Cellular Trafficking of the Pathogenic Prion Protein PrP
Scand
Phenotypic Characterisation of Deletion Mutants in the Hydrophobic Domain of the Normal Prion Protein PrP
CDissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)
des Fachbereichs Biologie der Universität Konstanz
vorgelegt von Nathalie Monika Veith
April 2008
Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/5772/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-57723
Tag der mündlichen Prüfung: 10.07.2008
Referenten: Prof. A. Bürkle (Gutachter und Prüfer)
Prof. E. Ferrando-May (Gutachterin und Prüferin) Prof. C. Stürmer (Prüferin)
Ich versichere hiermit, dass ich die vorliegende Arbeit mit dem Titel “Cellular Trafficking of the Pathogenic Prion Protein PrPSc and Phenotypic Characterisation of Deletion Mutants in the Hydrophobic Domain of the Normal Prion Protein PrPC” selbstständig verfasst und keine anderen Hilfsmittel als die angegebenen verwendet habe. Die Stellen, die anderen Werken dem Wortlaut oder dem Sinn nach entnommen sind, habe ich in jedem einzelnen Falle durch Angaben der Quelle kenntlich gemacht.
Die Arbeit wird nach Abschluss des Prüfungsverfahrens der Universitätsbibliothek Konstanz übergeben und ist durch Einsicht und Ausleihe somit der Öffentlichkeit zugänglich.
Konstanz, den……….. ……….
Nathalie Monika Veith
Table of content
1 Introduction ... 10
1.1 Prions ... 10
1.2 Biosynthesis and trafficking of PrPC ... 10
1.3 Structure of PrP ... 11
1.4 Function of PrPC ... 12
1.5 Mechanisms of prion toxicity ... 14
1.6 Trafficking of PrPSc ... 15
1.7 PrPSc specific antibodies ... 17
1.8 The TM1 domain of the prion protein ... 20
1.9 Deletion mutants in the TM1 domain ... 22
2 Objectives of the study ... 24
3 Material ... 25
3.1 Antibiotics ... 25
3.2 Antibodies... 25
3.3 Bacteria ... 26
3.4 Cell lines ... 26
3.5 Chemicals ... 26
3.6 Enzymes ... 28
3.7 Fluorescent dyes ... 29
3.8 Kits ... 29
3.9 Loading buffers ... 29
3.10 Marker ... 29
3.11 Media ... 29
3.12 Oligonucleotides ... 30
3.13 Plasmids ... 30
3.14 Software ... 31
3.15 Solutions and buffers... 31
3.16 Technical equipment ... 33
4 Methods ... 35
4.1 Cell culture ... 35
4.1.1 Culture of eukaryotic cells ... 35
4.1.2 Cryopreservation of eukaryotic cells ... 35
4.1.4 Infection of H6 cells with scrapie strain 22L ... 36
4.2 Immunocytochemistry ... 36
4.2.1 Immunofluorescence staining ... 36
4.2.1.1 Coating of coverslips or lumox dishes with poly-L-lysine (PLL) ... 36
4.2.1.2 Staining of PrPSc ... 36
4.2.1.3 Induction and staining of lipid bodies (LBs)... 37
4.2.1.4 Concanavalin A (ConA) stimulation ... 37
4.2.1.5 Staining of PrPSc in combination with trafficking organelles ... 38
4.2.1.6 Assay for the detection of PrPSc at the plasma membrane ... 38
4.2.1.7 Cell ELISA ... 39
4.2.1.8 Modification of the cell ELISA ... 39
4.2.2 Immunostaining for electron microscopy ... 40
4.2.2.1 Staining of PrPC and trafficking organelles in H6-22L cells upon crosslinking with antibodies ... 40
4.2.2.2 Staining of PrPSc and trafficking organelles in H6-22L ... 40
4.2.2.3 Staining of PrPSc and trafficking organelles in H6-22L upon treatment with ConA ... 41
4.3 Working with lentiviruses ... 41
4.3.1 Transfection of HEK293T for production of recombinant lentiviruses ... 41
4.3.2 Concentration of lentiviruses with ultracentrifugation... 42
4.3.3 Transduction of H6-22L cells with recombinant lentiviruses ... 42
4.3.4 FACS analysis ... 42
4.3.5 Calculation of virus titres ... 43
4.4 Molecular biological methods ... 44
4.4.1 Restriction analysis ... 44
4.4.2 Gel elution ... 45
4.4.3 Blunting of DNA fragments ... 45
4.4.4 Dephosphorylation ... 45
4.4.5 Purification of reaction mixes ... 45
4.4.6 Ligation ... 45
4.4.7 PNGaseF digestion ... 46
4.5 Microbiological methods ... 46
4.5.1 Preparation of chemo competent bacteria ... 46
4.5.2 Transformation ... 46
4.5.3 Colony Screening ... 47
4.5.4 Preparation of purified plasmids ... 47
4.6 Protein analysis ... 47
4.6.1 Preparation of cell lysates for western blots ... 47
4.6.2 SDS-PAGE ... 48
4.6.3 Western blot ... 48
4.6.4 Isolation of lipid bodies (LBs) ... 49
5 Results ... 50
5.1 Part I: Localisation of PrPSc in scrapie infected neuroblastoma cells ... 50
5.1.1 Staining of PrPSc with cell ELISA ... 50
5.1.2 Staining with PrPSc specific antibodies 15B3 and V5B2 ... 50
5.1.3 Detection of PrPSc in H6-22L cells after GdnHCl denaturation by ... immunofluorescence ... 55
5.1.4 Localisation of PrPSc in scrapie infected H6-22L cells at the plasma membrane by immunofluorescence ... 58
5.1.5 Internalisation of PrP from the cell surface with Concanavalin A (ConA) ... 62
5.1.6 Colocalisation of PrPSc with endosomal and lysosomal markers... 65
5.1.7 Visualisation of PrPSc in combination with endoplasmatic reticulum and Golgi apparatus ... 72
5.1.8 Electron microscopy analysis of PrPC and PrPSc in scrapie infected H6-22L ... 73
5.1.9 PrPSc and lipid bodies (LBs) ... 80
5.1.10 Scrapie infected H6-22L cells displayed less PrPC than uninfected N2a cells ... 83
5.2 Part II: Phenotypic analysis of deletion mutants within the hydrophobic region of the prion protein ... 85
5.2.1 Cloning of deletion mutants into the transfer plasmid pWPT ... 85
5.2.2 Production and titration of recombinant lentiviruses... 88
5.2.3 Expression of PrP deletion mutants in eukaryotic cells ... 90
5.2.4 Conversion of deletion mutants in PrPSc ... 92
6 Discussion ... 95
6.1 Part I: Localisation of PrPSc in scrapie infected neuroblastoma cells ... 95
6.1.1 Antibodies 15B3 and V5B2 ... 95
6.1.2 Experimental setup of PrPSc staining ... 97
6.1.3 PrPSc at the plasma membrane and in clathrin coated pits ... 99
6.1.4 Internalisation of PrPSc... 102
6.1.5 PrPSc in early endosomes and late endosomes/lysosomes ... 104
6.1.6 Exocytosis of PrP ... 106
6.1.8 PrPC present in N2a and H6-22L cells ... 110
6.2 Part II: Phenotypic analysis of deletion mutants within the hydrophobic region of the prion protein ... 111
6.2.1 Characterisation of deletion mutants in the hydrophobic core of the prion protein . 111 6.2.2 Experimental setup for investigation of the convertibility of deletion mutants... 113
7 Conclusion and perspective... 115
8 Summary... 117
9 Zusammenfassung ... 119
10 Literature ... 122
11 Acknowledgments/Danksagung ... 136
Abbreviations
aa amino acid
Ab antibody
AP alkaline phosphatase
BCIP 5-bromo-4-chloro-4-indolyl phosphate-p-
toluidin salt
BSA bovine serum albumin
ConA Concanavalin A
CJD Creutzfeldt-Jakob-Disease
CTB cholera toxin subunit B
dαg donkey anti goat
DIC differential interference contrast
ECL enhanced chemiluminescence
EM electron microscopy
ER endoplasmatic reticulum
FA formaldehyde
FFI Fatal Familial Insomnia
GA glutaraldehyde
gαm goat anti mouse
gαr goat anti rabbit
gα goat anti
GdnHCl guanidine hydrochloride
GdnSCN guanidine isothiocyanate
GPI-anchor glycosylphosphatidylinositol anchor
GSS Gerstmann-Sträussler-Scheinker Syndrom
h hour
HRP horseradish peroxidase
ICC immunocytochemistry
IF immunofluorescence
IHC immunohistochemistry
IP immunoprecipitation
LB lipid body
LSM laser scanning microscopy
mAb monoclonal antibody
min minutes
MOI multiplicity of infection
NBT nitroblue tetrazolium
o/n overnight
pAb polyclonal antibody
PBS phosphate buffered saline
PFA paraformaldehyde
PIPLC phosphoinositide-specific phospholipase C
PLL poly-L-lysine
PK proteinase K
PMSF phenylmethylsulfonylfluoride
PrP prion protein
PrPC normal, cellular prion proteins
rα rabbit anti
ROS reactive oxygene species
RT room temperature
s seconds
SEM standard error of the mean
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
electrophoresis
TSE Transmissible Spongiform Encephalopathies
WB Western blot
w/o without
wt wild type
Introduction 10
1 Introduction
1.1 Prions
Prions are the agents of Transmissible Spongiform Encephalopathies (TSEs) [1]. These are transmissible neurodegenerative diseases such as Creutzfeldt-Jakob-disease (CJD) in humans, scrapie in sheep and Bovine Spongiform Encephalopathy (BSE) in cattle. The brain of affected animals and humans show spongiform changes, loss of neurons and gliosis [2-5].
