Investigation on the interaction of dipeptidyl peptidase IV with the transactivator of transcription protein of Human Immunodeficiency Virus Type-1 [Elektronische Ressource] / presented by Felista Lemnyui Tansi
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Investigation on the interaction of dipeptidyl peptidase IV with the transactivator of transcription protein of Human Immunodeficiency Virus Type-1 [Elektronische Ressource] / presented by Felista Lemnyui Tansi

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Published 01 January 2010
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Investigation on the Interaction of Dipeptidyl Peptidase IV
with the Transactivator of Transcription Protein of Human
Immunodeficiency Virus Type-1




Dissertation to achieve the academic degree of
Doctor of Natural Sciences (Dr. rer. nat.)

Submitted to the
Department of Biology, Chemistry and Pharmacy
of the
Freie-Universität Berlin




Presented by
Felista Lemnyui Tansi
from Cameroon
2010


Charité-Universitätsmedizin Berlin
Campus Benjamin Franklin
Institute of Biochemistry and Molecular biology
Arnimallee 22
14195, Berlin-Dahlem



This work was performed under the supervision of Priv. Doz. Dr. Hua Fan
The experimental work was performed from April 2005 to December 2009 in the
Institute of Biochemistry and Molecular biology (AG Reutter / Fan) in Berlin


Part of the work has already been published:
Felista L. Tansi, Véronique Blanchard, Markus Berger, Rudolf Tauber, Werner
Reutter and Hua Fan (2010): Interaction of human dipeptidyl peptidase IV and
human immunodeficiency virus type-1 transcription transactivator in Sf9 cells.
Virology J 7, 267



First Reviewer: Priv. Doz. Dr. Hua Fan
Second Reviewer: Prof. Dr. Rupert Mutzel

Disputation: 11-11-2010 Declaration

I hereby declare that this thesis is the result of my original work carried out at the Institute of
Biochemistry and Molecular biology of the Charité-Universitätsmedizin in Berlin-Dahlem,
Germany. The research was an independent study under the supervision of Priv. Doz. Dr. Hua
Fan. This thesis has never been submitted in part or in whole for a degree at any institution.
References to other sources or people’s work have been duly cited and acknowledged.



Signed by author:
Felista Lemnyui Tansi………………………………………………………………………….


















“Dignity consists not in possessing honours, but in the
consciousness that we deserve them“


Aristotle








































Dedicated

to

my parents, Juliana and Henry
Thanks for not only giving me life, but also making me realise the sense of
being alive!

and to

my late aunt Angeline and her two daughters, my cousins Emma and
Apolonia
who all died suddenly within 6 weeks.
The shock of learning you left this world together, took my breath away.
Thanks to your love and encouragements while alive, I could pick up from
where you left me and become strong enough to complete the work you so
much wanted me to.
May your souls find perfect peace!
Table of contents | i

Table of Contents
1 Introduction ............................................................................................................................ 1
1.1 The Prolyl Oligo-Peptidase family............................................................................................ 1
1.2 Dipeptidyl Peptidase IV (DPPIV) ............................................................................................. 3
1.2.1 Occurrence and distribution of DPPIV ............................................................... 3
1.2.2 Structure of DPPIV ................................................................................................ 4
1.2.3 Biological functions of DPPIV .............................................................................. 7
1.2.3.1 DPPIV as a serine protease ............................................................................................. 7
1.2.3.2 DPPIV in the immune system....................................................................................... 10
1.2.3.2.1 DPPIV as a co-stimulator in T cell activation .................................................. 11
1.2.3.2.2 DPPIV as a regulator of chemokine function ................................................... 12
1.2.4 DPPIV interaction and binding partners........................................................... 14
1.2.4.1 Adenosine deaminase (ADA) ....................................................................................... 14
1.2.4.2 Collagen and Fibronectin .............................................................................................. 15
1.2.4.3 Plasminogen type-2 ...................................................................................................... 16
+ +1.2.4.4 Kidney Na /H ion exchanger 3 (NHE3) ..................................................................... 18
1.2.4.5 The C-X-C Chemokine Receptor 4 (CXCR4) .............................................................. 18
1.2.4.6 CD45 ............................................................................................................................. 19
1.2.4.7 Mannose-6-phosphate / insulin-like growth factor II receptor ..................................... 20
1.2.4.8 Mannose-binding protein (MBP) .................................................................................. 21
1.2.4.9 Caveolin-1 ..................................................................................................................... 22
1.2.4.10 CARMA1 ...................................................................................................................... 23
1.2.5 DPPIV and Diseases ............................................................................................. 25
1.2.5.1 DPPIV in Diabetes Mellitus Type-2 ............................................................................. 26
1.2.5.2 DPPIV in HIV infection and AIDS .............................................................................. 30
1.2.5.2.1 The HIV1 transactivator of transcription (HIV1-TAT) ................................... 32
1.2.5.2.2 Biological roles and effects of HIV1-TAT on its host ..................................... 34
2 Aim of Work ......................................................................................................................... 38
3 Results I: Expression, Purification and Characterization of TAT protein .................... 39
3.1 Expression and Purification of recombinant TAT proteins in E. coli................................. 39
3.1.1 Purification of GST-TAT-His protein ................................................................ 40
Table of contents | ii

