Requirements for cleavage and function of the transmembrane chemokine fractalkine/CX3CL1 [Elektronische Ressource] / vorgelegt von Michael Andrzejewski

Andrzejewski

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

“Requirements for cleavage and function of the
transmembrane chemokine fractalkine/CX3CL1”




Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der RWTH Aachen University zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigte Dissertation




vorgelegt von
Diplom-Biologe
Michael Andrzejewski
aus
Torun, Polen



Berichter:
Professor Dr. rer. nat. Andreas Ludwig
Universitätsprofessor Dr. rer. nat. Lothar Elling

Tag der mündlichen Prüfung: 21.12.2009


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. The results of this work were in part published in:


Schulte A, Schulz B, Andrzejewski MG, Hundhausen C, Mletzko S, Achilles J, Reiss K,
Paliga K, Weber C, John SR, Ludwig A.
Sequential processing of the transmembrane chemokines CX3CL1 and CXCL16 by
alpha- and gamma-secretases.
Biochem Biophys Res Commun. 2007 Jun 22;358(1):233-40.


Michael G. Andrzejewski, Franz M. Hess and Andreas Ludwig.
Proteolytic shedding of the transmembrane Chemokine CX3CL1 involves multiple
structural determinants.
Wiener klinische Wochenschrift, ISSN 1434-6869, SpringerWienNewYork, Vol. 120,
Supplement 1, 2008, p. 181

Michael G. Andrzejewski, Franz M. Hess and Andreas Ludwig.
The intracellular C-terminus of CX3CL1 contributes to trafficking and shedding of the
chemokine, but is not required for cell adhesion.
European Journal of Immunology, ISSN 0014-280, Vol. 39, No. S1, 2009, p. 646
























Table of contents I
TABLE OF CONTENTS

1 INTRODUCTION .....................................................................................................1
1.1 Mechanisms of inflammation ...............................................................................1
1.2 Chemokines and their receptors............................................................................4
1.2.1 Classification and structure of chemokines .....................................................4
1.2.2 Structure and signal transduction of chemokine receptors ..............................5
1.3 Transmembrane chemokines ................................................................................6
1.3.1 The transmembrane chemokine CX3CL1 .......................................................6
1.3.2 The function of CX3CL1-CX3CR1 interaction...............................................8
1.4 Proteolytic cleavage by ADAM proteins..............................................................9
1.4.1 The family of ADAM proteins ........................................................................9
1.4.2 ADAM10, ADAM17 and their substrates in inflammatory processes..........11
1.4.3 Shedding of CX3CL1 by ADAM10 and ADAM17 ......................................12
1.5 Issues and aims ...................................................................................................14
2 MATERIALS AND METHODS............................................................................15
2.1 Chemicals and reagents ......................................................................................15
2.2 Molecular biology...............................................................................................15
2.2.1 RNA isolation and synthesis of cDNA ..........................................................15
2.2.2 Polymerase chain reaction .............................................................................16
2.2.3 Agarose gel electrophoresis and DNA-purification.......................................18
2.2.4 Topo-cloning reaction....................................................................................18
2.2.5 Digestion, ligation and precipitation of plasmid DNA ..................................19
2.2.6 Transformation of competent E. coli cells.....................................................19
2.2.7 Small and large scale isolation of plasmid DNA...........................................20
2.2.8 DNA sequencing............................................................................................21
2.3 Cell culture..........................................................................................................21
2.3.1 Cell lines ........................................................................................................21
2.3.2 Isolation of peripheral blood mononuclear cells and neutrophils..................22
2.3.3 Transient transfection of mammalian cell lines.............................................23
2.3.4 Generation of stable cell lines........................................................................23
2.3.5 Generation of lentiviral derived shRNA HEK293 knockdown system.........23
2.3.6 Detection of the surface expression by flow cytometry.................................24
II Table of contents

2.3.7 Immunofluorescence ..................................................................................... 26
2.3.8 Adhesion assay.............................................................................................. 27
2.3.9 Transmigration assay 28
2.4 Protein chemistry................................................................................................ 29
2.4.1 Preparation of conditioned cell medium and cell lysates.............................. 29
2.4.2 Enzyme-linked Immuno-sorbant Assay (ELISA) ......................................... 30
2.4.3 The β-glucoronidase assay ............................................................................ 31
2.4.4 Shedding assay 31
2.4.5 Tandem protein purification.......................................................................... 32
2.4.6 Deglycosylation............................................................................................. 33
2.4.7 SDS-PAGE.................................................................................................... 34
2.4.8 Western blotting ............................................................................................ 35
2.5 Data illustrations and statistical analysis............................................................ 36
3 RESULTS ................................................................................................................ 38
3.1 Structural characterisation of mouse CX3CL1 .................................................. 38
3.1.1 Shedding of mouse CX3CL1 ........................................................................ 38
3.1.2 Structural determinants for shedding of mouse CX3CL1............................. 41
3.1.2.1 Cleavage sites for human CX3CL1 ............................................................ 41
3.1.2.2 Substitution of the cleavage region............................................................. 43
3.1.2.3 Substitution of the N-terminal region ......................................................... 45
3.1.2.4 Substitution and removal of the transmembrane and the C-terminal ............
region .......................................................................................................... 46
3.1.3 Generation and characterisation of HEK293 cells stably expressing wt and
truncated mCX3CL1 ........................................................................................ 55
3.2 The role of mouse CX3CL1 in adhesion and transmigration............................. 61
3.2.1 Adhesion of mouse CX3CL1 ........................................................................ 61
3.2.2 Transmigration of mouse CX3CL1............................................................... 62
4 DISCUSSION 66
4.1 Characterisation of mouse CX3CL1 .................................................................. 66
4.2 Analysis the shedding and trafficking of mouse CX3CL1 ................................ 68
4.3 The cytoplasmic tail in the function of m ................................... 73
5 CONCLUSIONS AND OUTLOOK...................................................................... 76
6 SUMMARY ............................................................................................................. 81
Table of contents III
7 REFERENCES ........................................................................................................83
8 ABBREVIATIONS..................................................................................................93
9 LIST OF FIGURES.................................................................................................97
10 SUPPLEMENTS......................................................................................................99
DECLARATION ...............................................................................................................108
ACKNOWLEDGEMENTS ..............................................................................................109
CURRICULUM VITAE ...................................................................................................110

Introduction 1
1 1 INTRODUCTION


1.1 Mechanisms of inflammation

The mamma