The disease leads to progressive dementia, ataxia and finally death.
The TSE agent is an unconventional one because it is devoid of nucleic acids, but its infectivity could be reduced by procedures that hydrolyse or modify proteins [6, 7].
Therefore, the hypothesis arose that the infectious agent consists exclusively of a protein [8, 9], of a proteinaceous infectious particle, hence called prion [9].
The infectious protein is called PrPSc (scrapie-associated prion protein) [10]. PrPSc is an abnormal isoform of the endogenous, cellular prion protein, PrPC. PrPC is converted into PrPSc through a process whereby a portion of its α-helical structure is refolded into β-sheets [11]. In contrast to PrPC, PrPSc is insoluble in mild detergents. It displays a strong tendency to aggregate into amyloid fibrils and is resistant to proteases [12].
1.2 Biosynthesis and trafficking of PrPC
The prion protein PrPC, which is encoded on chromosome 2 (mouse) or chromosome 20 (human), is a highly conserved cell membrane protein in vertebrates [13]. It is mainly expressed in neurons and glial cells but also in several other tissues. The mouse prion protein consists of 254 amino acids (aa) and contains an N-terminal signal peptide for translocation into the endoplasmatic reticulum (ER) and a C-terminal signal sequence for attachment of a glycosylphosphatidylinositol (GPI)-anchor. In the course of biosynthesis in ER and Golgi-apparatus the protein is N-glycosylated at two asparagine sites (Asn 180 and 196), a disulfide bond is formed between Cys 178 and Cys 213 and the GPI-anchor is attached [14-16]. After cleavage of the signal peptide the prion protein comprises aa 23-231.
At the N-terminal part (aa 59-90) five glycine/proline rich octarepeats are located which could bind Cu2+. Then, a hydrophobic core (aa 111-134) follows which seems to be important for the conversion of PrPC to PrPSc (see chapter 1.8). With the C-terminal GPI-anchor the protein is attached at the cell membrane in regions rich in cholesterol and glycosphingolipids
(so-called “lipid rafts”) [17-19]. PrPC can be constitutively internalised. A major pathway for internalisation of PrPC appears to use clathrin mediated endocytosis [20] but also caveolin related endocytosis is reported [21]. Clathrin mediated internalisation preferably takes place in neurons whereas caveolin mediated endocytosis is reported to occur in CHO cells. A part of the PrPC pool is recycled back to the cell surface while the majority is transported through early and late endosomes before it enters lysosomes for degradation [20, 22-24]. Forms of exocytosis via lipid bodies (LBs) in Jurkat T cells [25] or exosomes [26] were also reported.
1.3 Structure of PrP
Most structural analysis of PrP was performed with NMR analysis of recombinant proteins [27, 28]. While the N-terminal region (23-121) is flexible and does show any secondary structure, the C-terminal part of the protein shows a globular domain of three α-helices (aa 144-154, 173-194, 200-228) and two β-sheets (aa 128-131, 161-164). With FTIR spectroscopy Pan et al. [11] could show that PrPC displays 42 % α-helices and 3 % β-sheets. PrPSc instead displays 30 % α-helices and 43 % β-sheets.
Figure 1: On the left the NMR structure of PrPC is depicted containing three α-helices and two short β-strands.
On the right the hypothetical structural model of PrPSc is shown with its high content of β-sheets [27].
But the structure of PrPSc is quite difficult to resolve, because of its insolubility, tendency to form aggregates and pure isolation from brain extracts or cells. With electron crystallography it could be shown that helix 2 and 3 remain intact in PrPSc but the N-terminal half up to aa 160 is arranged as parallel β-sheets [29]. As PrPSc forms amyloid fibrils, a trimeric or hexameric arrangement of right- or left-handed β-helical models was proposed.
Spectroscopically, β-sheets and β-helices cannot be differentiated, so that the new model would not contradict earlier spectroscopic studies [30]. In 2004, Govaerts et al. preferred the trimeric model, where residues 89-175 were arranged in a left-handed parallel β-helical
Introduction 12
conformation, while the C-terminus (aa 176-227) retained the disulfide-linked α-helical conformation observed in the normal cellular isoform [31].
Figure 2: Domain swapped β-helical trimeric model of PrP 27-30 [31].
The stability of this trimer could be achieved by intermolecular interactions (domain- swapped trimeric prion model) [32]. This is not the final description of the structure of PrPSc but it is the best model currently available and takes into account both electron microscopic and spectroscopic data as well as the intermolecular stabilisation of the PrPSc structure [30].
In this β-helical model helix 1 had to be converted into β-sheets. But Watzlawik and co- workers found strong evidence that helix 1 was not converted into β-sheet during formation of PrPSc [33].
1.4 Function of PrPC
The function of PrPC is still under debate. Prnp-/- mice develop normal and do not show a neuropathological phenotype [34]. They are resistant to scrapie infection [35, 36]. Only minor changes have been reported like changes in circadian rhythm and sleep pattern [37].
Furthermore, electrophysiological and structural abnormalities in the hippocampus [38-42]
and alterations of intracellular calcium homeostasis [43] are notified. Prnp-/- mice are more susceptible to oxidative stress damages and less viable in culture as wild type cells [44-48]. In other Prnp-/- mouse lines, in which the prion-like protein Doppel (Dpl) is ectopically expressed in the brain as a result of an intergenic splicing event between the adjacent PrP and Dpl gene, progressive ataxia and loss of cerebellar Purkinje cells is reported [49-53].
Since PrPC is a GPI-anchored protein, the entire polypeptide chain is located on the extracytoplasmatic face of the lipid bilayer. So it stands to reason that PrPC could participate
to transmembrane signalling processes [54-58]. PrPC would need to interact with transmembrane adaptor proteins in order to transmit signals into the cytoplasm.
Antibody-mediated crosslinking is found to stimulate the activity of the non-receptor tyrosine kinase fyn in 1C11 cells [55]. This requires a direct interaction with the raft protein caveolin. The antibody-induced fyn activation leads to downstream stimulation of NADPH oxidase and extracellular-regulated kinases (ERKs) as well as production of reactive oxygen species (ROS) [56].The activities of several G protein-coupled serotonin receptors found on the surface are also altered by PrP crosslinking [59].The signalling pathway caused by antibody crosslinking are postulated to be pro-survival. Stuermer et al. (2004) showed that crosslinking of PrPC in Jurkat T cells cause cell signalling events. Due to antibody crosslinking and capping PrPC co-clusters with Thy-1, TCR/CD3, fyn, lck and LAT. The consequence is an increase of tyrosine and MAP kinases ERK 1/2 phosphorylation, actin polymerisation and an increase of Ca2+ concentration [58]. In other reports a potential role for the PI3 kinase/Akt signalling pathway had been suggested for the neuroprotective effect of PrPC [60, 61].
