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Originally published In Press as doi:10.1074/jbc.M410796200 on September 23, 2004

J. Biol. Chem., Vol. 279, Issue 51, 53306-53316, December 17, 2004
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Glycosylation Deficiency at Either One of the Two Glycan Attachment Sites of Cellular Prion Protein Preserves Susceptibility to Bovine Spongiform Encephalopathy and Scrapie Infections*

Erdmute Neuendorf{ddagger}§, Artur Weber{ddagger}, Armin Saalmueller¶, Hermann Schatzl||, Kurt Reifenberg**, Eberhardt Pfaff¶, and Martin Hermann Groschup{ddagger}{ddagger}{ddagger}

From the {ddagger}Institute for Novel and Emerging Infectious Diseases, Federal Research Centre for Virus Diseases of Animals, Isle of Riems 17943, Institute of Immunology, Federal Research Centre for Virus Diseases of Animals, Tübingen 72076, ||Institute of Virology, Technical University, Munich 80802, and **Centralized Animal Facility, University of Mainz, Mainz 55101, Germany

Received for publication, September 20, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The conversion into abnormally folded prion protein (PrP) plays a key role in prion diseases. PrPC carries two N-linked glycan chains at amino acid residues 180 and 196 (mouse). Previous in vitro data indicated that the conversion process may not require glycosylation of PrP. However, it is conceivable that these glycans function as intermolecular binding sites during the de novo infection of cells on susceptible organisms and/or play a role for the interaction of both PrP isoforms. Such receptor-like properties could contribute to the formation of specific prion strains. However, in earlier studies, mutations at the glycosylation sites of PrP led to intracellular trafficking abnormalities, which made it impossible to generate PrP glycosylation-deficient mice that were susceptible to bovine spongiform encephalopathy (BSE) or scrapie. We have now tested more than 25 different mutations at both consensus sites and found one nonglycosylated (T182N/T198A) and two monoglycosylated (T182N and T198A) mutants that rather retained authentic cellular trafficking properties. In vitro all three mutants were converted into PrPres. PrP mutant T182N/T198A also provoked a strong dominant-negative inhibition on the endogenous wild type PrP conversion reaction. By using the two monoglycosylated mutants, we generated transgenic mice overexpressing PrPC in their brains at levels of 2–4 times that of nontransgenic mice. Most interestingly, such mice proved readily susceptible to a challenge with either scrapie (Chandler and Me7) or with BSE. Incubation times were comparable or in some instances even significantly shorter than those of nontransgenic mice. These data indicate that diglycosylation of PrPC is not mandatory for prion infection in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prion diseases include Creutzfeldt-Jacob disease, Gerstmann-Straüssler-Scheinker syndrome, Kuru, and fatal familial insomnia in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE)1 in cattle, transmissible mink encephalopathy in mink, feline spongiform encephalopathy in cats, and chronic wasting disease in cervids. Prion diseases are characterized by incubation times of up to several years or decades. The post-translational conversion of cellular protease-sensitive prion protein (PrPC syn. PrPsen) into its protease-resistant isoform (PrPres) is the key feature in these infectious diseases (1). Cellular and pathological PrP are encoded by the same gene (Prnp) (2–5), have the same covalent structure, and are both glycoproteins with a glycophosphatidylinositol anchor (6). The cellular isoform is bound to the external surface of the plasma membrane, whereas PrPres accumulates in late endosomes and lysosomes (7, 8). Cellular PrP is rich in {alpha}-helices and is proteinase K-sensitive and soluble in detergents (9). In contrast, abnormal PrPres is rich in {beta}-sheets (10, 11), partially proteinase K-resistant, and is insoluble. Mouse PrP contains two consensus sites for N-linked glycosylation (12, 13). The first is located at codon 180 and the second at codon 196. In the fully matured protein both N-glycosylation sites are occupied. Analysis of hamster- or mouse-derived PrPres revealed that the glycan chains represent a mixture of bi-, tri-, and tetra-antennary complex-type sugars with 70% of the galactose residues sialyzed (12, 14, 15).

A variety of experimental scrapie strains has been isolated and characterized in rodents, which show marked differences in the clinical phenotype, pathology, and in the biochemical features of PrPres. The existence of different strains still raises the question of how such a diversity can be encoded by a single protein composed of a defined amino acid sequence. Variations in the protein conformation and in the glycosylation of PrP have been postulated as modulators of the conversion and/or disease process and of prion strain properties (16–19).

Prion strains can be distinguished (20) by PrPres glycosylation patterns (21). Although some scrapie strains such as Chandler produce predominantly heavily diglycosylated PrPres, in others (e.g. Me7) monoglycosylated PrPres is favored (22). Comparison of the glycoforms of normal and abnormal hamster PrP showed that both contain generally the same N-linked oligosaccharides but at different ratios (23). Moreover, it has recently been shown that the PrPC glycosylation can significantly affect the PrPres formation, if both are derived from different species (24).

To elucidate the role of the glycans in prion infection and pathogenesis, we sought to develop transgenic mice that express mono- or nonglycosylated PrPC with retained authentic PrP trafficking. It is generally accepted that the surface expression of PrPC is necessary for a de novo prion infection of cells (25–29). Earlier work (30, 31) showed that elimination of the first consensus site for N-glycosylation by substituting threonine by alanine at codon 182 leads to a PrPC retention in the cell with no expression on the plasma membrane. Transgenic mice carrying this mutation were resistant to a scrapie infection and showed only low expression levels of PrPC (19). On the other hand, the deletion of the second glycosylation site did not change the trafficking of the prion protein nor the fundamental susceptibility. However, expression levels that were achieved did not suffice to effectively support the infection cycle (19). Another approach was based on the direct mutation of the attachment sites (N180Q/N196Q) (32) in vitro. However, viable transgenic mice based on this concept have not been reported to date.

