digestion and Dephosphorylation 40
2.2.4 Ligation of DNA fragments 40
2.2.5 Transformation of E.coli with plasmid DNA 40
2.2.6 Selection of clones 41
2.2.7 Isolation of plasmid DNA from bacteria 41
2.2.8 Agarose gel electrophoresis 41
2.2.9 DNA extraction from agarose gels 42
2.2.10 DNA Sequencing 42
2.3 Biochemical Methods 42
2.3.1 SDS-Polyacrylamid-gel electrophoresis 42
2.3.2 Western Blot Analysis 44
2.3.2.1 Transfer of proteins to polyvinylidene fluoride (PVDF) membrane 45
2.3.2.2 Ponceau S staining 46
2.3.2.3 Immunochemical detection of proteins (HRP system) 46
2.3.2.4 Immunochemical detection of proteins (Licor System) 47
2.3.3 Biochemical assay to estimate the amount of secreted FGF2-GFP 47
2.3.4 Isolation of detergent-insoluble microdomains 47
2.3.5 Biotinylation of proteins associated with the cell surface 48
2.3.6 Preparation of cell free supernatant 50
2.3.7 Binding of FGF2 to heparin beads 50
2.3.8 Precipitation of FGF2-GFP from culture media using heparin beads 50
2.3.9 Binding of FGF2-GFP to CHO Cells 51 MCAT/TAM2
2.3.10 Immunoprecipitation of FGF2-GFP from growth medium 51
2.4 FACS Analysis 52
2.4.1 Antibody labelling of cells in suspension 52
2.4.2 Antibody labelling of Cells Attached to the culture plate 53
II Content


2.5 Production of stable Cell Lines 54
2.5.1 Retroviral Transduction 54
2.5.2 FACS Sort 54
2.6 Confocal Microscopy 55
3 RESULTS 56
3.1 Generation of model cell lines expressing FGF2-GFP in a doxicycline-dependent manner 57
3.1.1 Verification of the stable integration of FGF2-GFP into the genome of CHO cells 59
3.1.2 Characterization of CHO and CHO cells employing fluorescence microscopy, Western FGF2-GFP GFP
blotting and FACS analysis 60
3.2 Establishing an in vivo system to quantitatively asses FGF2-GFP secretion 61
3.2.1 Secreted FGF2-GFP is detected on the cell surface of CHO cells 62
3.2.1.1 Cell surface staining is removable by trypsin and heparin treatment 63
3.2.1.2 FGF2-GFP binding capacity to the cell surface 66
3.2.2 Characterization of FGF2-GFP secretion regarding kinetics, unspecific release and sensitivity to
ouabain 68
3.2.3 Biochemical analysis of FGF2-GFP secretion 70
3.2.4 Analysis of FGF2-GFP secretion by confocal microscopy 72
3.2.5 Secreted biosynthetic FGF2-GFP is targeted to non-lipid raft microdomains 73
3.2.6 Intercellular spreading of exported biosynthetic FGF2-GFP 76
3.2.7 Refinement of FACS processings in order to prevent unspecific release 78
3.3 Mutational analysis of FGF2-GFP targeting to its transport machinery 80
3.3.1 Selection and cloning of FGF2 mutants 81
3.3.1.1 Random mutagenesis 81
3.3.1.2 Point mutations 82
3.3.1.3 Truncations 85
3.3.1.4 C-terminal Truncations 86
3.3.2 Characterisation of FGF2 mutants with regard to export efficiency, binding to heparan sulfate
proteoglycans in vivo and to heparin in vitro 88
3.3.2.1 In vitro binding of FGF2 using heparin beads 88
3.3.2.2 In vivo binding of FGF2 to CHO cells 89
3.3.2.3 Quantification of FGF2-GFP export by flow cytometry 90
3.3.2.4 Biochemical secretion assay using botin to analyze FGF2 export from CHO cells 92
3.3.3 Analysis of mutants obtained by performing random Mutagenesis 94
3.3.3.1 Overview of mutations with regard to their amino acid changes 94
3.3.3.2 Experimental data for FGF2 mutants not impaired regarding export efficiency and binding to
III Content