Another pro-survival signalling cascade involves the interaction of PrPC with stress-inducible protein 1 (STI-1) [62-64]. Incubation of hippocampal neurons with recombinant STI-1 stimulates neurite outgrowth in a PrP-dependent manner, an effect requiring signalling through a mitogen-activated protein kinase (MAPK) pathway [64].
Neurite outgrowth is also an effect of interaction of PrPC with neuronal cell adhesion molecule N-CAM [65]. As N-CAM interacts with PrPC in hippocampal neurons, it is also possible that PrPC acts as a direct or indirect cell adhesion molecule upon interaction with N- CAM [66] and/or laminin [67]. In both cases the consequence is neurite outgrowth of cells.
Furthermore, retraction of neurons is caused by laser ablation of cell surface PrP [67].
Adhesion in N2a cells are reported to be PrP-dependent [68].
Several lines of evidence suggest that PrPC may play a role in protecting cells from oxidative stress [69]. The most compelling observation in this direction is that neurons cultured from Prnp-/- mice are more sensitive to ROS than neurons from wild-type mice [44, 45, 70]. PrPC itself can act to detoxify ROS. PrPC is said to possess a copper dependent superoxide dismutase (SOD) activity [45, 71, 72]. However, several publications argue against this hypothesis [73, 74]. Jones et al. (2005) failed to find SOD activity of PrPC above background level. It is more likely that PrPC acts indirectly to protect cells from ROS by upregulating the activities of other proteins, such as Cu-Zn-SOD, catalase or glutathione reductase [45-47, 71].
Introduction 14
PrPC is also a copper-binding protein [75-78]. Micromolar concentrations of copper stimulate endocytosis of cell surface PrP via clathrin-coated pits [79-81]. Based on the effect of Cu2+ on PrP trafficking, the protein can serve as a receptor for cellular uptake or efflux of copper ions [80]. PrPC may play a role in regulating copper release at the synapse [82, 83].
PrPC is preferentially concentrated along axons and in pre-synaptic terminals [84-90] but is also a subject to anterograde and retrograde axonal transport [88, 91]. It is reported that PrPC plays a regulatory role in synapse formation and function [37, 39-41, 92-94].
Another cytoprotective aspect of PrPC can be seen in its anti-apoptotic activity. PrPC prevents Bax-mediated cell death in human fetal neurons [95-97], in HpL3-4 (Prnp-/- cells) [98, 99], in MCF-7 cells [100], in cultured cerebellar granule neurons [101] and in N2a cells [102]. On the contrary, PrP has been reported to increase the susceptibility to staurosporine induced apoptosis mediated through p53 [103-105].
In addition, a long list of putative functions for PrPC can be mentioned. But in this chapter only the main possible functions of PrPC are described according to Westergard (2007) and Zomosa-Signoret (2008) [106, 107].
1.5 Mechanisms of prion toxicity
A key area of controversy is the toxicity of the disease associated isoform PrPSc itself.
Although PrPSc is associated with the pathology and infectivity of prion diseases, the link between PrPSc and neurotoxicity is unclear.
One hypothesis indicates that PrPC possesses a function which is lost upon conversion into PrPSc and this might contribute to prion induced neurodegeneration (Figure 3B). However, a loss of function seems to be incompatible with the observation that Prnp-/- mice behave normally and do not display a neuropathological phenotype [34]. Another possibility is that PrPSc subverts or modifies the normal function of PrPC, rather than causing a complete loss of PrPC function (Figure 3C). In this model the neuroprotective function of PrPC could be altered into a neurotoxic signal upon interaction with PrPSc. This hypothesis is supported by the finding that Prnp-/- cells are not susceptible to the toxic effect of PrPSc [108, 109]. They are also resistant to apoptosis induced by the synthetic peptide PrP 105-125, which mimics PrPSc [110]. Scrapie-infected mice expressing GPI-anchorless PrPC only show minimal neurological pathology indicating that PrPC has to be located at the plasma membrane for inducing toxicity [111]. PrPSc might produce this effect by crosslinking of cell surface PrPC,
which has been shown to induce apoptosis of CNS neurons in vivo [112], or by binding to and blocking of specific functional domains of PrPC. These functional domains could be aa 105- 125, as mice expressing PrPC with a deletion of aa 105-125 develop a severe neurotoxic phenotype [113]. Also the neurotoxic phenotype of transgenic mice expressing PrPΔ32- 121/134 supports the idea that specific domains of PrP are essential for its neuroprotective function and deletion of such domains causes a neurotoxic effect perhaps by altering interaction with signalling proteins [114].
The gain-of-function hypothesis describes that PrPSc possesses a novel toxic property which is not related to the normal physiological function of PrPC (Figure 3A). PrPSc may block axonal transport, interfere with synaptic transmission or physically damage cellular membranes.
This hypothesis mainly describes the toxic mechanisms in Alzheimer´s, Huntington´s or Parkinson´s disease, but the toxic gain of function do not hold true for inherited prion diseases. In familial prion diseases a mutation in the prion protein does not effect the biochemical or thermodynamic nature of PrP and the formation of aggregates is minimal [115].
The three hypotheses are described according to Caughey (2006), Harris (2006) and Westergard (2007) [106, 116, 117].
Figure 3: Models for the toxicity of PrPSc. (A) Toxic gain of function mechanism. PrPSc has a novel neurotoxic activity that is independent of the normal function of PrPC. (B) Loss of function mechanism. PrPC has a normal, physiological activity, in this case neuroprotection that is lost upon conversion into PrPSc. (C) Subversion of function mechanism. The normal, neuroprotective function of PrPC is altered by binding to PrPSc [117].
1.6 Trafficking of PrPSc
The trafficking of PrPSc is far unknown. One major problem is that the conversion site and hence the formation site of PrPSc has not been identified yet. Several studies indicate that PrPSc is formed after PrPC has reached the plasma membrane [118-122]. Thus, the formation
Introduction 16
of PrPSc could take place either at the plasma membrane or immediately after its internalisation in the endo-/lysosomal compartment [24, 118, 119, 122]. It is shown that the synthesis of PrPSc is inhibited by blocking endocytosis and internalisation of PrPC [118].
Additionally, PrPSc is cleaved at its N-terminus by endogenous proteases in acidic compartments after its synthesis [122, 123]. Since PrPSc is not completely degraded in lysosomes, its conversion to a protease-resistant state must occur prior to its exposure to proteases within the endo-/lysosomes. Thus, it is likely that PrPSc is generated at the cell surface or along the endocytic pathway before exposure to proteases. Furthermore, the expression of a dominant-negative version of the GTPase Rab4a, which inhibits recycling to the plasma membrane, increases the production of PrPSc in infected cells. It has been proposed that PrPSc formation does not require cell surface recycling and occurs in an intracellular compartment [124]. There is also evidence that lipid rafts are the sites of conversion. PrPC could be found along with PrPSc in rafts [19, 21, 125-128]. When cholesterol was depleted, PrPC degradation slowed down and generation of PrPSc was reduced [19].
Sphingolipid depletion showed the opposite, namely PrPSc synthesis was increased [18]. Due to these reports one can assume that regulation of biosynthesis of PrPSc is caused by a cholesterol-dependent mechanism in rafts. Baron et al. showed that an insertion of PrPSc into membranes is a prerequisite to induce conversion of PrPC [126]. Recombinant transmembrane forms of PrPC, that are not directed to rafts, do not serve as substrates for PrPSc formation in scrapie infected Neuroblastoma cells [19, 121]. Pharmacological compounds, which disrupt rafts, inhibit PrPScsynthesis [19, 129, 130]. Thus, rafts have attracted attention as a candidate site for the generation of PrPSc.
