Influence of single amino acid polymorphisms on the in vitro convertibility of goat prion protein [Elektronische Ressource] / vorgelegt von Elizabeth Ortega-Soto

Influence of single amino acid polymorphisms on the in vitro convertibility of goat prion protein [Elektronische Ressource] / vorgelegt von Elizabeth Ortega-Soto

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Influence of single amino acid polymorphisms on the in vitro convertibility of goat prion protein I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Elizabeth Ortega-Soto geboren am 24.04.1980 in Zacatlán Puebla, Mexiko Insel Riems, Juli 2010 Dekan: Prof. Dr. Klaus Fesser 1. Gutachter: Prof. Dr. Dr. h. c. Thomas Mettenleiter 2. Gutachter: Prof. Dr. Detlev Riesner Tag der Promotion: 23. 09. 2010 Table of Contents 1. Objectives of the work ___________________________________________________ 1 2. Introduction ___________________________________________________________ 3 2.1 Prio n diseases ___________________________________________________________ 3 2.1.1 Prion diseases in animals ________________________________________________________ 3 2.1.1.1 Scrapie in sheep and goats __________________________________________________ 3 2.1.1.2 Bovine Spongiform Encephalopathy in cattle ___________________________________ 4 2.1.1.3 Other prion diseases in animals ______________________________________________ 5 2.1.2 Prion diseases in humans ________________________________________________________ 6 2.1.2.

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Influence of single amino acid polymorphisms on
the in vitro convertibility of goat prion protein


I n a u g u r a l d i s s e r t a t i o n

zur
Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
an der Mathematisch-Naturwissenschaftlichen Fakultät
der
Ernst-Moritz-Arndt-Universität Greifswald


