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Molecular analysis of the unconventional export machinery of galectin-1, a {β-galactoside-specific [beta-galactoside-specific] lectin of the extracellular matrix [Elektronische Ressource] / presented by Claudia Seelenmeyer

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231 pages
Molecular Analysis of the Unconventional Export Machinery of Galectin-1, a β-Galactoside-specific Lectin of the Extracellular Matrix 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-Biologin Claudia Seelenmeyer born in Karlsruhe 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-Biologin Claudia Seelenmeyer born in Karlsruhe Oral examination: Molecular Analysis of the Unconventional Export Machinery of Galectin-1, a β-Galactoside-specific Lectin of the Extracellular Matrix Referees: Prof. Dr. rer. nat Walter Nickel Prof. Dr. rer. nat Michael Brunner List of Publications Engling, A., Backhaus, R., Stegmayer, C., Zehe, C., Seelenmeyer, C., Kehlenbach, A., Schwappach, B., Wegehingel, S., and Nickel, W. (2002). Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells. J Cell Sci 115, 3619-3631. Seelenmeyer, C., Wegehingel, S., Lechner, J., and Nickel, W. (2003). The cancer antigen CA125 represents a novel counter receptor for galectin-1.
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Molecular Analysis of the Unconventional
Export Machinery of Galectin-1,
a β-Galactoside-specific Lectin
of the Extracellular Matrix


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-Biologin Claudia Seelenmeyer
born in Karlsruhe

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-Biologin Claudia Seelenmeyer
born in Karlsruhe


Oral examination:


Molecular Analysis of the Unconventional
Export Machinery of Galectin-1,
a β-Galactoside-specific Lectin
of the Extracellular Matrix

















Referees:
Prof. Dr. rer. nat Walter Nickel
Prof. Dr. rer. nat Michael Brunner List of Publications

Engling, A., Backhaus, R., Stegmayer, C., Zehe, C., Seelenmeyer, C., Kehlenbach,
A., Schwappach, B., Wegehingel, S., and Nickel, W. (2002). Biosynthetic FGF-2 is
targeted to non-lipid raft microdomains following translocation to the extracellular
surface of CHO cells. J Cell Sci 115, 3619-3631.

Seelenmeyer, C., Wegehingel, S., Lechner, J., and Nickel, W. (2003). The cancer
antigen CA125 represents a novel counter receptor for galectin-1. J Cell Sci 116,
1305-1318.

Seelenmeyer, C., Wegehingel, S., Tews, I., Kunzler, M., Aebi, M., and Nickel, W.
(2005). Cell surface counter receptors are essential components of the
unconventional export machinery of galectin-1. J Cell Biol 171, 373-381.

























Wohin auch immer wir reisen,
wir suchen, wovon wir träumten,
und finden doch stets nur uns selbst.


Günter Kunert (*1929)





Contents I

Contents
ABBREVIATION 1
ZUSAMMENFASSUNG 5
ABSTRACT 7
1 INTRODUCTION 9
1.1 ER/Golgi-mediated protein secretion 9
1.2 Unconventional secretion 14
1.2.1 Galectins 17
1.2.2 Pro-angiogenic Growth Factors: FGF-1 and FGF-2 23
1.2.3 Leishmania hydrophilic acylated surface protein B (HASPB) 25
1.2.4 Cytokines: Interleukin-1β, Thioredoxin and Macrophage Migration Inhibitory Factor 26
1.3 Gal-1 receptors in different cell types 28
1.3.1 Biological functions of Gal-1 in different cell types 28
1.3.2 Galectin-1 receptors 30
1.4 Galectins and apoptosis 34
1.5 Aim of this work 36
2 MATERIAL AND METHODS 37
2.1 Material 37
2.1.1 Chemicals 37
2.1.2 Technical devices 40
2.1.3 Plasmids 41
2.1.4 DNA modifying enzymes 42
2.1.5 Primers and oligonucleotides 42
2.1.6 Bacteria and bacterial media 48
2.1.7 Eukaryotic cell lines 49
2.1.8 Eukaryotic cell culture media 50
2.1.9 Primary antibodies 51 Contents II

2.1.10 Secondary antibodies 51
2.2 Molecular biological methods 52
2.2.1 Bacterial transformation 52
2.2.2 Selection and amplification of plasmids 53
2.2.3 Plasmid preparation 53
2.2.4 Determination of DNA concentration 54
2.2.5 Agarose gel electrophoresis 54
2.2.6 DNA marker 55
2.2.7 Site-directed mutagenesis 56
2.2.8 Polymerase chain reaction 58
2.2.9 PCR purification 60
2.2.10 Gel extraction of DNA fragments 60
2.2.11 Restriction digests 60
2.2.12 DNA dephosphorylation 61
2.2.13 Ligation of DNA fragments 61
2.2.14 DNA sequencing 62
2.2.15 Short interfering RNAs in mammalian cells 62
2.3 Eukaryotic cell culture techniques 63
2.3.1 Maintaining cell lines 63
2.3.2 Freezing of eukaryotic cells 64
2.3.3 Thawing of eukaryotic cells 65
2.3.4 Viral transduction 65
2.3.5 Addition of doxicycline 67
2.4 Biochemical methods 67
2.4.1 Recombinant proteins 67
2.4.2 Preparation of cell lysates 68
2.4.3 Preparation of cell-free supernatants 68
2.4.4 Determination of protein concentration based on GFP fluorescence 69
2.4.5 Sample preparation for SDS polyacrylamide gel electrophoresis 69
2.4.6 SDS polyacrylamide gel electrophoresis 70
2.4.7 SDS-PAGE protein molecular weight standards 71
2.4.8 Western blot analysis 72
2.4.9 Immunochemical protein detection using the ECL system 73
2.4.10 Immunochemical protein detection using the LICOR system 74
2.4.11 Gal-1 affinity matrix and binding experiments employing subcellular fractions of S-HeLa cells 75
2.4.12 Protein identification employing MALDI-Tof mass spectrometry 76
2.4.13 Biotinylation of cell surface proteins 77 Contents III