In cell culture systems PrPSc localises in lysosomes [131], late endosomes [132, 133], cytosol [132, 134], at the plasma membrane [131, 132, 135], in exosomes [26, 136], seldom in the Golgi apparatus [134] and in the nucleus [137] and it could form aggresomes upon proteasome inhibition [132, 138]. In vivo, in mouse or human brain, PrPSc could be found in lysosomes [139-145], late endosomes [146] and at the plasma membrane [147].
Figure 4: Trafficking scheme for PrPC and PrPSc and possible pathways for PrPSc formation. PrPC (green dots) is synthesised at the ER, transits through the Golgi and reaches the plasma membrane. PrPC may undergo recycling or internalisation via clathrin coated pits or caveolae into early endosomes. PrPC is degraded in lysosomes. PrPSc (pink dots) could infect a new cell by conversion of PrPC either at the plasma membrane, in the endosome or in the lysosome. Upon proteasome inhibition PrPSc can be found in aggresomes. Large accumulations are also found in the cytosol inside the cell or in form of fibrils outside the cell.
1.7 PrPSc specific antibodies
An important tool for detection of PrPSc has yet been missing, a pathogen-specific antibody (Ab). Since PrPC and PrPSc share the same peptide sequence, it is difficult to generate an Ab which can distinguish between PrPC and PrPSc. Up to now this can be circumvented by hydrated autoclaving of brain tissues in citrate buffer followed by a treatment with concentrated formic acid before expose to prion protein specific Abs [145]. Moreover, in eukaryotic cells PrPSc could be treated with 6 M GdnHCl [134], 3 M GdnSCN [131] or 96 % formic acid [138] prior to staining . These procedures not only denature PrPSc to inactivate the infectious agent but uncover an Ab specific epitope that was buried before within the molecule [148].
The only difference known so far between PrPC and PrPSc lies in its biochemical and biophysical properties. PrPSc does not share the same secondary structure with PrPC. Thus, the host immune system´s ability to recognise PrPSc as foreign or pathological may be restricted to conformational epitopes. Additionally, the conformation and aggregation state
Introduction 18
of the PrPSc glycoprotein severely limits exposure of the polypeptide surface to Abs. These structural features and the high conservation of PrP peptide sequence in mammals have limited attempts to generate PrPSc specific Abs. Thus, years of intense efforts led to several reports of PrPSc specific Abs: 15B3 [149], Abs with tyrosine-tyrosine-arginine (YYR) motifs [150], V5B2 [151] and motif grafted Abs IgG 19-33 [152], IgG 98-112 and IgG 136-158 [153].
15B3, derived from Prnp-/- mice immunised with full-length recombinant bovine PrP, immunoprecipitates PrPSc from brain homogenates without PK digestion and denaturation it specifically recognises PrPSc in human, bovine and murine brain homogenates. Recently, it was observed that 15B3 precipitated both PrPSc and aggregated insoluble forms of PrP not necessarily being infectious [154]. These insoluble forms were produced partially by point mutations within the prion protein (P101L, D177N, E199K), by PG14, a prion protein with 9 octarepeats, by cytoplasmatic PrP, upon Cu2+ treatment and by purified recombinant PrP aggregates. In all cases 15B3 could precipitate the insoluble aggregate in brain homogenates and in cell culture. Ab 15B3 was reported to stain purified aggregates of recombinant PrP.
Another publication also revealed that 15B3 immunoprecipitated mutant PrP P101L [155].
15B3 is an immunglobuline of class M which are often designated to recognise the specific conformation of a molecule. This also holds true for 15B3. The 15B3 epitope consists of three distinct peptide sequences: 142-148, 162-170 and 214-226 (human sequence) which are distributed all over the prion protein sequence.
Figure 5: Mapping of the 15B3 epitope onto the NMR structure of PrPC. In yellow, epitope 1 with aa 142-248;
in violet, epitope 2 with aa 162-170 and in cyan, epitope 3 with aa 214-226 [149].
Induction of β-sheet structures in recombinant PrP is associated with increasing solvent accessibility of tyrosines which were hidden before within the protein. Conserved YYX motifs were conserved in human, sheep, mouse, hamster and bovine PrP. They could be found at amino acid residues 149-151, 162-164 and 225-227 (human sequence). Therefore, Paramithiotis et al. conclude that the increased solvent exposure of tyrosyl side chains in β-
sheet rich recombinant PrP might involve at least one bi-tyrosine motif. If recombinant β- sheet rich models share some structural features of PrPSc, antibody access to one or several YYR motifs may provide a PrPSc-selective conformational epitope. YYR Abs were derived from mice immunised with CYYRRYYRYY peptide or from rabbit immunised with CYYR peptide.
Positive IgM Abs (from mice, later transformed into chimeric IgG) and IgG (from rabbit) were tested in immunoprecipitation (IP), plate capture immunoassay and flow cytometry and recognise the pathological isoform of the prion protein PrPSc but not PrPC. But also recognision of misfolded PrP with YYR Abs could be observed like with 15B3 and motif grafted Abs [154].The epitopes of 15B3 and YYR partially overlap. YYR motifs could be found in the 15B3 epitope. The similarity of the epitopes may be the reason why both Abs are IgMs. Tyr repeats have been reported to define a dominant B-cell epitope [156], suggesting that the high IgM frequency within this region may be the result of a specific mouse immune response to YYR antigens since immunised rabbits with YYR repeats only reveal IgGs [150].
V5B2, raised against a human PrP sequence at 214-226, is IgG antibody and recognises PrPSc selectively without any pretreatment. Serbec et al. used V5B2 in dot blot, IP, ELISA and IHC of human brain tissues [151]. The mAb selectively detected PrPSc in sCJD and vCJD brain samples. For IHC, cryo sections of cerebellum were used immediately for staining, but also formaldehyde fixed, formic acid treated and paraffin embedded brain sections were used.
Cryo sections displayed staining of PrPSc plaques in sCJD cases, uninfected brain sections were not stained (Figure 6). But vCJD and sCJD brain samples fixed with formaldehyde and treated with formic acid, probably to reduce the infectivity of the samples and not the antigenecity, showed a more pronounced staining of PrPSc plaques. A 13-residue synthetic peptide (214-226 in human PrP sequence) was used for immunisation of mice. V5B2 favoured binding to oligomeric forms or to fibril like aggregates of the peptid in solution and to PrPSc aggregates itself [157]. Perhaps it acts like all other designated PrPSc specific Abs and recognises soluble PrP aggregates which do not have to be infectious.
Introduction 20
Figure 6: Staining of PrPSc in human brain with V5B2. (a) sCJD case with PrPSc plaques in the cerebellum, stained with V5B2. (b) Control brain stained with V5B2 shows no signs of staining [151].
For prion propagation a direct and specific interaction between PrPC and PrPSc is proposed.
PrPSc replications could be inhibited in cell culture and in vivo by certain PrP peptides, PrP specifc Abs or their Fab fragments [153, 158-162]. The inhibitory effect may be due to binding to distinct regions of PrPC that likely interact (in)directly with PrPSc and thus prevent assembly of the prion replicative complex. In another work the inhibitory PrP sequences/epitopes were grafted into a recipient Ab scaffold [152, 153]. Only three of them, namely IgG 19-33, IgG 89-112 and IgG 136-158 efficiently recognise mouse, human and hamster PrPSc, but not PrPC, in IP. As these Abs are designated to detect PrPSc exclusively they were also used for IHC in prion infected brain tissues [163] These finding suggests that residues 19-33, 89-112 and 136-158 within the prion protein are key components of the PrPC-PrPSc complex. They bind selectively PrPSc indicating that these three epitopes are hidden within the PrPC molecule. Upon conversion they are presented at the surface of PrPSc and could therefore react with motif grafted Abs. But is could recently be shown that these motif grafted Abs detect not only PrPSc but also aggregated insoluble PrP in brain tissue, in cell culture and in a purified form [154]. Since 15B3, Abs mit YYR motif and motif grafted Abs do not only recognise PrPSc but also insoluble PrP aggregates, the use of these Abs as PrPSc specific is limited. On the other hand the use of them is extended to other inherited prion diseases which do not necessarily develop infectivity and protease resistance of PrP. In both applications lack of PrPC staining is of great advantage.