heparin 95
3.3.3.3 Experimental data for FGF2 mutants impaired in binding and protein stability 105
3.3.3.4 Experimental data for a secretion deficient FGF2 mutant 112
3.3.3.5 Classification of FGF2-GFP mutants obtained by random mutagenesis with regard to secretion
efficiency, protein stability and binding to heparin 113
3.3.4 Analysis of mutants obtained by site-directed mutagenesis 114
3.3.4.1 Experimental data for mutants showing no phenotype regarding secretion efficiency and
affinity to heparin 116
3.3.4.2 Experimental data for FGF2 mutants showing a reduced expression level of FGF2-GFP 159
3.3.4.3 Experimental data for FGF2 mutants impaired in protein stability, binding to heparin and to
heparan sulfate proteoglycans 162
3.3.4.4 Experimental data for a double cysteine FGF2 mutant potentially deficient in secretion 166
3.3.4.5 Overview of mutants obtained by point mutation with regard to secretion efficiency, affinity to
heparin and protein stability 167
3.3.5 Truncations of N- and C-Terminus 168
3.3.5.1 Functional analysis of FGF2 mutants with N-terminal Truncations 168
3.3.5.2 Functional analysis of FGF2 mutants with C-terminal Truncations 175
3.4 Characterization of FGF2-GFP mutants differing from wild-type as identified by the screening
procedure 189
3.4.1 Analysis of C-terminal truncations with regard to unconventional secretion 189
3.4.2 Characterization of mutant rM 156 190
3.4.2.1 Quantification of secretion employing FACS Analysis 191
3.4.2.2 Biotinylation of surface proteins to assess the amount of secreted FGF2 192
3.4.2.3 Degradation experiment 193
3.4.2.4 Binding efficiency to HSPGs of FGF2-GFP , rM 156 and clone 36 195 wt
4 DISCUSSION 197
4.1 Generation of CHO cells expressing FGF2-GFP in a doxicycline-dependent manner as a tool for
the analysis of FGF2 secretion 198
4.2 Establishing an in vivo system to analyze unconventional secretion of FGF2 200
4.2.1 FGF2-GFP is localized on the cell surface of CHO cells 200
4.2.2 Characterization of FGF2-GFP localization on the cell surface 201
4.2.3 Functional characterization of the translocation mechanism 202
4.2.3.1 Kinetics of the translocation process of FGF2 202
4.2.3.2 Binding capacity of FGF2 for binding to heparan sulfate proteoglycans present on the cell
surface of FGF2-GFP 203
4.2.3.3 Unspecifically released FGF2-GFP does not contribute to the cell surface signal 203
IV Content


4.2.3.4 Inhibition of FGF2 secretion by ouabain 204
4.2.4 Intercellular spreading of FGF2-GFP 204
4.2.5 Refinement of FACS processing in order to prevent unspecific release of FGF2-GFP 205
4.3 Biosynthetic FGF2-GFP is localized to non-lipid raft microdomains following translocation 207
4.4 Screening of FGF2 mutants to elucidate targeting motifs for unconventional secretion 208
4.4.1 Characterization of FGF2 mutants obtained by random mutagenesis 210
4.4.2 Characterization of FGF2 mutants obtained by site-directed mutagenesis 211
4.4.3 Characterization of N-terminally truncated versions of FGF2 214
4.4.4 Characterization of C-terminally truncated versions of FGF2 215
4.4.5 Detailed analysis of mutant 156 with regard to secretion efficiency, protein stability, heparin and
heparan sulfate binding efficiency 218
4.4.6 Future perspectives 220
5 ABBREVIATIONS 223
6 REFERENCES 225
ACKNOWLEDGEMENTS 246
V Summary

Summary

The majority of secretory proteins is exported from mammalian cells by t