To reveal PrP glycosylation-deficient mutants with retained authentic subcellular trafficking, we systematically studied more than 25 different mutations at the consensus sites. Among those we found only three mutants lacking either one or both glycosylation sites and with no detectable change in their expression topology in neuroblastoma, Chinese hamster ovary, and murine fibroblast cells. In scrapie-infected neuroblastoma cells, even mutants with hampered or blocked trafficking turned out to be convertible into their proteinase K-resistant isoform, but only mutants highly expressed on the surface promoted a de novo infection of cells. In addition, although being convertible itself, the unglycosylated mutant T182N/T198A showed a remarkable dominant-negative inhibition of the endogenous murine PrP conversion process in ScN2a cells. Similar effects have been reported in the literature and were explained by conversion inefficiencies of PrPC across species barriers (amino acids 112–187) (33) and/or an interference with the "protein X"-binding site, a proposed chaperone-like factor facilitating murine PrP conversion (amino acids 167, 171, 214, and 218) (34–37). Finally, we produced transgenic mice overexpressing these glycosylation-deficient PrP mutants and that were proven to be susceptible to challenge with BSE and scrapie prion strains.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of Glycosylation Consensus Site Mutants—Mutants of mAb 3F4 epitope-tagged mouse PrP-encoding constructs were designated T182X (where X indicates every possible amino acid) without or in combination with T198A. Mutations were introduced by site-directed PCR mutagenesis. The 3F4-tagged mouse ORF cDNA including 406 bp of the 3'-untranslated region cloned into the vector pcDNA3.1/Zeo(+) (Invitrogen, AF286076 [GenBank] ) served as template. The following primers were used: ngly182.rev, 5'-GGT GGT GGT GAC CGT GTG CTG CTT GAT GGC aAT ATT GAC GC-3' (bp 533–573 moORF; mutations are shown in boldface; at position bp 543 (lowercase letter) silent mutagenesis for an additional SspI restriction site); musnarxxho.rev, 5'-CCC CCC TCG AGG GCG CCA TCC CCA AAC AGT GGC TTC TTT GGT TG-3' (bp 1138–1170 3'-untranslated region moPrnp, introducing an XhoI restriction site (boldface) at position bp 407 downstream of moORF); ngly198.seq, 5'-GCA CAC GGT CAC CAC CAC CAC CAA GGG GGA GAA tTT CGC CGA GAC CG-3' (bp 555–601moORF; mutations are marked boldface; at position bp 588 (lowercase letter) silent mutagenesis for an additional ApoI restriction site); mu5.seq, 5'-GCG AAC CTT GGC TAC TGG CTG-3' (bp 4–24); PI.rev, 5'-GGT GGT GGT GAC CGT GTG CTG CTT GAT SWW GAT ATT GAC GC-3'; PII.rev, 5'-GGT GGT GGT GAC CGT GTG CTG CTT GAT SSS GAT ATT GAC GC-3'; PIII.rev, 5'-GGT GGT GGT GAC CGT GTG CTG CTT GAT SSW GAT ATT GAC GC-3'; PIV.rev:, 5'-GGT GGT GGT GAC CGT GTG CTG CTT GAT SWS GAT ATT GAC GC-3' (bp 533–573 moORF, S indicates G and C; W indicates A and T). For generation of T182X mutants, mu5.seq was used in combination with PI.rev, PII.rev, PIII.rev, or PIV.rev. By using these primers we did not obtain every amino acid at codon 182. Therefore, we used the following primers in combination with mu5.seq: Val182.rev, Gln182.rev, Glu182.rev, Asp182.rev, Lys182.rev, Met182.rev, Arg182.rev, or Trp182.rev corresponding to PI.rev-PIV.rev but encoding the specific amino acid. Amplicon and template were then cleaved by Bsu36I and BsteII, and the mutated PCR products were ligated into the vector again. The T198A mutation was introduced by primers ngly198.seq and musnarxxho.rev. Product and template were cleaved by BsteII and NarI. The T182A/T198A and T182N/T198A constructs were obtained by introducing the T198A mutation into the T182A or T182N cDNA by using Bsu36I and BsteII cleavage sites. For conversion experiments, PrP 3F4 epitope expressing mutants encoding T182A, T182N, T198A, T182A/T198A, and T182N/T198A were cloned into the retroviral vector pSFF (Z22761 [GenBank] ) (38) using BamHI and XhoI cleavage sites. For generation of transgenic mice, the corresponding mutants were cloned into the vector phgPrP (43, 44) by XmaI and NarI. Before microinjection the pBluescript backbone was removed by SalI and NotI digestion. The fidelity of each construct was confirmed by sequencing. All restriction enzymes were purchased from New England Biolabs (Schwalbach, Germany).

Cloning of an N-terminal Deletion Mutant—This construct, missing the N-terminal signal sequence (amino acids 1–22), was produced by SOE-PCR (splicing by overlap extension) using the following primers: N-sig.seq, 5'-gaA TCA GTC ATC ATG AAG CGG CCA AAG CCT GGA GGG-3'; CMV.seq, 5'-cat atg cca agt acg ccc cct att gac g-3'; BsteII.rev, 5'-CAT CGG TCT CGG TGA AGT TCT CCC CCT TGG-3'; N-sig.rev, 5'-CGC TTC ATG ATG ACT GAT tct aga tct gc-3'. Three consecutive PCRs were performed as follows. The first used primers CMV.seq and N-sig.rev, and the second used primers N-sig.seq and BsteII.rev. A 3F4-tagged mouse ORF served as a template. Finally, the obtained PCR products were used as templates for the last PCR with primers CMV.seq and BsteII.seq. The fidelity was confirmed by sequencing.

Cell Lines—Murine neuroblastoma (N2a) and Chinese hamster ovary (CHO) cells were purchased from ATCC (Manassas, VA). Scrapie-infected neuroblastoma cells as well as murine fibroblast cell lines Pa 317 (41) and {Psi}2 cells (42) (which were derived from NIH3T3 cells) (43) were generously supplied by B. Chesebro and S. Priola (Hamilton, MT). N2a cells were grown in MEM-NEAA, CHO cells in a 3:1 mixture Dulbecco's MEM and F-12, and murine fibroblast cells in RPMI 1640 and ScN2a cells in Opti-MEM or MEM-NEAA. Media were enriched with 10% FCS and contained penicillin/streptomycin. All cell culture reagents were purchased from Invitrogen. Cells were grown in an atmosphere of 5% CO2.

Transient or Stable Expression of Mutant PrP in Cell Cultures—N2a and CHO cells were transfected using LipofectAMINE (Invitrogen) and murine fibroblasts were transfected by CaPO4 (Stratagene) according to the manufacturer's directions. For every construct at least two independent transfections were performed. The transfection efficiency was measured by FACS analysis using pEGFPn3 (Clontech, U57609 [GenBank] ). For N2a cells, transfection rates of about 70% were achieved. Antibiotic resistant clones of N2a and CHO cells were selected by using 500 µg/ml Zeocin (Invitrogen), and when necessary the cells were subcloned and then maintained in 250 µg/ml Zeocin. For every construct we performed at least two transfections.

FACS Analysis—Living cells (5 x 105) were resuspended in 100 µlof ice-cold PBS containing 2% FCS and were incubated (20 min, 4 °C) with mAb3F4 1:100. The cells were washed and incubated (20 min, 4 °C) with IgG GAM2a-FITC 1:200 or IgG GAM-PE 1:50 in cold PBS, 2% FCS, then washed twice, and resuspended in 200 µl of PBS, 2% FCS, and then 10,000 cells were analyzed by FACStarPlus (BD Biosciences). Calculations were made using the "Cellquest" program (BD Biosciences) on a Macintosh Quadra 650. Fluorescein (FITC)- and phycoerythrin (PE)-conjugated second antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). To every FITC-labeled sample, 30 µl of DNA-binding propidium iodide solution (1 mg/ml) (Sigma) was added prior to measurement in order to stain dead cells for an electronic exclusion. (The signal of FITC is measured at 530 nm, whereas propidium iodide has its emission at about 570 nm.) For every construct we performed at least two transient transfections. As a control, cells were FACS-analyzed by adding a conjugate only.

PrP Conversion in ScN2a Cells—PrP in vitro conversion assays were conducted by using a retroviral expression system for exogenous PrP (43). ScN2a cells (3 x 105) were plated in medium and grown overnight. This medium was removed and replaced by another medium containing 4 µg/ml Polybrene (Sigma). After 2 h a retroviral supernatant in different dilutions was added and incubated overnight. The cells were split on a T-25 flask (for PrPC detection), a T-75 flask (for PrPres detection), and a 24-well plate (for immunohistochemistry) and were grown to confluence and treated as follows.

For the detection of PrPC, cells from a confluent T25-flask were washed with PBS and resuspended with lysis buffer (0.32 M sucrose, 0.5% Nonidet P-40, 0.5% deoxycholate) to prepare a 10% cell homogenate, which could be then further analyzed by SDS-PAGE and immunoblotting.