vorgelegt von
Elizabeth Ortega-Soto

geboren am 24.04.1980
in Zacatlán Puebla, Mexiko

Insel Riems, Juli 2010

































Dekan: Prof. Dr. Klaus Fesser


1. Gutachter:
Prof. Dr. Dr. h. c. Thomas Mettenleiter

2. Gutachter:
Prof. Dr. Detlev Riesner


Tag der Promotion:
23. 09. 2010

Table of Contents


1. Objectives of the work ___________________________________________________ 1
2. Introduction ___________________________________________________________ 3
2.1 Prio n diseases ___________________________________________________________ 3
2.1.1 Prion diseases in animals ________________________________________________________ 3
2.1.1.1 Scrapie in sheep and goats __________________________________________________ 3
2.1.1.2 Bovine Spongiform Encephalopathy in cattle ___________________________________ 4
2.1.1.3 Other prion diseases in animals ______________________________________________ 5
2.1.2 Prion diseases in humans ________________________________________________________ 6
2.1.2.1 Creutzfeldt-Jakob Disease (CJD) _____________________________________________ 6
2.1.2.2 Kuru ___________________________________________________________________ 6
2.1.2.3 Gerstmann-Sträussler-Scheinker disease (GSS) and Fatal Familiar Insomnia (FFI) ______ 7
2.2 The Prion Hypothesis: Two different isoforms of one protein ____________________ 7
C2.2.1 PrP ________________________________________________________________________ 7
C2.2.1.1 Structural characteristics of PrP _____________________________________________ 7
C2.2.1.2 PrP fun c t i on ____________________________________________________________ 9
Sc2.2.2 Characteristics of PrP ________________________________________________________ 10
Sc2.2. 3 PrP as the causal agent of TSEs ________________________________________________ 11
Sc2.2.4 Replication of PrP ___________________________________________________________ 12
2.3 Genetics in TSE ________________________________________________________ 13
2.3.1 Influence of mutations of human PrP gene in prion diseases ___________________________ 14
2.3.2 Polymorphisms in sheep that modulate the susceptibility to scrapie ______________________ 14
2.3.3 PRNP polymorphism in goats ___________________________________________________ 16
Sc2.4 P r P propagation models ________________________________________________ 19
3. Materials and methods __________________________________________________ 22
3.1 M at e r i al _______________________________________________________________ 22
3.1.1 Bacteria, cells and plasmids ____________________________________________________ 22
3.1.2 Primers for PCR _____________________________________________________________ 22
3.1.3 E n z y m e s ___________________________________________________________________ 24
3.1.3.1 DNA modification enzymes _______________________________________________ 24
3.1.3.2 Protein modification enzymes ______________________________________________ 24
3.1.4 Antibodies __________________________________________________________________ 24
3.1.5 Prion strains and isolates _______________________________________________________ 25
3.1.5.1 Mouse-passaged strains ___________________________________________________ 25
3.1.5.2 Isolates ________________________________________________________________ 25
3.2 Methods _______________________________________________________________ 26
3.2.1 Molecular biological methods ___________________________________________________ 26
3.2.1.1 Insertion of point mutations by PCR _________________________________________ 26
3.2.1.2 Digestion of DNA with restriction enzymes ___________________________________ 27
3.2.1.3 Preparation of the vector for ligation _________________________________________ 27 Page I
3.2.1.4 Ligation of DNA fragments ________________________________________________ 28
3.2.1.5 Production of competent cells ______________________________________________ 28
3.2.1.6 T r a n s for m a t i on of E. coli __________________________________________________ 29
C3.2.1.7 Selection of constructs carrying the DNA of interest (PrP from goat) _______________ 29
3.2.1.8 Preparation of plasmid DNA from a bacterial culture ____________________________ 29
3.2.2 Biochemical methods _________________________________________________________ 30
C3.2.2.1 Expression of PrP in E . c ol i M15 ___________________________________________ 30
C3.2.2.2 Purification of the bacterial PrP ____________________________________________ 30
3.2.2.3 SDS polyacrylamide gel electrophoresis (SDS-PAGE) ___________________________ 30
3.2.2.4 Western blot (immunoblot) ________________________________________________ 31
3.2.2.5 Protein quantification (method by Roth) ______________________________________ 31
3.2.2.6 CD-Spectra ____________________________________________________________ 31
3.2.3 Cell culture _________________________________________________________________ 32
3.2.3.1 N2a cell culture _________________________________________________________ 32
3.2.3.2 Freezing and thawing cells ________________________________________________ 32
3.2.3.3 Transfection of N2a cells __________________________________________________ 32
3.2.4 Fluorescent activated cell sorting (FACS)__________________________________________ 33
3.2.5 Deglycosylation of glycoproteins ________________________________________________ 33
3.2.5.1 Cell homogenates________________________________________________________ 33
C3.2.5.2 Deglycosylation of PrP __________________________________________________ 33
3.2.5.3 Phospatidyl-inositol Phospholipase C release __________________________________ 34
3.2.6 Experimental part of the work ___________________________________________________ 34
C3.2.6.1 Biotinylation of recombinant PrP ___________________________________________ 34
Sc3.2.6.2 Isolation of PrP from different sources ______________________________________ 35
C Sc 3.2.6.3 Cell-fr e e c on ve r s i on of P r P into PrP with different strains ______________________ 35
3.2.6.4 Generation of plasmids MoPrP.Xho N146S and N146D __________________________ 36
C C3.2.6.5 Construction of pcDNA3.1Zeo-PrP plasmids for expression of caprine PrP in N2a cells 37
4. Results _______________________________________________________________ 39
C4.1 Prokaryotic expression of recombinant PrP ________________________________ 39
4.1.1 Cloning of PRNP haplotypes ___________________________________________________ 40
4.1.2 Expression and purification of recombinant prion proteins_____________________________ 42
C4.2 Characterization of recombinant PrP _____________________________________ 43
C4.2.1 Antigenic characterization of PrP _______________________________________________ 43
C4.2.2 CD-Spectra of PrP ___________________________________________________________ 44
Sc4.3 Characterization of PrP is o l at e s __________________________________________ 44
C Sc4.4 In vitro conversion of PrP into PrP using different strains ___________________ 47
4.4.1 Mouse-passaged strains ________________________________________________________ 47
4.4.1.1 Cell-free conversion with Me7 _____________________________________________ 47
4.4.1.2 Cell-free conversion with BSE/Bl6 __________________________________________ 56
4.4.1.3 Biotinylation of recombinant prion protein ____________________________________ 58
4.4.2 Scrapie isolates ______________________________________________________________ 63
4.4.3 BSE isolates ________________________________________________________________ 66
4.5 Dominant negative effect _________________________________________________ 69
4.6 Generation of transgenic mice carrying N146D and N146S polymorphisms _______ 70
C4.7 Expression of PrP i n N 2 a ________________________________________________ 71
4.7.1 Cloning of PRNP in pBluescript 3.1 zeo ___________________________________________ 71 Page II
C4.7.2 Expression of PrP in N2a cells _________________________________________________ 72
C4.7.3 Expression of PrP in cell membranes ____________________________________________ 73
C4.7.4 G l yc os yl a t i on of P r P in cell culture ______________________________________________ 73
5. Discussion ____________________________________________________________ 75
C5.1 Effect of caprine polymorphisms on the in vitro conversion of PrP _____________ 75
5.1.1 Cell free conversion using mouse adapted Me7 scrapie prions __________________________ 76
5.1.2 Cell-free conversion using mouse-adapted BSE prions _______________________________ 78
5.1.3 Cell-free conversion with biotinylated PrP haplotypes with scrapie and BSE isolates ________ 79
C5.1.4 Dominant negative effect of resistant haplotypes on the conversion of PrP _______________ 81
C5.2 Molecular mechanisms of PrP conversion and the influence of PrP polymorphisms 82
C5.2.1 Characterisation of eucaryotically expressed PrP ___________________________________ 84
5.2.2 Generation of transgenic mice ___________________________________________________ 85
5.3 Conclusion ____________________________________________________________ 85
5.4 Perspectives ___________________________________________________________ 86
6. Abstract ______________________________________________________________ 87
7. References ____________________________________________________________ 89
8. Appendix ____________________________________________________________ 100
8.1 Amino acid alignment of prion proteins from selected mammal species _________ 100
C8.2 Relative cell free conversion rates of caprine PrP variants by Me7 prions ______ 101
8.3 Relative conversion rates obtained in the dominant negative effect experiment ___ 102
8.4 Buffers and fluids used in this studies _____________________________________ 103
8.4.1 Molecular biology solutions ____________________________________________________103
8.4.2 Buffers for biochemical methods ________________________________________________104
8.4.2.1 Protein expression and purification __________________________________________104
Sc8.4.3 PrP pur i f i c a t i on _____________________________________________________________105
8.4.4 IVC buffers and solutions ______________________________________________________106
8.4.4.1 SDS-PAGE and Western blot buffers ________________________________________107
8.4.5 Cell culture _________________________________________________________________109
8.4.5.1 C e l l gr ow ______________________________________________________________109
8.4.5.2 Lysis buffers and glycosidase digestion. ______________________________________110
8.5 Instruments and laboratory equipment ____________________________________ 111
8.6 Reactives _____________________________________________________________ 112
8.6.1 Chemicals __________________________________________________________________112
8.6.2 DNA markers _______________________________________________________________114
8.6.3 P r ot e i n markers ______________________________________________________________114
Sc8.7 Safety regulations of PrP agent _________________________________________ 115