3.1.2 Purification of GST-TAT protein ....................................................................... 42
3.1.3 Purification of His-TAT-His fusion protein....................................................... 43
3.1.4 Purification of TAT protein without fusion tags ............................................... 43
3.1.5 Purification of TAT fusion protein with reagents that prevent protein
aggregation ........................................................................................................................... 45
3.2 Expression and purification of TATGFP in stably transfected CHO cells ........................ 46
3.3 Expression and purification of TAT protein in Sf9 cells ...................................................... 48
3.3.1 Cloning, preparation and analysis of TAT-recombinant baculovirus ............ 48
3.3.2 Expression of recombinant TAT protein in Sf9 cells ........................................ 49
3.3.3 Purification of Sf9-TAT and TATV5His protein from Sf9 cells ...................... 50
Summary I: Expression and purification of recombinant TAT protein......................... 51
3.4 Characterization of purified recombinant TAT protein ...................................................... 52
The purified recombinant TAT protein have low endotoxin levels ................................................... 52
3.4.1 Evaluation of the biological activity of purified recombinant TAT protein ... 52
3.4.1.1 The purified recombinant TAT-protein transactivate the viral LTR promoter ............ 53
3.4.1.2 Purified TAT protein influences CXCR4-localisation in stably transfected cell
lines 54
3.4.1.2.1 Analysis of CXCR4GFP expression in CHO and Hek293 cell lines ............... 54
3.4.1.2.2 Purified TAT protein causes vesicular accumulation of CXCR4GFP ............. 55
3.4.2 Effects of recombinant TAT protein on binding and inhibition of human-
DPPIV 56
3.4.2.1 Recombinant TAT did not bind DPPIV in pull-down, immunoprecipitation or
SPR 56
3.4.2.2 Recombinant TAT retards the cleavage of a chromogenic substrate by DPPIV .......... 56
3.4.2.3 Recombinant TAT retards the cleavage of natural substrates by DPPIV ..................... 58
3.4.2.3.1 Recombinant Sf9-TAT, TAT10xHis and His-TAT-His retard cleavage of
GLP1 by human-DPPIV .................................................................................................... 60
3.4.3 The HIV1-TAT reveal properties typical of intrinsically unstructured proteins
62
3.4.3.1 Gel-filtration spectra reveal an intrinsically unstructured nature of TAT protein ........ 62
3.4.3.2 Electron micrographs of recombinant TAT reveal a mixture of oligomeric
structures 64
Table of contents | iii

Summary II: characterization of purified recombinant TAT protein ............................ 69
4 Results II: Interaction of TAT and DPPIV in CHO and Sf9 cells ................................... 70
4.1 Co-expression of TAT and DPPIV in CHO cells leads to DNA-fragmentation ................. 70
4.2 Co-expression of TAT and DPPIV in CHO cells leads to membrane inversion ................ 72
Summary III: TAT induces apoptosis in CHO-DPPIV cell lines .................................... 74
4.3 Interaction of HIV1-TAT and human-DPPIV in Sf9 cells ................................................... 75
4.3.1 The level of human-DPPIV is increased upon co-expression with HIV1-TAT75
4.3.2 Co-expression of TAT and DPPIV alters the cell surface expression of DPPIV in
Sf9 cells 77
4.3.3 HIV1-TAT co-localizes with human-DPPIV in co-infected Sf9 cells .............. 79
4.3.4 Human-DPPIV and HIV1-TAT co-immunoprecipitate from co-infected Sf9
cells 81
4.3.5 Serine-phosphorylation of TAT in Sf9 cells is reduced due to co-expression with
DPPIV 82
4.3.6 The HIV1-TAT protein induces tyrosine-phosphorylation of human-DPPIV84
4.3.7 Human-DPPIV protein expressed in Sf9 cells is O-glycosylated ..................... 87
4.4 Purification and characterization of TAT/DPPIV protein complex Sf9 cells .................... 88
4.4.1 Purification by immuno-affinity chromatography ........................................... 88
4.4.2 Purification by size-exclusion FPLC .................................................................. 88
4.4.3 The purified TAT/DPPIV protein retained TAT and DPPIV specific activities90
4.4.4 Purified TAT/DPPIV protein revealed a heterogeneous distribution on 2D gels
92
4.4.5 Crystallization screen of the purified TAT/DPPIV protein complex .............. 93
Summary IV: DPPIV and TAT associate in co-infected Sf9 cells ................................... 94
5 Results III: Subproject Interaction of DPPIV and Caveolin-1 ........................................ 95
5.1 Amplification of Caveolin-1 cDNA from different human organs ...................................... 95
5.2 Expression and purification of GST-tagged Cav1 protein ................................................... 96
5.2.1 Purification of GSTCav1 protein by affinity chromatography ....................... 96
5.2.2 Purification of GSTCav1 protein by SE-FPLC ................................................. 97
5.3 GSTCav1 binds to human-DPPIV in immunoprecipitation test ......................................... 98
5.4 GSTCav1 binds to DPPIV directly in SPR tests ................................................................... 99
6 Discussion ............................................................................................................................ 101
Table of contents | iv