For the detection of PrPres, cells from a confluent T-75 flask were harvested using 0.25% trypsin and pelleted at 800 rpm for 5 min. The cells were then resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate), and debris was removed by mild centrifugation. The samples were digested with proteinase K (PK 25 µg/ml, 37 °C, 1 h), and PMSF was added to stop digestion followed by a final centrifugation at 265,000 x g for 1 h at 4 °C. After resuspension and heat denaturation in sample buffer (final concentration 4% SDS, 100 mM Tris-HCl, 175.2 mM sucrose, 1% {beta}-mercaptoethanol, 0.02% bromphenol blue, pH 7.4), one-fourth from each sample was run on a 16% SDS-polyacrylamide gel followed by immunoblotting. Untransduced cells were loaded as a control, because initial experiments revealed no discrepancy between mock- and non-transduced cells (data not shown).

Intrinsic Proteinase K Resistance of PrPC—Appropriate cell lysate equivalents were treated with one of the following procedures (at 37 °C): 25 µg/ml PK for 1 h, 1 µg/ml PK for 30 min, 0.5 µg/ml PK for 30 min, or 3.3 µg/ml PK for 10 min. The digestions were stopped by heat denaturation at 95 °C for 10 min. Samples were then pretreated with guanidinium HCl (final concentration 3 M) for 10 min at room temperature, and proteins were acetone-precipitated (final concentration 50%). The resulting pellets were resuspended in sample buffer, electrophoresed (16% gels), and immunoblotted using mAb 3F4 (44).

Deglycosylation of PrPC—Where indicated, cell lysates were treated with 10 units/ml N-glycosidase F or 0.26–0.36 units/ml endoglycosidase H (both reagents were from Roche Applied Science) for 16 h at 37 °C before the samples were electrophoresed followed by immunoblotting. To avoid incomplete digestion, higher endoglycosidase H concentrations were used as described in earlier protocols (31).

Biotinylation of Cell Surface Proteins—Cell membrane proteins were biotinylated as described (45) using Sulfobiotin-X-NHS (Calbiochem), which is not able to enter intact cells and was immunoprecipitated with the mAb 3F4 as described (46). Trypan blue staining (Serva, Heidelberg, Germany) was carried out to exclude that sulfobiotin-X-NHS was labeling intracellular proteins because of cytoplasmic leakages. As additional control, we performed an immunoprecipitation using mAb {beta}-actin for detection of intracellular actin. N2a cells were biotinylated, and the reaction was stopped or not stopped and afterward immunoprecipitated by {beta}-actin. Only data from experiments were used in which no signal was detectable in the stopped sample.

Immunoprecipitated PrPC was run on 13 or 16% SDS-polyacrylamide gels and blotted onto Immobilon P membranes (Millipore, Bedford, MA), and biotinylated PrP was detected with streptavidin-horseradish peroxidase (1:1.000) followed by visualization with enhanced chemiluminescence (ECL, Amersham Biosciences).

PI-PLC Release—N2a cells (80% confluent) from a T-75 flask were washed three times with MEM-NEAA and incubated with PI-PLC (0.1 units/ml in MEM-NEAA) (Roche Applied Science) at 37 °C for 4 h. Following this treatment the supernatant was separated from floating cells by centrifugation (800 rpm, 5 min), and the proteins were then methanol-precipitated. The resulting pellet was resuspended in electrophoresis sample buffer. Cells were harvested and lysed (as described above for the detection of PrPC). The cell lysate and PI-PLC released proteins were analyzed on 13% SDS-PAGE followed by immunoblotting. Identically treated tissue cultures without the addition of PI-PLC served as controls.

Verification of Disulfide Bonds—Cells of a confluent T-75 flask were lysed in N-ethylmaleimide (Calbiochem) lysis buffer (0.5% Triton X-100, 1 mM EDTA, pH 6.8, 20 mM N-ethylmaleimide, 1 mM PMSF in PBS). After a mild centrifugation, supernatant proteins were heat-denatured at 95 °C for 5 min either under nonreducing (by adding SDS-PAGE sample buffer: 400 mM Tris-HCl, pH 6.8, 6% w/v SDS, 20% glycerin, 2 mM EDTA, pH 6.8, 0.01% bromphenol blue) or reducing conditions (sample buffer plus 5 µl dithiothreitol (300 mM dithiothreitol)). Reduced and nonreduced samples were separated by 12% SDS-PAGE and immunoblotted. One lane was spared because of probable diffusion of dithiothreitol in the gel.

Solubility Assay—Cells from a T-75 flask were dissolved in 3 ml of solubility buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate), and debris was removed by mild centrifugation. PMSF (0.81 mM) was added, and the samples were pelleted by ultracentrifugation at 265,000 x g, 4 °C for 1 h. The pellets were resuspended in sample buffer, and supernatants were precipitated as described above.

Western Blotting—Samples were separated by SDS-PAGE, and the proteins were transferred onto Immobilon P membranes (Millipore) and blocked in 5% non-fat dry milk in PBS, 0.1% Tween 20 for 30 min. The membranes were incubated overnight in non-fat dry milk in PBS, 0.1% Tween 20 with either mAb 3F4 (1:3.000), Ra5/7 (1:2.000), or mAb SAF70 (1:1.500). The membranes were washed three times in washing buffer (PBS, 0.1% Tween 20) followed by incubation with GAM-PO (goat anti-mouse IgG peroxidase, 1:3.000) or SAR-PO (sheep anti-mouse IgG peroxidase, 1:3.000) (Dianova, Hamburg, Germany) for 45 min. After another three washing steps, signals were visualized with ECL (Amersham Biosciences). Mouse monoclonal antibody 3F4 (44) raised against PrP 27–30 from scrapie-infected hamster was a gift from Dr. Michael Beekes. Mouse monoclonal antibody SAF70, detecting hamster and mouse PrP, was a gift from Dr. J. Grassi (47). Polyclonal anti-peptide serum Ra5/7 from rabbits, raised against amino acids 95–110 of sheep PrP, has been described previously (48).

Overlay Experiments—Brain tissue from mice infected with scrapie strains Chandler, Me7, and 79A were obtained from terminal stage C57Bl and CD1 mice. The brains were homogenized in Opti-MEM (10% w/v), subjected to ultrasonification, and heated for 10 min at 70 °C. ScN2A cells were cloned twice and selected for PrPres negative cells. Such cells were then transfected with 1 ml of retroviral supernatant as described above and grown in T-75 flasks to a confluence of 70–80%. Either 200 µl of Me7, 400 µl of 79A, or 400 µl of Chandler homogenates (10%), which contained comparable PrPres amounts as determined by immunoblotting, were used for infection. The cells were washed three times with PBS and overlaid with 5 ml of Opti-MEM plus 10% FCS containing 400 or 200 µl of brain homogenate. The cells were incubated for 4 h, and afterward 10 ml of medium was added per flask. The cells were grown for 4 days to confluence and treated as described for PrPres. For each construct every infection experiment was performed at least twice. Cells overlaid with 400 µl of brain homogenate of a non-infected C57Bl mouse served as a control.