Page III
Figure Index
CFigure 1: Schematic representation of PrP . ..........................................................................8
ScFigure 2: Model proposed for PrP . ..................................................................................... 10
Figure 3: Mutagenic PCR in two steps. ................................................................................. 27
Figure 4: Vector pQE40. ....................................................................................................... 28
Figure 5: Immunological detection in the cell-fr e e co nver s io n. ............................................. 36
Figure 6: Obtaining of MoPrP.Xho-INRQ and N146D. ........................................................ 37
Figure 7: pcDNA3.1/Zeo (+) vector. ..................................................................................... 38
CFigure 8: Position of different polymorphisms of caprine PrP . ............................................ 40
Figure 9: Generation of caprine prion variants via two-step PCR mutagenesis. ..................... 42
CFigure 10: Recombinant caprine PrP after expression in E. coli M15. ................................. 42
CFigure 11: Western blot of recombinant PrP s with different antibodies. .............................. 43
CFigure 12: CD-Spectra of different mutants of PrP . ............................................................. 44
Figure 13: Antigenic characterization of scrapie and BSE isolates. ....................................... 45
Figure 14: Triplot diagram from glycosylation pattern of used TSE strains. .......................... 46
Figure 15: Cell-free conversion of the prion protein (haplotype INRQ) after incubation with
Me7. ..................................................................................................................................... 47
Figure 16: Cell-free conversion of M112T after incubation with Me7. .................................. 48
Figure 17: Conversion of M137I with Me7. .......................................................................... 49
Figure 18: Conversion of L141F with Me7. .......................................................................... 49
Figure 19: Cell-free conversion of haplotype I142M after incubation with Me7. ................... 50
resFigure 20: Conversion of H143R into PrP after incubation with Me7. ................................ 50
Figure 21: Inhibition of cell-free conversion of N146S after incubation with Me7. ............... 51
Figure 22: Inhibition of cell-free conversion of N146D after incubation with Me7. ............... 51
Figure 23: Cell-free conversion of R151H with Me7............................................................. 52
Figure 24: Conversion of R211Q with Me7. ......................................................................... 52
Figure 25: Inhibition of conversion of Q215R with Me7. ...................................................... 53
Figure 26: Inhibition of cell-free conversion of Q222K after incubation with Me7. ............... 54
Figure 27: Inhibition of cell-free conversion of M142K222, R143K222 and Q211K222 after
incubation with Me7. ............................................................................................................ 54
Figure 28: Relative conversion rates of caprine polymorphisms with Me7. ........................... 55
resFigure 29: Conversion of INRQ into PrP with BSE/Bl6 and Me7. ..................................... 57
C resFigure 30: Effect of caprine polymorphism in the conversion of PrP into PrP w i t h
BSE/Bl6. .............................................................................................................................. 57
CFigure 31: Biotin-tagged PrP . .............................................................................................. 59
CFigure 32: Ce ll-free conversion of biotinylated PrP (INRQ-bio) after incubation with Me7. 60
C Figure 33: Cell-free conversion of biotinylated PrP harbouring different polymorphisms after
incubation with Me7. ............................................................................................................ 61
Figure 34: Conversion of biotinylated PrP (INRQ) with BSE/Bl6. ........................................ 61
Figure 35: Cell-free conversion of biotinylated PrP haplotypes with Me7 and BSE/Bl6. ....... 62
ScFigure 36: Conversion of the biotin tagged PrP (INRQ) with ovine PrP . ............................ 64
Figure 37: Ce ll-free conversion of biotinylated PrP haplotypes with ovine scrapie seeds. ..... 64 Page IV
ScFigure 38: Cell-free conversion of biotinylated PrP (INRQ) with caprine PrP .................... 65
Figure 39: Cell-free conversion of biotinylated PrP haplotypes with caprine scrapie seeds .... 65
Figure 40: Cell-free conversion of INRQ-bio with BSE isolates from cattle and sheep.......... 66
Figure 41: Cell-free conversion of caprine polymorphisms after incubation with ovine BSE. 67
Figure 42: Cell-free conversion of 8 different biotinylated caprine polymorphisms by 6
d iffer e nt T SE st r ains. ............................................................................................................ 68
Figure 43: Dominant negative effect of N146S, N146D and Q222K in the conversion of
INRQ. .................................................................................................................................. 69
Figure 44: Generation of MoPrP.Xho-N146S and MoPrP.Xho-N146D. ................................ 70
Figure 45: Generation of pcDNA-N146S and pcDNA-N146D. ............................................. 71
CFigure 46: Stable expression of PrP (full length) in N2a cells. ............................................. 72
CFigure 47: Expression of PrP in the cellular membrane of transfected N2a cells. ................. 73
CFigure 48: Analysis of the PrP glycosylation. ...................................................................... 74