1.8 The TM1 domain of the prion protein
Alternatively to the GPI-anchored version of the prion protein at the plasma membrane, two transmembrane versions have been described, whereby the highly conserved hydrophobic domain (aa 111-134) is spanning the membrane of the endoplasmatic reticulum [164]. The hydrophobic part is also called transmembrane domain (TM1). These transmembrane forms
of the prion protein are called NtmPrP or CtmPrP depending on whether the N-terminus or the C-terminus of PrP is located in the lumen of the ER. In terms of evolution, the TM1-domain is the most highly conserved region within PrP [165]. Consequently one can assume a very important role of the TM1 domain for the function of the prion protein. PrPC interacts with stress-inducible protein 1 (STI-1) through residues 113-129 of PrP leading to neuroprotection in hippocampal neurons through the MAPK pathway [64] (see chapter 1.4). The functional importance of the TM1 domain is confirmed by findings that mice transgenic for PrP lacking residues 32-121 or 32-134 (PrP∆32-121; PrP∆32-134) suffer from severe ataxia and neuronal death within 2-3 months after birth [166]. The neurodegenerative phenotype suggests that specific domains of PrP are essential for its neuroprotective function and that deletion unmask a neurotoxic activity consistant with the subversion of function theory (see chapter 1.5). Recent studies showed an even more dramatic phenotype in transgenic mice carrying a deletion in the central domain of the prion protein (PrP∆94-134) [167]. This phenotype was rescued by coexpression of full-length PrPC. Transgenic mice carrying the deletion PrP∆114- 121 showed no obvious phenotype, but the toxicity of PrP∆94-134 was enhanced by coexpression of PrP∆114-121 while the toxicity of PrP∆32-134 was diminished by coexpression of PrP∆114-121. Deletion of the entire central domain generates a strong recessive-negative mutant of PrPC, whereas removal of residues 114-121 creates a partial agonist with context-dependent action. Another work showed that transgenic mice with deletion of residues 105-125 in the prion protein develop spontaneously a severe neurodegenerative illness that was lethal within one week after birth [113]. This phenotype, too, was rescued in a dose dependent manner by coexpression of wild type PrP. In vitro, the peptide 105-125, which mimicks the toxicity of PrPSc, looses it neurotoxicity and β-sheet structure upon exchange of hydrophobic amino acids in the TM1 region into hydrophilic amino acids [168]. During the conversion of PrPC to PrPSc the region between amino acids 90- 120 seems to undergo a conformational change, because antibodies directed against this region readily recognise PrPC but not PrPSc [169]. Intriguingly, in vivo studies showed that such antibodies inhibit PrPSc accumulation [160]. Furthermore, peptides from the region 106-128 and 109-141 show in vitro an inhibition of the conversion of PrPC to PrPSc dependent on the central hydrophobic sequence AGAAAGA (aa 112-119). The more of the central hydrophobic domain is present in the peptide the higher is the inhibition of conversion [170]. Especially the presence of residues 119 and 120 were crucial for an efficient inhibitory
Introduction 22
effect. Thus, residues in the vicinity of aa 106-141 of PrP are critically involved in the intermolecular interactions that lead to PrPSc formation. But Féraude et al. (2005) described that inhibition of PrPSc formation by crosslinking of Abs to PrPC is more likely due to sequestration of PrPC from the cell surface and PrP trafficking events, because the epitopes lay in different regions suggesting a non-conformation dependent process [161].
1.9 Deletion mutants in the TM1 domain
Mutants of the prion protein are not converted into PrPSc when parts of the TM1 region were missing [171]. This effect was also shown for other deletion mutants where amino acids 114-121 [172] or amino acids 112-119 [173] were missing. In the presence of the deletion mutants (PrP∆112-119 and PrP∆114-121) a reduction of endogenous PrPSc formation in scrapie infected Neuroblastoma cells was observed [172]. The deletion mutants could not be converted into PrPSc and they inhibit the conversion of endogenous wild type PrPC into PrPSc. This effect is termed “dominant negative” regarding the PrPSc accumulation and highlights once again the crucial importance of the TM1 region, particularly the central hydrophobic domain, for prion infection.
The Bürkle group is interested in further investigation of the TM1 domain, especially of the mutant PrP∆114-121 (hence called 114Δ8). First, it was hypothesised that the dominant- negative character of 114Δ8 could be due to structural differences between wild type PrP (wt) and 114Δ8. Indeed, 114Δ8 shows an additional short antiparallel β-sheet which stabilises the protein and probably represents an energy barrier that prevents conversion of 114Δ8 into PrPSc [174].In another sudy, the different TM forms (NtmPrP, CtmPrP) in wt and 114Δ8 should be examined, because differences in membrane topology could shed light on the dominant-negative behaviour of 114Δ8 and on the different behaviour of wt and 114Δ8.
Here, also smaller deletion mutants within the TM1 domain were examined. Expression of
CtmPrP is linked to neurodegenaration [164, 175]. All mutations displayed reduced formation of TM topologies in comparison to wt. Additionally, 114Δ8 did not exhibit the α-cleavage product C1 [176]. The α-cleavage takes place during the normal metabolism of PrP. It is cleaved at amino acids 110/111 resulting in a small N1 fragment and the 17 kDa C1 fragment. The role of C1 in PrP metabolism is still unknown.
Furthermore, the putative differences in PrP functions were analysed. As PrPC is supposted to have neuroprotective functions, presumably by protection from ROS, the aim was to
elucidate a possible role of PrPC (wt and 114Δ8) in antioxidative defence in vitro. Mouse neuroblastoma cells (N2a), cells from whole brain of 114Δ8-transgenic (tg) mice (114Δ8-tg mice described in [167, 177]) and cerebellar granule neurons (CGN) isolated from 114Δ8-tg mice were examined for their intracellular ROS level. Upon overexpression of wt and 114Δ8 in N2a cells the ROS level is lowered at the same level in both cases. CGN and whole brain cells do not show any differences in ROS level comparing wt and 114Δ8. Thus, the antioxidant function is not altered by expression of 114Δ8. It is more likely that the protective effect is caused by the octarepeat region rather than its TM1 domain [178].
The dominant-negative character of 114Δ8 could also be shown in vivo. Mice tg for 114Δ8 were created [167, 177] and inoculated with RML scrapie strain. Prnp-/- mice carrying the 114Δ8 deletion were resistant to scrapie infection, while a prolonged survival was observed in wt mice carrying the deletion mutant 114Δ8 [177]. Currently, KO cell lines stable expressing PrP wt or 114Δ8 are created. These cell lines should be infected with the scrapie strain 22L to confirm that 114Δ8 is not convertible into PrPSc (Hanf, diploma thesis).
Objectives of the study 24
2 Objectives of the study
The aim of the project was twofold and therefore the results are divided into two parts. The first part deals with the trafficking and putative conversion sites of PrPSc and the second part elucidates the phenotypic character of deletion mutants in the TM1 domain.
Trafficking of the pathogenic prion protein PrPSc in scrapie infected neuroblastoma cells With light and electron microscopy PrPSc should be visualised in different compartments. In a first step, the appropriate method to stain PrPSc in scrapie infected neuroblastoma cells should be established. Since none of the so far available Abs distinguish between PrPC and PrPSc, several Abs thought to be PrPSc specifc should be used for immunofluorescence staining. Here, the mAbs 15B3 [149]and V5B2 [151] were tested for significant staining of PrPSc. Another possibility to stain PrPSc was after treatment with 6 M GdnHCl [134] either in combination with or without PK digestion. After establishment of a staining protocol for PrPSc in light and electron microscopy the trafficking process of PrPSc, which is largely unknown, should be investigated with special emphasis on the endosomal/lysosomal pathway by driving internalisation of PrPSc.