Generation and Characterization of Transgenic Mice—Suitable ORF mutations encoding non- or monoglycosylated PrPC were cloned into the vector phgPrP that comprised the murine prion gene but lacked intron 2 (39). Fertilized oocytes were taken from PrP0/0 mice (40), microinjected with DNA of the relevant phgPrP mutant according to standard procedures, and reimplanted into NMRI mice. For the detection of transgenic mice, we applied the protocol of Bueler et al. (40). Vector phgPrP and PrP0/0 mice were generously supplied by C. Weissman, London, UK.

Founder animals were mated with PrP0/0 mice and the offspring examined for PrPC expression. 10% homogenates were prepared in lysis buffer by using an Ultra-Turrax (Janke and Kunkel, Germany) and were analyzed on SDS-PAGE followed by immunoblotting. The protein expression levels in the brains were determined by using a photo imager with the software "Tina" (Raytest, Straubenhard, Germany) and were compared with the normal expression level of nontransgenic mice. The Prnp copy number was estimated accordingly by comparing PCR amplifications to corresponding standard samples.

BSE and Scrapie Challenge of Transgenic Mice—Hemizygous transgenic mice were inoculated intracerebrally with 30 µl of C57Bl mouse brain homogenates (in saline solution) containing 10% BSE or scrapie (Me7 and Chandler). Negative and positive controls included transgenic and conventional C57Bl mice inoculated either with non-infectious or with BSE or scrapie mouse brain homogenates. These mice were checked three times per week until the onset of clear clinical symptoms and were culled. Their brains were later sampled and analyzed by immunoblotting or immunohistochemistry for demonstrating the accumulation of PrPSc.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Deletion of the First Glycosylation Site by Codon 182 Mutation—In earlier studies the deletion of the first glycosylation consensus site led to aberrant trafficking of PrPC in cells (19, 30, 31). However, these studies left the question unanswered as to whether these differences were because of the introduced point mutation at position 182 or because of the missing glycosylation of PrP itself. Because wild type PrPC synthesized in the presence of tunicamycin was readily present on the cell surface (31), the first explanation seemed to be more likely. Our initial experiments therefore intended to generate a mutant of the first glycosylation consensus site of PrPC exhibiting unaltered intracellular trafficking.

For this purpose we substituted the codon for threonine at position 182 with codons for any other amino acid. We expressed these 19 mutants as well as a wild type PrP control in N2a cells, and we analyzed the effects by immunoblotting to reveal expression rates and glycosylation patterns (Fig. 1) and by FACS analysis to test for the presence of proteins on the cell surface (Fig. 2). All mutants were based on a template that encoded a 3F4 epitope-tagged mouse PrP. This allowed the detection of mutated PrP against the background of wild type mouse PrP. Mutations at the consensus site were introduced by using a set of wobble primers and PCR mutagenesis. All constructs were cloned into the expression plasmid pcDNA3.1Zeo(+) and transfected into neuroblastoma (N2a) cells, and stably expressing cells were selected by Zeocin. For FACS analysis, we used living, non-permeabilized cells so that the antibodies only detected surface-expressed PrPC (see "Experimental Procedures"). Of the 19 different mutants at codon 182, we found only two that were still accessible extracellularly, T182S and T182N. However, only mutant T182N was monoglycosylated (Figs. 1 and 4a) because serine instead of threonine can also function as a glycosylation consensus site (Fig. 1). This mutant therefore served as a supplementary positive control in addition to mAb 3F4 epitope-tagged wild type PrP (data not shown).



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  		FIG. 1.
Expression of PrPT182X mutants in neuroblastoma cells. Nineteen different T182X mutants of PrPC were expressed in N2a cells using the pcDNA3.1zeo expression system. After Zeocin treatment stably mutant PrP-overexpressing cells were selected. Cell lysates were analyzed by immunoblot using mAb 3F4 as a detection antibody and visualized using a chemiluminescence system. Note that all mutations except for one abolish PrP glycosylation at the first consensus site. Only the threonine to serine mutation (different subclones are named f5, c4, and d3) still functions as a recognition site. Untransfected N2a cells (N2a), transfections using wild type 3F4 epitope-tagged PrP encoding constructs, and hamster PrP (brain lysate) served as controls. 0, 1, and] = non-, mono-, and diglycosylated PrPC. M = molecular mass marker.

 



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  		FIG. 2.
Cell surface expression of PrPC glycosylation mutants. T182N, T182A, and T198A single and double mutants of 3F4 epitope-tagged murine PrPs were expressed in N2a cells (pcDNA3.1zeo expression system). Stably transfected cells were selected using Zeocin, and mutant PrPCs overexpressing N2a cell clones were harvested after subcloning. FACS analysis was carried out on non-permeabilized cells using mAb 3F4 as a primary antibody and phycoerythrin-coupled secondary antibodies. Dead cells were excluded by propidium iodide staining. Each analysis was performed with the addition of secondary antibodies (dark line) and without (light line) in order to discriminate between specific and non-specific intrinsic fluorescence. By using this extremely sensitive technique, all single and double mutants were detected on the cell surface. Comparing the extent of shifting revealed differences in the levels of surface expression. A {Delta}N signal sequence deletion mutant, which is incapable of reaching the cell membrane, was used as a negative control, whereas wild type 3F4-tagged PrP served as a positive control.

 



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  		FIG. 4.
Processing and maturation of PrPC mutants. a and b, PNGaseF mutants of PrPC were expressed in N2a cells using the pcDNA3.1zeo expression system. After Zeocin treatment and subcloning, stably mutant PrP-overexpressing cells were selected. Cell lysates were analyzed by immunoblot using mAb 3F4 as detection antibody and a chemiluminescence visualization system. Asparagine linkage of PrP glycosylation was confirmed by enzymatic treatment of cell lysates using PNGaseF (+). Transfections using wild type 3F4 epitope-tagged PrP-encoding constructs as well as hamster PrP (brain lysate) served as controls. c, Endo H. Stable mutant PrP-expressing fibroblast cells were lysed, and the lysates were treated with endoglycosidase H. Lysates were then immunoblotted, and PrPC signals were visualized by using mAb 3F4. Mid-Golgi matured PrPC is resistant to deglycosylation, and therefore PrP banding signals do not shift toward the non-glycosylated form. M = molecular mass marker. 0, 1, and 2 = non-, mono-, and diglycosylated PrPC, respectively.

 
Generation of Nonglycosylated, Surface-expressed PrPC—To generate a nonglycosylated PrPC mutant with retained intracellular trafficking, the T182N encoding substitution was combined with a T198A mutation by which the second N-linked glycosylation consensus site was also eliminated (choosing the same approach as for the first consensus site would have introduced an asparagine at position 198 which in combination with a threonine at position 200 would have generated a new consensus site (196NFNET200)). This new construct T182N/T198A was then transfected into N2a cells, and the subcloned cells were analyzed by immunoblot and FACS analysis. It was shown that this mutant PrPC was completely unglycosylated (Fig. 4b) while still being detectable on the cell surface (Fig. 2). This result demonstrated that the glycosylation is not mandatory for surface presentation of PrPC.

Comparative Analysis of Novel Mutants with the Earlier Described Glycosylation Site Mutants in Different Cell Lines—To compare better the mutants described here (T182N and T182N/T198A) with those reported earlier in literature (T182A, T198A, and T182A/T198A) (19, 30, 31), expression plasmids were also cloned for T182A, T198A, and T182A/T198A. Moreover, three different cell lines, N2a, CHO, and murine fibroblast cells, were transfected with each of these five mutants to determine specific effects on the glycosylation and trafficking. As it turned out, the choice of the cell line used for these studies had in general no impact on the results that were obtained (Table I).


