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Analysis of coding microsatellite mutations in MSI colorectal carcinomas and characterization of their effects on the cellular glycosylation machinery [Elektronische Ressource] / vorgelegt von Nina Röckel

139 pages
INAUGURAL - DISSERTATION zur Erlangung der Doktorwürde der Naturwissenschaftlich-Mathematischen Gesamtfakultät der Ruprecht - Karls - Universität Heidelberg vorgelegt von Diplom-Biologin Nina Röckel aus Zweibrücken Tag der mündlichen Prüfung: ................................................ ANALYSIS OF CODING MICROSATELLITE MUTATIONS IN MSI COLORECTAL CARCINOMAS AND CHARACTERIZATION OF THEIR EFFECTS ON THE CELLULAR GLYCOSYLATION MACHINERY Gutachter: Prof. Dr. Bernhard Dobberstein Prof. Dr. Jürgen Kopitz Meinen Eltern Table of contents I. LIST OF FIGURES VI II. LIST OF TABLES VIII 1. ABSTRACT 1 1.1 Zusammenfassung 2 2. INTRODUCTION 4 2.1 Colorectal Cancer 2.2 Molecular Pathogenesis of MSI and CIN 2.3 Microsatellite Instability (MSI) in Colorectal Cancer 6 2.3.1 Molecular background of MSI tumors 2.3.2 Clinicopathological characteristics of MSI tumors 8 2.4 Eucaryotic glycosylation 9 2.4.1 N-glycosylation and the secretory pathway 10 2.4.2 O-glycosylation 14 2.4.3 Glycosaminoglycans: Components of Proteoglycans 15 2.4.4 How, where and why are proteins glycosylated? 18 2.5 Glycans involved in tumorigenesis 20 2.5.1 N-glycans and cancer 2.5.2 O-glycans and cancer 21 2.5.3 Proteoglycans and cancer 22 2.5.4 Glycans and MSI CRC 23 2.
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INAUGURAL - DISSERTATION
zur
Erlangung der Doktorwürde
der
Naturwissenschaftlich-Mathematischen Gesamtfakultät
der
Ruprecht - Karls - Universität
Heidelberg


















vorgelegt von Diplom-Biologin
Nina Röckel
aus Zweibrücken


Tag der mündlichen Prüfung: ................................................


ANALYSIS OF CODING MICROSATELLITE MUTATIONS IN
MSI COLORECTAL CARCINOMAS AND
CHARACTERIZATION OF THEIR EFFECTS ON THE
CELLULAR GLYCOSYLATION MACHINERY
































Gutachter: Prof. Dr. Bernhard Dobberstein
Prof. Dr. Jürgen Kopitz
















Meinen Eltern















Table of contents

I. LIST OF FIGURES VI
II. LIST OF TABLES VIII
1. ABSTRACT 1
1.1 Zusammenfassung 2
2. INTRODUCTION 4
2.1 Colorectal Cancer
2.2 Molecular Pathogenesis of MSI and CIN
2.3 Microsatellite Instability (MSI) in Colorectal Cancer 6
2.3.1 Molecular background of MSI tumors
2.3.2 Clinicopathological characteristics of MSI tumors 8
2.4 Eucaryotic glycosylation 9
2.4.1 N-glycosylation and the secretory pathway 10
2.4.2 O-glycosylation 14
2.4.3 Glycosaminoglycans: Components of Proteoglycans 15
2.4.4 How, where and why are proteins glycosylated? 18
2.5 Glycans involved in tumorigenesis 20
2.5.1 N-glycans and cancer
2.5.2 O-glycans and cancer 21
2.5.3 Proteoglycans and cancer 22
2.5.4 Glycans and MSI CRC 23
2.6 Aims of the work 25
3. RESULTS 27
3.1 Glycogenes involved in MSI-H colorectal tumorigenesis 27
I Table of contents

3.2 LMAN1 mutational inactivation in MSI-H colorectal
tumorigenesis 31
3.2.1 LMAN1 transcript is present, but LMAN1 protein is not
detectable in biallelic-mutated cells 31
3.2.2 Complete or partial loss of LMAN1 protein expression
in MSI-H CRC tumors 33
3.3 LMAN1 expression changes the phenotype in MSI-H CRC cells 36
3.3.1 Stable LMAN1 expression does not change cell
viability and growth 36
3.3.2 LMAN1 expression changes the glycan profile
on the cell surface 38
3.4 Analyses of LMAN1 cargo proteins relevant in MSI-H CRC 41
3.4.1 Alpha-1-antitrypsin secretion is impaired in
LMAN1-deficient cells 41
3.4.2 Protein retention in the ER/Golgi fraction changes with
LMAN1 expression 43
3.4.3 The LMAN1 carbohydrate domain shows high affinity
for LMAN1 substrates 45
3.5 XYLT2 loss of function mutation in MSI-H colorectal
tumorigenesis 50
3.5.1 Variation of XYLT2 and XYLT1 expression within
MSI-H CRC cell lines
3.5.2 XYLT2 reconstitution changes xylose incorporation in
XYLT2 deficient MSI-H CRC cell lines 51
4. DISCUSSION 54
4.1 Glycogenes show high mutation frequencies in MSI-H CRC 54
II Table of contents