Table Index

CTable 1: Susceptibility against classical scrapie of different genotypes of ovine PrP ............ 15
CTable 2: PrP polymorphisms in goats .................................................................................. 16
Table 3: Silent mutations in goat PRNP ................................................................................ 18
Table 4: Oligonucleotides used in the first-step of mutagenic PCR ....................................... 23
Table 5: Constructs generated and expressed in E. coli ......................................................... 39
Table 6: Length of DNA products after first-step PCR mutagenesis ...................................... 41
Sc ScTable 7: Molecular weight of the unglycosylated PrP fragments and the PrP glycoform
proportion ............................................................................................................................. 46
CTable 8: Effect of different caprine PrP polymorphisms in the in vitro co nver s io n w it h Me7
............................................................................................................................................. 56
Table 9: Semi-quantitative analysis of cell-free conversion of caprine polymorphisms with
BSE/Bl6 and Me7 ................................................................................................................. 58
Table 10: Semi-quantitative determination of the influence of biotinylation of different
Ccaprine variants in the cell-free conversion of PrP with Me7 and BSE/Bl6 .......................... 63
Table 11: Semi-quantitative analysis of cell-free conversion of biotinylated PrP haplotypes
with BSE/Bl6, Me7, ovine scrapie, caprine scrapie, BSE, ovine BSE and caprine BSE ......... 68
CTable 12: Correlation of the effect of different caprine PrP polymorphisms with in vivo
observations ......................................................................................................................... 78

Page V
Abbreviations

Am ino acids
A Alanine
C C yst e ine
D Aspartic acid
E Glutamic acid
F Phenylalanine
G Glycine
H Histidine
I Isoleucine
K Lysine
L Leucine
M Methionine
N Asparagine
P Proline
Q Glutamine
R Arginine
S S er ine
T Threonine
V Valine
W Tryptophan
Y T y r o s i n e


Used in this work
PrP Prion protein
CPrP Cellular prion protein
ScPrP Scr a p i e -associated Prion Protein
resPrP Proteinase K resistant Prion Protein
AH Q Sheep PrP A H Q 136 151 171
AR H Sheep PrP A R H 136 151 171
AR Q Sheep PrP A R Q (wild-t y p e ) 136 151 171
VRQ Sheep PrP V R Q 136 151 171
INRQ G o at P r P I N R Q (wild-type) 142 146 211 222
IVC In vitro co nver s io n or cell-free conversion
PK Proteinase K
Ab Ant ibo d y
ma b Monoclonal antibody
pab P o lyc lo na l a nt ibo d y
wt Wild-t y p e
rec Recombinant
Page VI