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  		TABLE I
Properties of PrPC glycosylation mutants expressed in three different cell lines

3F4 epitope-tagged murine PrP mutants were either stably or transiently expressed in neuroblastoma (N2a), CHO, or 3T3 derived fibroblast ({Psi}2/PA 317) cells.

 
Most surprisingly, as shown by FACS analysis and biotinylation studies, not only the newly generated mutants but also the earlier mutants were expressed on the cell surface, although at quite different levels. PrP mutants T182N, T198A, and T182N/T198A trafficked clearly onto the cell surface of subcloned N2a, CHO, and murine fibroblast cells, whereas only lower surface presentation levels were observed for PrP mutants T182A and T182A/T198A. Subcloned cells expressing 3F4-tagged PrPC lacking an N-terminal sequence were used as a additional negative controls in FACS analysis. As the N-terminal signal sequence is mandatory for the protein translocation into the endoplasmic reticulum, this mutant remained in the cytosol (Fig. 2). FACS results were reconfirmed by biotinylation of cell surface proteins followed by PrPC immunoprecipitation using mAb 3F4, immunoblotting, and detection of biotinylated proteins using streptavidin conjugated to peroxidase. Whereas mutants T182N, T198A, and T182N/T198A were comparable with those of 3F4 epitope-tagged wild type PrP, surface expression rates of mutants T182A and T182A/T198A were considerably lower (Fig. 3). To exclude false-positive results because of dead and therefore permeable cells, we used DNA-binding propidium iodide (FACS analysis) or trypan blue staining (biotinylation assay) as markers for dead cells (see "Experimental Procedures"). When we tested the panel of glycosylation mutants in the different cell lines, no significantly diverging results were found in respect to surface expression and immunoblot profiles.



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  		FIG. 3.
Left and right, cell surface biotinylation of glycosylation-deficient PrPC mutants. Mutants were stably expressed in N2a cells, and the outer membrane proteins were biotinylated using sulfobiotin-X-NHS which is not able to enter intact cells. The absence of {beta}-actin biotinylation and trypan blue-permeable cells (<1%) was also monitored. Following cell lysis, PrPC was immunoprecipitated with mAb 3F4 and separated on 13% (left) or 16% (right) SDS-polyacrylamide gels and blotted onto Immobilon P membrane. Biotinylated PrP was detected by a streptavidin-horseradish peroxidase/chemiluminescence visualization system. Note that mutants T182N, T198A, and T182N/T198A were detected at abundant amounts on the cytoplasmic membrane, whereas mutants T182A and T182A/T198A were detected at much lower levels. Due to experimental variations between gel runs, the quantitation of the results shown on left and right is hardly possible. M = molecular mass marker.

 
To determine whether these constructs were attached to the cytoplasmic membrane by a glycophosphatidylinositol anchor, transfected cells were treated with PI-PLC, which cleaves the phosphatidylinositol-diacylglycerol linkage specifically. With one exception, all cell surface-exposed constructs were released by PI-PLC. Only mutant T198A was resistant to PI-PLC-treatment, which was surprising as the double mutant T182N/T198A was releasable (Table I). Therefore it must be assumed that the monoglycosylated protein is either anchored differently or, more likely, the cleavage site is sterically inaccessible to the enzyme.

To confirm that sugar residues were linked to asparagine, glycoproteins were treated with enzyme N-glycosidase F, which specifically cleaves off N-linked carbohydrate moieties. Independent from the cell line used, all three monoglycosylated mutants showed a shift in their molecular mass to that of nonglycosylated PrPC (Fig. 4, a and b).

To determine processing deficits, monoglycosylated PrPC mutants were also analyzed by endoglycosidase H treatment. Carbohydrate moieties N-linked to PrPC are excessively trimmed and processed during their passage through the Golgi apparatus to complex-type oligosaccharides. Mannosidase II in the mid-Golgi compartment renders them resistant to endoglycosidase H cleavage. Therefore, no shift or only a partial shift of di- or monoglycosylated bands to the low molecular mass range of nonglycosylated PrP revealed disturbance in mid-Golgi processing. The following results were obtained: T182A showed low partial resistance, T182N high partial resistance, and T198A total resistance to endoglycosidase H (Fig. 4c). These results indicate that presumably only a few T182A mutants passed through the mid-Golgi apparatus, whereas the processing of T182N PrPC was only scarce and that of T198A PrPC was not disturbed at all.

Proteinase K Sensitivity and Solubility of Glycosylation Mutants—To exclude an intrinsic proteinase K resistance in the mono- or nonglycosylated PrPC molecules, mutant PrP expressed in N2a, CHO, or murine fibroblast cells was subjected for different incubation times to digestions with various concentrations of proteinase K. In general, no particular partial or complete PK resistance was detected (data not shown).

It has been reported (31) that all glycosylation consensus site mutants based on the T/A substitution show detergent insolubility. To reveal whether this characteristic applies also to the newly generated non- and monoglycosylated mutants, a solubility assay was carried out. In accordance with earlier work, these new glycosylation mutants could also be detected after ultracentrifugation in the pellet as well as in the supernatant fractions, whereas wt3F4PrP could be detected in the supernatant only (Fig. 5). Therefore, it must be assumed that the depletion of the glycan side chains leads generally to an increased tendency of the non- and monoglycosylated PrP mutants to aggregate (49, 50). In addition, we observed when developing blots with mAb SAF 70, which detects murine and 3F4-tagged PrP, that fully glycosylated murine PrP, which is also expressed in N2a cells, is not involved in this aggregation process (data not shown). Therefore, an interaction with resident cell PrPC could be excluded.



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  		FIG. 5.
Solubility of PrP glycosylation mutants expressed in N2a cells. 3F4-tagged wild type PrP and corresponding mutants were expressed in N2a cells. Confluent cell layers were harvested and lysed using detergents, and the lysate was ultracentrifuged. Concentrated supernatant proteins (a) and the pellet fraction (b) were immunoblotted using mAb 3F4 to reveal possible insolubilities of PrP mutants. Note that wild type PrPC is only detectable in the supernatant fraction and hence mostly or rather fully soluble, whereas all glycosylation-deficient mutants were found in both fractions. It must therefore be concluded that the attached glycans contribute to the solubility of PrPC. M = molecular mass marker.

 
Conversion of PrPC Glycosylation Mutants into Their Proteinase K-resistant Isoforms—After the characterization of mutant PrPC, we wanted to know if these mutants were convertible into their proteinase K-resistant isoforms. Therefore, we performed conversion experiments in Chandler-infected ScN2a cells using a retroviral system for expression of mutant PrPC. For this purpose replication-defective murine retroviruses encoding mutant PrP molecules were cloned and generated using vector pSFF (38, 43, 52) and packaging cell lines (41, 42). These retroviruses were then transduced into ScN2a cells. The advantage of this system is the high transfection efficiency (as revealed by immunocytochemistry), which leads to a reliable overexpression of exogenous mutant PrP in each cell (53, 54). Cell lysates intended for PrPres detection were digested with proteinase K (25 µg/ml, 1 h, 37 °C), ultracentrifuged, and separated by SDS-PAGE. Those intended for PrPC detection were analyzed without pretreatment. The expression and conversion of mutated exogenous PrP in ScN2a cells were selectively detected by immunoblotting by using mAb 3F4, because mAb SAF70 or polyclonal antibodies Ra5/7 stain total (endogenous wild type plus exogenous) murine PrP.