6.1 Purification of recombinant HIV1-TAT proteins ............................................................... 101
6.2 HIV1-TAT protein reveal properties of intrinsically unstructured proteins ................... 102
6.3 HIV1-TAT inhibits the proteolytic cleavage of GLP1 by human-DPPIV ........................ 104
6.4 HIV1-TAT protein induced apoptosis in CHO cells in a DPPIV-dependent manner ..... 105
6.5 Co-expression of human-DPPIV and HIV1-TAT in Sf9 insect cells ................................. 106
6.5.1 Co-expression of human-DPPIV and HIV1-TAT leads to an increase in
membrane and total DPPIV protein in Sf9 cells ............................................................. 106
6.5.2 Human-DPPIV and HIV1-TAT associate in Sf9 cells .................................... 107
6.5.3 HIV1-TAT protein induced tyrosine-phosphorylation of DPPIV in Sf9 cells109
6.5.4 Human-DPPIV protein expressed in Sf9 cells is o-glycosylated .................... 110
6.5.5 Purification of HIV1-TAT in complex with DPPIV and crystallization screen
111
6.6 Cloning, expression and purification of Caveolin-1 ............................................................ 113
6.6.1 Interaction of DPPIV and Caveolin-1 .............................................................. 114
7 Summary ............................................................................................................................. 115
7.1 Zusammenfassung .................................................................................................................. 117
7.2 Future prospects ............................................................................Error! Bookmark not defined.
8 Materials ............................................................................................................................. 119
8.1 Vectors..................................................................................................................................... 119
8.2 Cell lines .................................................................................................................................. 119
8.2.1 Bacteria cell lines ................................................................................................ 119
8.2.2 Insect cell lines .................................................................................................... 119
8.3 Media and cell Culture .......................................................................................................... 119
8.3.1 Media for Bacteria culture ................................................................................ 119
8.3.2 Media for Insect cell culture ............................................................................. 120
8.4 Mammalian cell culture ......................................................................................................... 121
8.4.1 Media and reagents for cell culture .................................................................. 121
8.4.2 Mammalian cell lines and respective culture media ....................................... 121
8.5 Primer and oligonucleotides used for PCR and Sequencing.............................................. 121
8.6 Kits, Marker and Enzymes ................................................................................................... 122
8.6.1 Kits....................................................................................................................... 122
Table of contents | v

8.6.2 Marker ................................................................................................................ 122
8.6.3 Enzymes .............................................................................................................. 122
8.7 Antibodies ............................................................................................................................... 123
8.8 Synthetic peptides .................................................................................................................. 123
8.9 Solutions for the purification and analysis of nucleic acids and proteins ......................... 123
8.9.1 Solutions for small scale purification of plasmids from Bacteria .................. 123
8.9.2 Solutions for the electrophoretic analysis of nucleic acids ............................. 123
8.9.3 Solutions for the purification and analysis of proteins ................................... 124
8.9.3.1 Buffers for cell lysis and solubilisation of Protein ..................................................... 124
8.9.3.2 Solutions and buffers for SDS PAGE and western blot ............................................. 125
8.10 Chemicals and other materials ............................................................................................. 126
8.11 Instruments and Apparatuses ............................................................................................... 126
9 Methods ............................................................................................................................... 128
9.1 Molecular biology methods ................................................................................................... 128
9.1.1 Cloning and preparation of plasmids for protein expression ........................ 128
9.1.1.1 Cloning of human Caveolin-1 in pGEX4T-2 vector .................................................. 128
9.1.1.2 Cloning of TAT in pFastBac1 vector ......................................................................... 128
9.1.1.3 Cloning of TAT in pGEX4T-3, pRSET-B, pQE-60 and pEGFP-N1 ......................... 129
9.1.1.4 Cloning of CXCR4 in pFastBac1 and pEGFP-N1 ..................................................... 131
9.1.2 Plasmid preparation .......................................................................................... 131
9.1.3 Determination of DNA concentration .............................................................. 132
9.1.4 Enzymatic modification of DNA with restriction endonuclease .................... 132
9.1.5 Dephosphorylation of DNA ............................................................................... 132
9.1.6 Ligation of DNA fragments ............................................................................... 132
9.1.7 Polymerase Chain Reaction (PCR) .................................................................. 133
9.1.7.1 Reverse Transcription-Polymerase Chain Reaction (RT- PCR) ................................ 134
9.1.8 DNA Sequencing ................................................................................................ 135
9.1.9 Agarose gel electrophoresis ............................................................................... 135
9.1.10 Extraction of DNA from agarose gels by gel elution ....................................... 136
9.1.11 Preparation of competent E. coli cells .............................................................. 136
9.1.12 Transformation of competent E. coli cells with plasmid DNA ...................... 137
9.1.13 Expression of recombinant protein in E. coli .................................................. 137