4.2 Genetic alterations in LMAN1 might play a role in MSI
colorectal tumorigenesis 55
4.2.1 LMAN1 transporter function in the cell
4.2.2 LMAN1 involvement in MSI-H tumorigenesis 56
4.2.3 The screening for new LMAN1 cargo proteins 61
4.2.4 LMAN1 re-expression changes the cell surface
glycoprotein pattern 65
4.3 Consequences of XYLT2 mutations in MSI-H colorectal tumors 68
4.4 Candidate genes with lower mutation frequency or
no mutations in their cMNRs 71
4.5 Conclusions and Perspectives 72
5. MATERIALS 74
5.1 Instruments
5.2 Consumables, reagents and chemicals 75
5.3 Commercially available kits 77
5.4 Enzymes, bacteria, antibodies, markers and vectors 77
5.5 Oligonucleotides 78
5.6 Buffers 81
6. METHODS 83
6.1 Molecular Biology methods 83
6.1.1 Isolation of genomic DNA and RNA
6.1.2 Oligonucleotide design
6.1.3 Standard Polymerase Chain Reaction (PCR) 83
6.1.4 Sequencing 84
III Table of contents

6.1.5 cMNR Frameshift mutation analysis 85
6.1.6 Reverse transcription PCR
6.1.7 Restriction digest 86
6.1.8 Cloning of DNA fragments
6.2 Biochemical methods 90
6.2.1 Protein concentration
6.2.2 Polyacrylamide Gelelectrophoresis 91
6.2.3 Western blot analysis 92
6.2.4 Silver staining of polyacrylamide gels
6.2.5 2D-gel electrophoresis 93
6.2.6 Protein expression and purification 94
6.2.7 Alpha-1-antitrypsin ELISA 97
6.2.8 ER-Golgi Fractionation 98
6.3 Radioactive labelling
6.3.1 Liquid scintillation counting (LSC)
1256.3.2 LMAN1 binding to I −A1AT
36.3.3 Metabolic labelling with H–Xylose 99
6.3.4 Pulse Chase Experiment 100
6.4 Cell Culture experiments 101
6.4.1 Human cancer cell lines
6.4.2 Cell culture
6.4.3 Transfection methods
6.4.4 Cell Proliferation Assay 103
6.4.5 Lectin-FACS analysis

IV Table of contents

6.5 Human tissues 104
6.5.1 Immunohistochemistry
6.5.2 Microdissection of Hemalaun- and Eosin-stained tissues 105
6.5 Database analysis 105
7. REFERENCES 107
8. APPENDIX 125
8.1 Abbrevations
8.2 Own publications, presentations and posters 126
9. ACKNOWLEDGEMENTS 128

V Table of contents

I. LIST OF FIGURES
Figure 2.1 Characteristics of the two major pathways in colorectal cancer. 5
Figure 2.2 Role of the MMR system in maintaining microsatellite length. 7
Figure 2.3 Overview of glycan functions. 10
Figure 2.4 Model for the quality control of glycoprotein folding. 11
Figure 2.5 Major N-glycans present in mammals. 13
Figure 2.6 Biosynthesis and structure of O-GalNAc structures. 15
Figure 2.7 Structure of disaccharide repeat units in Glycosaminoglycans. 16
Figure 2.8 N- and O-glycans involved in tumor progression. 21
Figure 2.9 Proteoglycans involved in tumor angiogenesis. 23
Figure 3.1 Strategy for the identification of candidate genes and the
statistical model for the predicition of MSI-H target genes. 28
Figure 3.2 Frameshift mutation analysis of candidate glycogenes in MSI-H
colorectal cancer cell lines. 29
Figure 3.3 Frameshift mutation frequencies of a subset of candidate
glycogenes in primary MSI-H colorectal cancers. 30
Figure 3.4 LMAN1 expression in MSI-H colorectal cancer cell lines. 32
Figure 3.5 Immunohistochemical detection of LMAN1 protein in MSI-H
colorectal tumors. 33
Figure 3.6 Molecular analysis of intratumoral LMAN1-deficient areas. 34
Figure 3.7 Western blot analysis of stably transfected LoVo cells
constitutively expressing LMAN1 protein. 36
VI Table of contents

Figure 3.8 Cell Proliferation Assay of stably transfected LoVo cells. 37
Figure 3.9 FACS histograms and Box & Whisker Plots for lectins PHA-L
and JAC. 40
Figure 3.10 A1AT secretion in LMAN1-proficient and LMAN1-deficient
cell lines. 42
Figure 3.11 RT-PCR analysis of A1AT. 43
Figure 3.12 Pulse Chase Experiment with parental LoVo cells and LMAN1-
transfected LoVo cells. 44
Figure 3.13 Full length LMAN1 purification using mannose-sepharose. 47
Figure 3.14 LMAN1-CRD purification using mannose- and A1AT-sepharose. 49
Figure 3.15 XYLT enzyme activity during proteoglycan synthesis. 50
Figure 3.16 XYLT1 and XYLT2 expression in colorectal cancer cell lines
and control cells. 51
3Figure 3.17 Metabolic H-xylose labelling in MSI-H colorectal
cancer cell lines. 52
3Figure 3.18 Metabolic H-xylose labelling in HDC9 cells upon XYLT2 re-
expression. 53
Figure 4.1 LMAN1 transporter function in the cell. 55
Figure 4.2 XYLT metabolism in mammals. 68
Figure 6.1 LMAN1 constructs. 86
Figure 6.2 Radioactive binding assay for verification of functional
LMAN1 binding. 98

VII

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