All mutants were highly expressed in ScN2a cells (Fig. 6c), and no influence on the endogenous mouse PrPC production was detectable (Fig. 6d). Moreover, almost all mutants were efficiently converted into their proteinase K-resistant isoform except for mutant T182N/T198A which was only detected in small amounts (Fig. 6a). When looking at the total PrPres levels in the transduced ScN2a cells, with the exception of T182N/T198N, none of the glycosylation mutants lead to a stronger reduction of PrPres than the wild type 3F4PrP that was used as a control (Fig. 6b). However, in the case of an abundant PrPres expression of mutant T182N/T198A, the accumulation of endogenous murine PrPres was dramatically suppressed (Fig. 6b). To characterize further this phenomenon, ScN2a cells were transduced with different dilutions of retrovirus-containing supernatants. This experiment revealed that the unglycosylated mutant T182N/T198A shows a dominant-negative inhibition, whereas wt3F4-mouse PrP and mutant T182A/T198A does not. In other words, conversion was only detectable when this construct was expressed at low levels (Fig. 7, a and b). In the presence of high concentrations of mutated proteins, no conversion was found, and the detectable amounts of endogenous mouse-PrP were dramatically reduced. This effect has never been described before in context with mutations in this region of the protein. (Such an effect has been described for mutants carrying the 3F4 epitope alone (55, 56), but under the experimental procedures presented here we saw no impact on wt3F4. During the course of the experiment, we mentioned that this effect provoked by the 3F4 epitope alone is dependent from the PrPres load of ScN2a cells. By applying ScN2a cells harboring much PrPres, no effect on wt3F4 could be seen, but in ScN2a cells expressing less PrPres, we saw this effect (data not shown).



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  		FIG. 6.
Conversion of PrP glycosylation mutants in ScN2a cells. 3F4-tagged PrP mutants were expressed in ScN2a cells by using recombinant replication-deficient retroviruses (pSFF system). For demonstrating PRPC or PrPres, cell lysates either pretreated without (c and d) or with proteinase K (25 µg/ml, 37 °C, 1 h followed by ultracentrifugation) (a and b) were separated by SDS-PAGE. Exogenous PrP was detected by probing the membrane with mAb 3F4 (a and c). For the detection of total (exogenous and endogenous) PrP, polyclonal antibodies Ra 5/7 were used. Nontransduced ScN2a-cells were used for controls. Every PRPC-mutant was expressed (c) without any inhibitory effect on the synthesis of endogenous PRPC (d). Note also that every mutant was efficiently converted into its PK-resistant isoform, except mutant T182N/T198A which was converted only in minimal amounts (a). The total PrPres accumulation in the infected neuroblastoma cells was not influenced by Wt3F4, T182A, T182N, T198A, and T182A/T198A (b), again with exception of mutant T182N/T198A which depressed the accumulation of total PrPres dramatically (b). 0, 1, and 2 = non-, mono-, and diglycosylated PrPC. M = molecular mass marker.

 



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  		FIG. 7.
Dominant-negative inhibition of mutant T182N/T198A tested in permanent scrapies-infected neuroblastoma cells. ScN2a cells were transduced using serial dilutions of recombinant virus containing supernatant (from 1 = pure to 1:16 dilution). All lysates were treated with proteinase K. For wild type 3F4 and mutant T182A/T198A, there was no difference in the PrPres accumulation as a consequence of the resulting lower exogenous PRPC expression (a and c) and no effect on the endogenous PrPres deposition in the ScN2a cells (b and d). However, PrPres derived from mutant T182N/T198A was detected only at lower exogenous PRPC expression rates (i.e. higher supernatant dilution rates). Hence the conversion of exogenous PrP was accompanied by an inhibitory effect on the endogenous PrPres accumulation All results shown were from one experiment, and the blot membranes were exposed together on one film. 0, 1, and 2 = non-, mono-, and diglycosylated PrPC. M = molecular mass marker.

 
The absence of the disulfide bridge can also hinder the conversion process of PrPC (57). Therefore, we analyzed whether mutant T182N/T198A, where the mutation is located in an area close to the disulfide bridge, still possessed such an intramolecular bond (Fig. 8). As such a linkage stabilizes the orientation of helices II and III, proteins where the disulfide bridge is disrupted are more extended and therefore migrate slower in SDS-PAGE. As a result, a slight bandshift can be observed when native proteins with an intact disulfide bridge are compared with dithiothreitol-denatured disulfide bridge proteins (Fig. 8). Our results for PrPC mutant T182N/T198A as well as for all other mutants expressed in N2a and murine fibroblasts clearly indicate the presence of disulfide bonds in these molecules (data not shown).



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  		FIG. 8.
Verification of disulfide bridge. The existence of disulfide bonds was verified by mAb 3F4 immunoblotting of cell lysate proteins from mutant PrP-expressing N2a cells that had been electrophoresed under reducing (R) or nonreducing (N) conditions using dithiothreitol as a reducing agent. As can be seen, reduced PrPC lacking the disulfide bond and therefore being bulkier in shape is running slower in the acrylamide gel (12%) than oxidized PrPC. This slight bandshift between the nonreduced (N) an reduced (R) sample indicates the presence of a disulfide bridge. M = molecular mass marker.

 
De Novo Conversion into PrPres Applying Overlay Experiments—Having clarified the convertibility of every mutant, we then wanted to simulate a natural scrapie infection, where the infectious agent first has to enter the cell. Therefore, transduced neuroblastoma cells expressing murine (wt3F4) PrP or T182N, T182A, T198A, T182A/T198A, and T182N/T198A PrP mutants were challenged with a scrapie mouse brain homogenate (Me7). Cell lysates were subjected to PK digestion, ultracentrifugation, and immunoblotting using either mAb 3F4 for the detection of de novo converted mono- and nonglycosylated mutants or antibody Ra5/7 for the detection of total PrPres. It was shown that 3F4-tagged murine PrPC as well as mutants that showed wild type-like surface expression in tissue culture (T182N, T198A, and T182N/T198A) were effectively converted into PrPres (Fig. 9). It was therefore demonstrated that for a de novo conversion of mutated proteins in cells, the target protein (PrPC) must be present in sufficient amounts on the cell surface.



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  		FIG. 9.
De novo conversion into PrPres applying overlay experiments. Neuroblastoma cells were first transduced with recombinant replication-deficient retrovirus encoding wild type and glycosylation-deficient PrP mutants and subsequently overlaid with a 10% mouse brain homogenate of a terminally diseased mouse infected with scrapie strain Me7 (m). Overlaying the cells with brain homogenate of a non-infected C57Bl mouse served as control (c). The amount of PrPres per flask is shown on the 3rd and 4th lanes of blot a (using Ra 5/7). Confluent cells were rinsed extensively and lysed, and the lysates were treated with proteinase K. Exogenous PrPres was revealed by immunoblotting using mAb 3F4 (b). For every construct the results of two independent experiments are shown. As can be seen, wild type 3F4 tagged PrPC, and mutants T182N, T198A, and T82N/T198A PrP were converted into PrPres, whereas mutants T182A and T182A/T198A PrP failed to be converted. Arrows indicate the position of non- and mono-glycosylated PrP, respectively. M = molecular mass marker.