Phenotypic characterisation of deletion mutants in the hydrophobic domain (aa 114-121) of the prion protein PrPC
In view of the dominant negative effect of PrP∆114-121 shown by Hölscher et al. in 1998 [172], a set of additional, more subtle deletion mutations in this region (PrP∆114-115, PrP∆114-117, PrP∆114-119, PrP∆114-121, PrP∆116-119, PrP∆116-121 and PrP∆118-121) had been created in order to investigate their conversion into PrPSc in a scrapie infected cell culture system and to determine the minimal deletion size that would lead to a significant/maximal inhibition of conversion. Such quantitative characterisation should be performed using lentiviral gene transfer of the PrP deletion mutants into scrapie infected cell culture.
3 Material
3.1 Antibiotics
Description Source
Ampicillin, cat. no. A2804 Sigma-Aldrich, Deisenhofen, Germany Penicillin/Streptomycin, cat. no. 15140-122 Invitrogen, Karlsruhe, Germany 3.2 Antibodies
Description Source
6H4, monoclonal mouse anti prion protein, raised against human aa 144-152, cat. no.
01-010
kind gift of Dr. A. Räber, Prionics, Zürich, Switzerland
12F10, monoclonal mouse anti prion protein, raised against human aa 141-159
kind gift of Prof. Dr. W. Bodemer, German Primate Centre, Göttingen, Germany
15B3, mouse anti prion protein PrPSc, raised against full length bovine PrP
kind gift of Dr. A. Räber, Prionics, Zürich, Switzerland
Kan72, rabbit anti prion protein PrPC, raised against mouse aa 89-103
Prof. A. Bürkle, Molecular Toxicology, University of Konstanz [172]
V5B2, monoclonal mouse anti prion protein PrPSc, raised against human aa 214-226
kind gift from V. Curin Serbec, Blood Transfusion Center of Slovenia, Ljubljana, Slovenia
3F4, mouse anti prion protein PrPC, raised against human and hamster aa 108-111
Millipore, Schwalbach, Germany Clathrin HC, polyclonal goat anti Clathrin
heavy chain, cat. no. sc-6579
Santa Cruz,Heidelberg, Germany EEA1, polyclonal goat anti early endosome
antigen 1, cat. no. sc-6415
Santa Cruz,Heidelberg, Germany Limp2, polyclonal goat anti Limp2, marker of
late endosome/lysosome, cat. no. sc-25867
Santa Cruz, Heidelberg, Germany ERp29, polyclonal rabbit anti ERp29, marker
of the endoplasmatic reticulum,cat. no.
ab11420
Abcam, Cambridge, UK
GM130, monoclonal mouse anti GM130, a Golgi matrix protein, cat. no. 610822
BD Bioscience, Heidelberg, Germany mouse anti alpha Tubulin, cat. no. T5168 Sigma-Aldrich, Deisenhofen, Germany goat anti mouse A488 IgG (H+L), cat. no.
A11001
Invitrogen, Karlsruhe, Germany goat anti rabbit A488 IgG (H+L), cat. no.
A11070
Invitrogen, Karlsruhe, Germany goat anti mouse A546 IgG (H+L), cat. no.
A11030
Invitrogen, Karlsruhe, Germany goat anti rabbit A546 IgG (H+L), cat. no.
A11010
Invitrogen, Karlsruhe, Germany donkey anti goat Cy3 F(ab) specific, cat. no.
705-166-147
Jackson ImmunoResearch, Suffolk, UK goat anti mouse IgM Cy3 Jackson ImmunoResearch, Suffolk, UK goat anti mouse HRP, cat. no. P0447 Dako, Hamburg, Germany
Material 26
goat anti rabbit AP, cat. no. A3687 Sigma-Aldrich, Deisenhofen, Germany goat anti mouse AP, cat. no. A3562 Sigma-Aldrich, Deisenhofen, Germany goat anti mouse IgM AP, cat. no. sc-2978 Santa Cruz, Heidelberg, Germany
pA-Au5nm, Protein A gold conjugate 5 nm University of Utrecht, Dept. Cell Biology, School of Medicine, Utrecht, The Netherlands
pA-Au10 nm, Protein A gold conjugate 10 nm University of Utrecht, Dept. Cell Biology, School of Medicine, Utrecht, The Netherlands
goat anti mouse F(ab)2-Au5 nm BioTrend, Köln, Germany goat anti mouse F(ab)2-Au10 nm BioTrend, Köln, Germany 3.3 Bacteria
Description Source
Escherichia coli DH5α
Genotype F- Ф80 lacZΔM15 Δ(lacZYA-argF) U169 deoR recA1 endA1 hsdR17 (rk-
, mk+
) gal- phoA supE44 λ- thi-1 gyrA96 relA1 (Hanahan 1983)
Invitrogen, Karlsuhe, Germany
3.4 Cell lines
Cell line Desription
Hek 293T human epithelial kidney cells, transformed
by SV 40 T antigen, semi-adherent
N2a mouse neuroblastoma cells, adherent
H6 clone H6 of mouse neuroblastoma cells,
adherent, highly susceptible for scrapie infection [179]
H6-22L clone H6 of N2a cells, infected with scrapie
strain 22L 3.5 Chemicals
Description Source
2-mercaptoethanol Sigma-Aldrich, Deisenhofen, Germany
2-propanol Riedel-de-Häen, Seelze, Germany
4-nitroblue tetrazolium chloride (NBT) Boeringer, Mannheim, Germany 5-bromo-4-chloro-4-indolyl phosphate-p-
toluidin salt (BCIP)
Roth, Karlsruhe, Germany
Agarose (SeaKEM) Biozym, Hess. Oldendorf, Germany
Agarose beads (anti mouse IgM μ chain specific)
Sigma-Aldrich, Deisenhofen, Germany Ammoniumpersulfat (APS) Serva, Heidelberg, Germany
Aqua Poly Mount Polysciences, Eppelheim, Germany
Bacto-Agar Becton-Dickinson, Heidelberg, Germany
Bacto-Tryptone Becton-Dickinson, Heidelberg, Germany
Bacto-Yeast Becton-Dickinson, Heidelberg, Germany
BCA Reagent A Pierce, Bonn, Germany
BCA Reagent B Pierce, Bonn, Germany
Benzil Sigma-Aldrich, Deisenhofen, Germany
Boric acid Riedel-de-Häen, Seelze, Germany
Bovine Serum Albumine Sigma-Aldrich, Deisenhofen, Germany
Bromphenol blue Sigma-Aldrich, Deisenhofen, Germany
BSA-c BioTrend, Köln, Germany
Calcium chloride Merck, Darmstadt, Germany
CasyClean Schärfe System, Reutlingen, Germany
CasyTon Schärfe System, Reutlingen, Germany
Complete Protease Inhibitor Roche, Mannheim, Germany
Concanavalin A Sigma-Aldrich, Deisenhofen, Germany
Coomassie Brilliant Blue R250 Roth, Karlsruhe, Germany
Cytochalasin B Sigma-Aldrich, Deisenhofen, Germany