2.4.14 Immunoprecipitation of proteins 79
2.4.15 Galectin binding to lactose-coupled beads 80
2.4.16 Galectin binding to the cell surface of CHO cells 80
2.4.17 Stability analysis of Galectin-GFP fusion proteins in conditioned media derived from CHO cells 81
2.5 Flow cytometry 81
2.5.1 Sample preparation for FACS analysis 81
2.5.2 Plate labelling technique 83
2.5.3 FACS sorting 84
2.6 Confocal microscopy 85
2.6.1 Sample preparation for confocal microscopy 85
2.6.2 Immunostaining of cell surface proteins for confocal microscopy 85
3 RESULTS 87
3.1 Identification of Gal-1 interacting proteins potentially involved in the export process of
human Gal-1 87
3.1.1 Identification of CA125 as a Gal-1 counter receptor 89
3.1.2 Specificity of CA125-mediated Galectin binding 93
3.1.3 CA125-C-TERM binding to Gal-1 depends on O-linked β-galactose-terminated oligosaccharide
chains 98
3.1.4 Despite lacking a N-terminal signal peptide, CA125-C-TERM is transported to the cell surface
of CHO and HeLa cells 101
3.1.5 CA125-C-TERM is transported to the cell surface via the ER/Golgi-dependent secretory pathway 103
3.1.6 Correlation of endogenous CA125 expression with increased cell surface expression of endogenous
Gal-1 in CHO and HeLa cells 108
3.1.7 CA125 expression does not stimulate Gal-1 export 112
3.2 Establishment of experimental systems to study unconventional secretion of Gal-1 114
3.2.1 Generation of cell lines 115
3.2.2 Quantitative analysis of export of reporter constructs as analyzed by flow cytometry 118
3.2.3 Export of reporter constructs as analyzed by a cell surface biotinylation assay 120
3.2.4 Quantitative analysis of Galectin binding to cell surfaces using flow cytometry 122
3.2.5 Biochemical analysis of Galectin binding to counter receptors using lactose-coupled beads 124
3.3 Mutational analysis of the export-targeting motif in human Gal-1 126
3.3.1 Random mutagenesis of Gal-1 126
3.3.2 Site-directed mutagenesis 127
3.3.3 Characterization of Gal-1 mutants regarding export and binding to β-galactosides 128 Contents IV

4 DISCUSSION 176
4.1 Identification and characterization of CA125 as a Gal-1 counter receptor 177
4.2 Specificity of CA125 binding to Galectins 179
4.3 CA125 expression does not stimulate Gal-1 export 181
4.4 Analysis of Gal-1 and CGL-2 regarding export to the cell surface and binding to β-galactosides 182
4.5 Mutational analysis of the export targeting motif of Gal-1 and CGL-2 185
4.5.1 Galectin mutants deficient in binding to β-galactosides are also deficient in export from
CHO cells 187
4.5.2 Characterization of N- and C-terminal truncated forms of Gal-1 189
4.6 Detailed analysis of Gal-1-GFP 191 R112H
4.7 Potential models for the unconventional secretion of Gal-1 192
4.8 Future perspectives 196
REFERENCES 198
ACKNOWLEDGEMENT 221 Abbreviation 1

Abbreviation

Abbreviation
ABC ATP binding cassette
APC allophycocyanin
Ac Acetate
APS ammonium peroxo disulphate
ARF ADP-ribosylation factor 1
ATP adenosin triphosphate
BFA brefeldin A
bp basepairs
cDNA Complementary DNA
CDB cell dissociation buffer
CHO Chinese hamster ovary (cells)
CRD carbohydrate recognition domain
COP Coat proteine
C-terminal carboxy terminal
DMSO dimethyl sulphoxide
DNA desoxyribonucleic acid
E.coli Escherichia coli
e.g. exempli gratia
ECL enhanced chemoluminescence
ECM Extracellular matrix
EDTA ethylenediaminetetraacetic acid
En2 engrailed 2
ER endoplasmatic reticulum
et al. et altera
FACS fluorescence activated cell sorting
FCS fetal calf serum
FGF2 fibroblast growth factor 2
FGFR fibroblast growth factor receptor