 
Generation and BSE and Scrapie Challenge of PrP Glycosylation-deficient Transgenic Mice—PrP mutants with wild type-like characteristics (T182N, T198A, and T182N/T198A) were used for the generation of transgenic mice. For this reason, PrP0/0 mice were microinjected with the corresponding constructs. The genome of the offspring was screened by PCR for the presence of the transgene and the transgene copy number (Table II). The protein expression was analyzed in a qualitative and quantitative manner by immunoblotting (Fig. 10). These transgenic mice overexpressed prion protein in their brains at levels of up to four times that of wild type mice. We then challenged these transgenic mice with mouse BSE or scrapie (Me7 and Chandler) strains. As expected, transgenic mice carrying mutations at either one of the glycosylation sites developed scrapie (Table III) and accumulated PrPSc in their brains as detected by immunoblotting (Fig. 11) or immunohistochemistry. However, following BSE and Chandler scrapie challenge, the incubation times were substantially shorter in the first consensus site mutant (i.e. 160–240 days) than in mice lacking the second glycosylation site (i.e. 305–410 days). This was true for both of the two challenged transgenic mouse lines of each construct. Most interestingly, compared with the other two strains, Me7 displayed a substantially prolonged incubation time in both transgenic lines carrying the mutation at the first glycosylation consensus site, whereas no such effect was observed with the mutation at the second site. Moreover, based on clinical observations, transgenic mice lacking the first or second PrPC glycosylation site developed distinct disease patterns, even when inoculated with the same infectious strain. Although the first site mutant mice died within hours after onset of clinical disease showing severe excitation and kyphosis, second site mutant mice became apathic and died eventually after a prolonged clinical phase. Unfortunately, no transmission results are as yet available for transgenic mice lacking both glycosylation sites (T182N/T198A).


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  		TABLE II
Generation of transgenic mouse lines expressing non- or monoglycosylated PrPC

 



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  		FIG. 10.
Transgenic mice overexpressing glycosylation-deficient murine PrP. For each mutant, 10% brain homogenates from two different transgenic mouse lines (roman numbers) were electrophoresed and immunoblotted using mAb 3F4. An equivalent volume of hamster brain homogenate was used as a control. Note that mutants T182N and T198A PrPC lack the diglycosylated band, whereas mutant T182N/T198N PrP only displays unglycosylated PrPC. M = molecular mass marker.

 


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  		TABLE III
BSE and scrapie challenge of transgenic mice overexpressing PrPC glycosylation mutants

Transgenic mice inoculated intracerebrally with BSE or scrapie (Chandler and Me7) were checked for their health status every 2 days. Animals showing clinical symptoms (e.g. hind limb paresis, excitation, kyphosis, abnormal tail tonus, behavioral changes, and/or weight loss) were sacrificed. Incubation times were determined as mean survival times of the mice having been euthanized or found dead. S.E. values were calculated and are given in parentheses.

 



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  		FIG. 11.
Infection of transgenic mice overexpressing glycosylation-deficient murine PrPC with murine BSE and scrapie strain Chandler. Hemizygous transgenic mice T182N expressing mutated PrPC and lacking the first glycosylation consensus site were inoculated intracerebrally with 10% brain homogenates of either BSE or scrapie strain Chandler diseased mice. Following onset of clinical disease, mice were sacrificed and the PrPSc accumulation in their brains verified by immunoblotting using mAb 3F4 as detection antibody and chemiluminescence detection. Where indicated, samples were treated with proteinase K (50 µg/ml at 55 °C for 1 h). Corresponding samples of hamster scrapie 263K and molecular mass markers were run as controls. M = molecular mass marker. +, after PK digestion; –, without PK digestion.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Diglycosylation of PrPc Is Not Mandatory for the Prion Infection Cycle in Vitro and in Vivo—In the experiments described here, we have studied the role of PrPC glycosylation in the molecular pathogenesis of prion infections. By using a systematic approach, we revealed that the PrP mutation T182N, which eliminates the first glycosylation consensus site, does not interfere with the transport of PrPC to the cell membrane. Mono- and even nonglycosylated (by additional T198A ablation of the second consensus site) PrPCs were expressed on the cell surface and were convertible in scrapie-infected neuroblastoma cells into PrPres. Moreover, the expression of these mono- and nonglycosylated PrPC mutants in neuroblastoma cells challenged by scrapie strain (Me7) lead to a de novo accumulation of mono- and nonglycosylated PrPres. This is in contrast to cloned glycosylation-deficient mutants that have been described in literature (T182A and T182A/198A). They were not convertible in our overlay experiments. This in vitro result goes along with earlier published in vivo results (18); transgenic mice expressing mutant T182A or T182A/T198A were not susceptible to scrapie infection. (Therefore, these results give rise to the speculation that not only our monoglycosylated mice will be infectious.) However, these earlier described constructs did prove to be convertible in neuroblastoma cells already infected with scrapie. In these studies, the deletion of the first glycosylation consensus site by the single point mutation threonine/alanine at codon 182 leads to aberrant trafficking and synthesis of the prion protein (19, 30, 31). Our data now support evidence reported previously that sufficient PrPC expression on the cytoplasmic membrane is a prerequisite for the susceptibility of cells (25, 26, 28, 35). This is supposed to be the reason why transgenic mice carrying a PrP T182A mutation are completely or partially scrapies-resistant (19). Based on these findings we generated glycosylation-deficient transgenic mice which proved to be BSE0 and scrapies-susceptible. Our data underline that neither the PrP glycosylation at the first nor at the second site are essential for the trafficking process and full infection cycle of prions in vivo. The results of the ongoing infection experiments are needed to learn more about the situation in which neither of the glycosylation sites are occupied.

Synthesis and Processing of Consensus Site Mutated PrPC—A number of earlier studies have addressed questions on the synthesis, processing, cellular trafficking, and the metabolism of PrPC glycosylation mutants. Variable results regarding the transport of mono- and nonglycosylated PrPC have been reported in the literature (30–32, 58). Whereas Rogers et al. (30) and Korth et al. (32) working with T182A mutations revealed no surface expression in transiently transfected CV1 and N2a cells, Lehmann et al. (31) observed this mutant on the cell surface of stable-transfected, subcloned CHO cells.

These discrepancies are presumably due to the different techniques that were used. During our experiments we found little surface expression in transfected Zeocin-treated, uncloned cell populations, whereas surface expression of the same mutant was easily detectable in CHO and N2a subcloned cells and also in uncloned murine fibroblast cells. One explanation could be that due to the high overexpression rate of the subcloned and selected cell lines that were used, their ability to control protein sorting was exhausted, which resulted in an accumulation of glycosylation-deficient PrPC which would normally be eliminated by the proteasome. The second explanation could be the high sensitivity of our assay that was achieved by using phycoerythrin (PE)-labeled conjugates as a secondary antibody. This antibody label is 20 times more sensitive than FITC, which was used in the references cited above. For the sake of comparison we also performed FACS analysis by using FITC conjugates, but we did not detect any surface expression of the T182N constructs. Moreover, in order to exclude false-positive results, an N-signal sequence deletion mutant (3F4-tagged) PrP was used as a control. To reveal possible cell line-specific effects, we tested three different cell lines (N2a, CHO, and murine fibroblast cells) in parallel but found no substantial differences.