Deoxycholate disodium salt Sigma-Aldrich, Deisenhofen, Germany
Digitonin Sigma-Aldrich, Deisenhofen, Germany
Dimethylformaide (DMFA) Sigma-Aldrich, Deisenhofen, Germany Dimethylsulfoxide (DMSO) Sigma-Aldrich, Deisenhofen, Germany di-potassium hydrogenphosphate Riedel-de-Häen, Seelze, Germany di-sodium hydrogenphosphate Roth, Karlsruhe, Germany
Dithiothreitol (DTT) Sigma-Aldrich, Deisenhofen, Germany Desoxynucleotidetriphosphate (dNTPs) MBI-Fermentas, St. Leon-Rot, Germany
Ethanol 99.8 % Riedel-de-Häen, Seelze, Germany
Ethidiumbromide Sigma-Aldrich, Deisenhofen, Germany
Ethylene glycol tetraacetic acid (EGTA) Sigma-Aldrich, Deisenhofen, Germany Ethylenediamine-tetraacetic acid disodium
salt dihydrate (EDTA)
Roth, Karlsruhe, Germany
Fetal Calf Serum (FCS) Biochrom/Seromed, Berlin, Germany
Glucose Merck, Darmstadt. Germany
Glutaraldehyde Sigma-Aldrich, Deisenhofen, Germany
Glycerin Acros, Geel, Belgium
Glycin Roth, Karlsuhe, Germany
Guanidine hydrochloride Sigma-Aldrich, Deisenhofen, Germany
Guanidine thiocyanide Roth, Karlsruhe, Germany
HEPES Roth, Karlsruhe, Germany
Hoechst 33342 Invitrogen, Karlsruhe, Germany
Hydrochloric acid 37 % Riedel-de-Häen, Seelze, Germany
Hydrogen peroxide Merck, Darmstadt, Germany
Lipofectamine Invitrogen, Karlsruhe, Germany
L-Glutamine Invitrogen, Karlsruhe, Germany
LR-Gold London Resin, London, UK
Luminol Sigma-Aldrich, Deisenhofen, Germany
Magnesium chloride Riedel-de-Häen, Seelze, Germany
Methanol Riedel-de-Häen, Seelze, Germany
Milk powder Rapilait, Sulgen, Switzerland
MilliQ water Millipore, Schwalbach, Germany
Nitroblue tetrazolium (NBT) Sigma-Aldrich, Deisenhofen, Germany Nonidet P-40 (NP 40) Sigma-Aldrich, Deisenhofen, Germany
Material 28
Oleic acid Sigma-Aldrich, Deisenhofen, Germany
Paraformaldehyde Riedel-de-Häen, Seelze, Germany
p-coumaric acid Sigma-Aldrich, Deisenhofen, Germany
Percoll GE Healthcare, München, Germany
Phenylmethylsulfonylfluoride (PMSF) Sigma-Aldrich, Deisenhofen, Germany
PIPES Sigma-Aldrich, Deisenhofen, Germany
Poly-L-lysine hydrobromide (cat. no. P1399) Sigma-Aldrich, Deisenhofen, Germany
Ponceau S Roth, Karlsruhe, Germany
Potassium chloride Riedel-de-Häen, Seelze, Germany
Potassium dihydrogenphosphate Riedel-de-Häen, Seelze, Germany Rotiphorese Gel 30 (30 % acrylamide/0.8 %
bisacrylamide)
Roth, Karlsruhe, Germany
Saponin Sigma-Aldrich, Deisenhofen, Germany
Sarcosyl Sigma-Aldrich, Deisenhofen, Germany
Sodium acetate Merck, Darmstadt, Germany
Sodium azide Merck, Darmstadt, Germany
Sodium chloride Roth, Karlsruhe, Germany
Sodium dihydrogenphosphate Merck, Darmstadt, Germany
Sodium hydroxide Riedel-de-Häen, Seelze, Germany
Sodium hydrogencarbonate Riedel-de-Häen, Seelze, Germany Sodiumdodecylsulfate (SDS) Roth, Karlsruhe, Germany
Sucrose Merck, Darmstadt, Germany
Tetramethylethylenediamine (TEMED) Serva, Heidelberg, Germany Trichloracetic acid (TCA) Roth, Karlsruhe, Germany
Triton X-100 Sigma-Aldrich, Deisenhofen, Germany
Trizma base (Tris) Sigma-Aldrich, Deisenhofen, Germany
Trypan blue (0.4 %) Sigma-Aldrich, Deisenhofen, Germany Trypsin-EDTA solution 0.25 % Sigma-Aldrich, Deisenhofen, Germany
Tween 20 Sigma-Aldrich, Deisenhofen, Germany
3.6 Enzymes
Enzyme Source
DpnI, 10 U/µl New England Biolabs (NEB), Frankfurt am
Main, Germany
KpnI, 10 U/µl NEB, Frankfurt am Main, Germany
MluI, 10 U/µl MBI Fermentas, St. Leon-Rot, Germany
MspI, 10 U/µl MBI Fermentas, St. Leon-Rot, Germany
SacI, 10 U/µl MBI Fermentas, St. Leon-Rot, Germany
SalI, 10 U/µl MBI Fermentas, St. Leon-Rot, Germany
Calf Intestine Alkaline Phosphatase (CIP), 10 U/µl
NEB, Frankfurt am Main, Germany Phusion Polymerase, 2 U/µl Finnzymes, Espo, Finland
PNGaseF (Peptide: N-Glycosidase F), 500 U/µl
NEB, Frankfurt am Main, Germany T4-Ligase, 5 U/µl MBI Fermentas, St. Leon-Rot, Germany T4-DNA-Polymerase, 5 U/µl MBI Fermentas, St. Leon-Rot, Germany Proteinase K, 30 U/mg, cat. no. P2308 Sigma-Aldrich, Deisenhofen, Germany
Proteinase K, 5-15 U/mg, cat. no. P8044 Sigma-Aldrich, Deisenhofen, Germany 3.7 Fluorescent dyes
Description Source
Bodipy 499/508 Invitrogen, Karlsruhe, Germany
Choleratoxin subunit B Conjugate-A555 Invitrogen, Karlsruhe, Germany Concanavalin A-A594 Invitrogen, Karlsruhe, Germany 3.8 Kits
Description Source
Amersham ECL Advance WesternBlotting Detection Kit, cat. no. RPN 2135
GE Healthcare, München, Germany Invisorb Spin Plasmid Mini Two, cat. no.
10101403
Invitek, Berlin, Germany PhoenIXTM Filter Maxiprep Kit, cat. no. 2075-
600
MP Biomedicals, Heidelberg, Germany Qiagen EndoFree Plasmid Mega Kit, cat. no.
12381
Qiagen, Hilden, Germany Qiaquick Gel Extraction Kit, cat. no. 28706 Qiagen, Hilden, Germany MinElute Reaction Cleanup Kit, cat. no.
28204
Qiagen, Hilden, Germany
3.9 Loading buffers for proteins:
5 x Laemmli loading buffer 100 mM Tris-HCl, pH 8.0 12.5 % SDS
25 % β-Mercaptoethanol 5 % Glycerol
bromphenol blue for DNA:
10 x DNA loading dye 1 mg/ml Bromphenol blue
50 % Sucrose 1 mM EDTA 3.10 Marker
Description Source
MassRuler DNA Ladder Mix MBI Fermentas, St. Leon-Rot, Germany GeneRuler 1kb DNA Ladder MBI Fermentas, St. Leon-Rot, Germany Prestained Protein Molecular Weight Marker MBI Fermentas, St. Leon-Rot, Germany Biotinylated SDS-PAGE Standard Broad
Range
BioRad, München, Germany
3.11 Media for eukoryotic cells:
Description Source
Material 30
DMEM high glucose, w/o Pyruvate, cat. no.
41966-029
Invitrogen (Gibco), Karlsruhe, Germany DMEM high glucose, with Pyruvate, cat. no.