Many glycoproteins require an intact N-glycosylation for cell surface expression (59–62). In accordance with Korth et al. (32), we could now show that PrPC is different in this respect and is rather similar to the canine histamine H2 receptor that retains its authentic trafficking after deletion of the N-glycosylation (63). Therefore, it seems that the peptide moiety plays an important role for the glycopeptide export and for directing it to a specific transport pathway (64). We believe that the distinguished surface expression of our T182N mutant is because of its retained stability as compared with the T182A mutant. In the past, spectroscopic and thermodynamic stability experiments revealed that the mutant T182A displays an extreme tendency to aggregate, resulting in a significantly reduced intrinsic stability and an extraordinary reduced cooper-ativity (65). These properties were considered to result from a destabilization of PrPC due to the loss of the two buried hydrogen bonds in the threonine side chain, to residues in the hydrophobic core (Tyr-161 and Cys-178), and to the loss of van der Waal's interactions of the absent side chain atoms within the protein. Asparagine and threonine are both neutral amino acids with two free carbon atoms in their side chain that are capable of building hydrogen bonds. Therefore, it is reasonable that only the T182N mutation produced a stable monoglycosylated PrPC molecule. Although the same would apply for a glutamine exchange mutant at this position, it must be due to the additional carbon atoms in the side chain giving the protein a bulkier shape, which leads to the destabilization of the analogous glycosylation mutant. Other than mutant T182N PrP, mutant T182A PrPres and PrPC were lacking the nonglycosylated band, which also indicates a reduced stability. Petersen et al. (45) demonstrated that the loss of glycosylation can lead to a decreased half-life time of PrPC. We found that the monoglycosylated band of T182N was running slower and broader in SDS-PAGE, indicating a more heterogeneous glycosylation pattern, which might also contribute to a higher stability of the molecule. Following synthesis, molecule aggregates are usually retained in the mid-Golgi compartment and are then eliminated by the cellular control machinery: either directly in the mid-Golgi compartment or after retrograde transport and final decomposition by the proteasome (66, 67). Due to the overexpression of the T182N mutant, some molecules must have escaped the control system and were transported onto the cell surface.

We were able to show that the destabilization of the T182A mutant was not due to the loss of the disulfide bridge. Comparing all possible mutants at codon 182 through the application of the Swiss model program (www.expasy.ch) underlined the importance of hydrogen bonds in the molecule. Similar to threonine, alanine, asparagine, and glutamine can build hydrogen bonds to cysteine 178, but only threonine and asparagine were able to build hydrogen bonds to tyrosine 161. Therefore, we postulate that the intramolecular binding between tyrosine161 (placed in {beta}-sheet 2) and the {alpha}-helix 2 is a key position for the biological behavior of PrPC.

Electrospray mass spectrometry on murine PrPres revealed that oligosaccharides attached to Asn-180 are largely composed of neutral and poorly sialylated bi- and triantennary structures within a mass range of 1.660–2.340 Da (14). In contrast, the more sialyzed and acidic glycan components at Asn-196 mainly range between 2.000 and 3.020 Da and consist of tri- and tetraantennary structures. Corresponding immunoblot patterns were revealed for the glycosylation mutants described here; PrPres and PrPC missing the first glycosylation site (T182A and T182N) produced a broader monoglycosylated band of higher molecular weight in comparison to the construct missing the second glycosylation site (T198A). The monoglycosylated band of mutant T182N was broader and had a higher molecular weight than mutant T182A. As this effect was independent from the cell line used, we propose that this is not due to a postulated different enzymatic set of the individual cell line but to the polypeptide chain itself indicating that the mutant T182N may be more stable and therefore able to bind more sugar groups.

In Vitro Convertibility of PrPC Glycosylation-deficient Mutants—The PrP conversion has been proposed to involve two steps: initial PrPC binding to PrPres followed by the folding reaction itself. Each of our glycosylation mutants was convertible into its PK-resistant isoform in scrapie-infected ScN2a cells. However, for the mutant T182N/T198A, this process was rather inefficient. Although this mutant was convertible, it was also capable of inhibiting the endogenous conversion process. This effect is described in the literature as dominant-negative inhibition (33, 35, 37, 55, 56, 68). Considering the results for the other mutants, this effect cannot be explained by the loss of glycosylation. Furthermore, this effect was not due to the single point mutation, because the corresponding single mutants T182N and T198A reacted like Wt3F4. One explanation for the observed inhibition of T182N/T198A PrP may be aberrant binding. Theoretically, there are three different binding partners: exogenous PrPC, endogenous PrPC, and endogenous PrPres. Aberrant binding could either mean autoaggregation or the interaction of endogenous with exogenous PrPC. However, autoaggregation would not explain inhibitory effects on endogenous PrPres because endogenous PrPC was not found in aggregates of exogenous PrPC in the performed solubility assays; therefore the trans-dominant inhibition must occur in the folding reaction itself. The most likely explanation is that the double mutant binds more tightly to endogenous PrPres than endogenous PrPC but requires binding to more than one PrPres molecule for conversion. Therefore, excessive exogenous PrPC molecules in the cell would outcompete PrPres binding and eventually hinder the conversion of either endogenous or exogenous PrPC.

Another less likely explanation is that T182N/T198A PrPC binds to PrPres, and due to being a rigid and stable molecule it reconverted endogenous PrPres to PrP* and PrPC. Another possible but also unlikely explanation is that T182N/T198A-PrPC is processed differently as wild type-PrPC, caused by a diverging translocation at the endoplasmic reticulum. Finally, depending on the expression level, different proportions of ctm, ntm, or sec forms are generated, some of which are possibly more or less resistant to conversion (51). This is the first description of the dominant-negative inhibition of mutant T182N/T198A, as the mutations were neither located in the forecasted region of the postulated protein X-binding site nor in the area assumed to be involved in species barrier effects (35, 36).


   FOOTNOTES
 
* This work was supported in parts by grants from the German Ministries for Education and Research and for Consumer Protection, Nutrition, and Agriculture and by the European Union commission. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Schaarschmidstrasse 6, 80995 Munich, Germany. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Institute for Novel and Emerging Infectious Diseases, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a, 17943 Greifswald, Isle of Riems, Germany. Tel.: 49-38351-7-0; Fax: 49-38351-7-191; E-mail: martin.groschup{at}rie.bfav.de.

1 The abbreviations used are: BSE, bovine spongiform encephalopathy; PrP, prion protein; CHO, Chinese hamster ovary; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; mAb, monoclonal antibody; ORF, open reading frame; moORF, mouse ORF; MEM, minimum Eagle's medium; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PK, proteinase K; PI-PLC, phosphatidylinositol-phospholipase C. Back


   ACKNOWLEDGMENTS
 
We thank Sue Priola and Bruce Chesebro (Hamilton, MT) for generously supplying us with the pSFF retroviral expression system (pSFF, packaging cells, and the knowledge) and Michael Beekes (RKI, Berlin, Germany) for contributing mAb 3F4 hybridoma supernatant. Mouse scrapie strains were kindly provided by C. Weissmann (London, UK) (Chandler), and M. E. Bruce (Edinburgh, Scotland, UK) (79A and Me7), and were propagated in mice at the Federal Research Centre. Excellent technical assistance was provided by Roswitha Fischer and Anett Buenten and methodological input by Ina Vorberg and Harriet Mella.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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