41966-039
Invitrogen (Gibco), Karlsruhe, Germany OptiMEM, cat. no. VX31985054 Invitrogen (Gibco), Karlsruhe, Germany for prokaryotic cells:
Media Composition
LB-Medium 0.5 % Yeast extract
1 % Tryptone 1 % NaCl
pH 7.5, autoclave
LB-Agar LB-Media + 1.5 % Agar
SOC Medium 2 % Tryptone
0.5 % Yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2
10 mM MgSO4
20 mM Glucose autoclave
2YT Medium 1 % Tryptone
1.6 % Yeast extract 0.5 % NaCl2
autoclave 3.12 Oligonucleotides
Description Sequence (5´→3´)
pWPT2264f, forward primer for sequencing AAC CGG TGC CTA GAG AAG GT pWPT3415r, reverse primer for sequencing GAA AAT GAA AGC CAT ACG GG 3.13 Plasmids
pCMV-wt and deletion mutants -114Δ2, - 114Δ4, -114Δ6, -114Δ8, -116Δ4, -116Δ6, - 118Δ4, -120Δ2
H. Niemann, DKFZ, Heidelberg, Germany
pCMV-wt-3F4 and deletion mutants -114Δ2- 3F4, -114Δ4-3F4, -114Δ6-3F4, -114Δ8-3F4, - 116Δ4-3F4, -116Δ6-3F4, -118Δ4-3F4
B. Thaa, Molecular Toxicology, Konstanz, Germany
pWPT-GFP, transfer plasmid for lentiviral gene transfer
D. Trono, School of Life Science, Lausanne, Switzerland
pWPT-wt-3F4, and deletion mutants -114Δ2- 3F4, -114Δ4-3F4, -114Δ6-3F4, -114Δ8-3F4, - 116Δ4-3F4, -116Δ6-3F4, -118Δ4-3F4
this work
psPAX2, packaging plasmid for lentiviral gene transfer
D. Trono, School of Life Science, Lausanne, Switzerland
pMD2.G, envelope plasmid for lentiviral gene transfer
D. Trono, School of Life Science, Lausanne, Switzerland
pEGFP-N1 BD Bioscience, Heidelberg, Germany
pCMS-PrP-EGFP S. Ramljak, University Hospital Göttingen, Germany
3.14 Software
Softare Supplier
AIDA Image Analysis Software 3.10 Raytest, Straubenhardt, Germany AxioVision AxioVs40 V4.3.0.101 Zeiss, Oberkochen, Germany
CarelDraw 12 Corel Corporation
Clone Manager 7 Scientific and educational software Cary, NC, USA
EMBOSS http://www.ebi.ac.uk/emboss
EndNoteX Thomson ISI Research Soft
Graph Prism 5.0 GraphPad Software Inc
ImageJ 1.39 Wayne Rasband, National Institute of
Health, USA
LSM 5 Image Examiner Zeiss, Oberkochen, Germany
MS Office 2007 Microsoft Corporation
Photoshop CS Adobe Systems Inc.
Primer 3 http://www.frodo.wi.mit.edu
3.15 Solutions and buffers
Buffer Composition
ECL solution solution 1:
Luminol (250 mM in DMSO) 50 µl p-courmaric acid (90 mM in DMSO) 22 µl Tris-HCl (1 M, pH 8.5) 0.5 ml Milli Q H2O 4.4 ml solution 2:
H2O2 (30 %) 3 µl Tris-HCl (1 M, pH 8.5) 0.5 ml Milli Q H2O 4.4 ml mix solution 1+1 before use
EDTA solution for trypsin-free detachment of eukaryotic cells
0.625 mM EDTA 0.125 mM EGTA 30 mM Tris 0.25 M Sucrose sterile filtration
FACS buffer 0.5 % FCS
2 mM NaN3
in 1 x PBS
Freezing medium for eukaryotic cells 70 % growth medium with supplements 20 % FCS
10 % DMSO
Freezing medium for prokaryotic cells 25 % Glycerin (100 %)
Material 32
75 % Bacteria from Mini or Maxiprep 2 x HBS for CaPO4 transfection 0.283 M NaCl
0.025 M HEPES 1.5mM Na2HPO4 pH 7.05 exactly sterile filtration 1 x HBSS (Mg2+, Ca2+ free) 5.33 mM KCl
0.441 mM KH2PO4
4.17 mM NaHCO3 137.93 mM NaCl 0.338 mM Na2HPO4
5.56 mM D-Glucose
High salt washing buffer 9 g NaCl
100 ml 0.5 M Tris pH 7.6 ad 1000 ml H2o bidest 10 x Laemmli running buffer for SDS-PAGE 250 mM Tris
1.92 M Glycine 1 % SDS
dilute to 1 x with Milli Q H2O
Lysis buffer 10 mM Tris-HCl pH 7.5
100 mM NaCl 10 mM EDTA 0.5 % Triton X-100
0.5 % Deoxycholate sodium salt
10 x PBS 1.37 M NaCl
100 mM Na2HPO4
30 mM KH2PO4
Percoll-Sucrose light:
14 % Percoll
20 % 5 x Sucrose buffer 66 % Milli Q H2O heavy:
68 % Percoll
20 % 5 x Sucrose buffer 12 % Milli Q H2O
Semi Dry Blot buffer 25 mM Tris
192 mM Glycine 0.1% SDS 10 % Methanol 5 x Separating gel buffer 1.86 M Tris
7 mM SDS pH 8.8
2 x Stacking gel buffer 0.25 M Tris
7 mM SDS pH6.8
Sucrose solution 8 % Sucrose
in 0.2 x HBSS (Mg2+, Ca2+ free)
Sucrose-Relaxation buffer 270 mM Sucrose 20 mM KCl 1 mM EGTA 5 mM HEPES pH 7.0
+ 5 µg/ml Cytochalasin B in DMSO + some crumbs DNAse I
5 x sucrose buffer 1.35 M Sucrose
100 mM KCl 25 mM HEPES pH 7.0
Substrate solution for Cell ELISA 50 mM Glycin-NaOH pH 9.7 4 mM MgCl2
50 µg/ml BCIP 100 µg/ml NBT
TB buffer 10 mM PIPES
15 mM CaCl2
250 mM KCl
pH adjusted to 6.7 with KOH, then addition of 55 mM MnCl2
10 x TBE 0.9 M Tris
0.9 M Boric acid 0.025 M EDTA pH 8.5
TNE 150 mM NaCl
50 mM Tris-HCl pH 7.5 5 mM EDTA
TNT 150 mM NaCl
10 mM Tris pH 8.0 0.05 % Tween 20 3.16 Technical equipment
Equipment Description and source
Centrifuges Centrifuge 5810R (Eppendorf, Hamburg,
Germany)
Centrifuge 5415R (Eppendorf, Hamburg, Germany)
Labofuge 400 (Heraeus, Hanau, Germany Megafuge 1.0R (Heraeus, Hanau, Germany) Ultracentrifuge LE-80K (Beckman Coulter, Krefeld, Germany)
Cell counter Casy TT Schärfe System, Reutlingen, Germany Cell culture plastic ware Corning, Schiphol-Rijk, Netherlands Centrifuge tubes (15, 50 ml) Corning, Schiphol-Rijk, Netherlands Centrifuge tubes, thick wall for
ultracentrifugation, cat. no. 355631
Beckman, Krefeld, Germany
Material 34
Coverslips Menzel, Braunschweig, Germany
Documentation of agarose gels Gel Jet Imager (Intas, Göttingen, Germany)
Electrophorese chambers for DNA:
Agagel Mini Biometra (Biomed, Göttingen, Germany)
ComPhor L Midi (BioRad, München, Germany)
for proteins:
Hoefer MiniVE Vertical Electrophoresis system blot module (Amersham Biosciences, Freiburg, Germany)
Semidry transfer unit Hoefer TE77 (Amersham Biosciences, Freiburg, Germany) Luminescence Image station LAS 1000 Pro (Fujifilm, Düsseldorf, Germany)
Lumox dishes Greiner-bio-one, Frickenhausen, Germany
Microscopes Confocal microscope: LSM 510 Meta (Zeiss,
Oberkochen, Germany)
Fluorescent microscopes: Axiovert S100 TV and 200M (Zeiss, Oberkochen, Germany)
Object slides Menzel, Braunschweig, Germany
Photometer Photometer 2100 pro (Amersham
Biosciences, Freiburg, Germany)
Pipettes Abimed, Langenfeld, Germany
Pipette tips Sarstedt, Nümbrecht, Germany
Power supplies Model 200 (BioRad, München, Germany)
Power Pac 300 (Amersham Biosciences, Freiburg, Germany)
Reaction tubes (0.5-1.5 ml) Sarstedt, Nümbrecht, Germany
Thermomixer Thermomixer comfort (Eppendorf, Hamburg,
Germany
Transfer membrane PVDF membrane Hybond P (Amersham
Biosciences, Freiburg, Germany)
Vortex Genie 2 VWR, Darmstadt, Germany
