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Originally published In Press as doi:10.1074/jbc.M407360200 on October 28, 2004

J. Biol. Chem., Vol. 280, Issue 1, 685-694, January 7, 2005
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Trapping Prion Protein in the Endoplasmic Reticulum Impairs PrPC Maturation and Prevents PrPSc Accumulation*

Alessio Cardinale{ddagger}, Ilaria Filesi{ddagger}, Vito Vetrugno§, Maurizio Pocchiari§, Man-Sun Sy¶, and Silvia Biocca{ddagger}||

From the {ddagger}Department of Neuroscience, University of Tor Vergata, Via Montpellier 1, 00133 Roma, Italy, the §Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, 00161 Rome, Italy, and the Institute of Pathology and Department of Neuroscience, School of Medicine Case Western Reserve University, Cleveland, Ohio 44120

Received for publication, July 1, 2004 , and in revised form, October 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The conversion of the normal cellular prion protein (PrPC) into the abnormal scrapie isoform (PrPSc) is a key feature of prion diseases. The pathogenic mechanisms and the subcellular sites of the conversion are complex and not completely understood. In particular, little is known on the role of the early compartment of the secretory pathway in the processing of PrPC and in the pathogenesis of prion diseases. In order to interfere with the intracellular traffic of endogenous PrPC we have generated two anti-prion single chain antibody fragments (scFv) directed against different epitopes, each fragment tagged either with a secretory leader or with the ER retention signal KDEL. The stable expression of these constructs in PC12 cells allowed us to study their specific effects on the synthesis, maturation, and processing of endogenous PrPC and on PrPSc formation. We found that ER-targeted anti-prion scFvs retain PrPC in the ER and inhibit its translocation to the cell surface. Retention in the ER strongly affects the maturation and glycosylation state of PrPC, with the appearance of a new aberrant endo-H sensitive glycosylated species. Interestingly, ER-trapped PrPC acquires detergent insolubility and proteinase K resistance. Furthermore, we show that ER-targeted anti-prion antibodies prevent PrPSc accumulation in nerve growth factor-differentiated PC12 cells, providing a new tool to study the molecular pathology of prion diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The normal cellular prion protein (PrPC)1 is a copper-binding sialoglycoprotein, present in a variety of tissues but mainly expressed in the central nervous system. The precise physiological function of PrPC remains enigmatic but a wealth of experimental data demonstrates its essential role in the susceptibility and in the pathogenesis of transmissible spongiform encephalopathies (TSEs) or prion diseases (1-4). During the biogenesis, PrPC is co-translationally directed into the secretory pathway to reach the cell surface. The maturation of PrPC is achieved by the attachment of two N-linked complex carbohydrate moieties, and the addition of a GPI anchor at its C terminus (1). All three forms of prion diseases, hereditary, sporadic, and acquired by infection, share the same pathogenic mechanism based on the conversion of the normal cellular prion protein, PrPC, into the pathogenic scrapie PrP isoform, PrPSc (5). Spectroscopic studies reveal that approximately half of the {alpha}-helical structure of PrPC is converted to {beta}-sheets structure in PrPSc. After conversion, the aggregated PrPSc becomes detergent-insoluble and partially resistant to proteolytic digestion (6, 7). Proteolytic treatment of this form removes 60-70 residues from the N-terminal region of the protein, yielding a protease-resistant core called PrP27-30, whereas PrPC is completely digested (5).

Studies in cell culture have suggested that the conversion occurs post-translationally on the cell surface or in the endocytic pathway even though the precise subcellular sites and the molecular mechanisms of this process remain still poorly understood (8-10). In particular, little is known on the processing of prion protein in the endoplasmic reticulum and on the role of this compartment in the PrPC-PrPSc conversion. In cellular models of inherited prion disorders, misfolded mutant prion proteins accumulate in the ER, and activate the ER stress cytotoxic response, resulting in neuronal cell death (11). Recent studies have also shown that a small percentage of nascent wild-type PrPC and at least two pathogenic mutants are subjected to retrograde transport from the ER, accumulation in the cytoplasm and degradation by the ubiquitin-proteasome system (12-15). Accumulation of PrPC in the cytoplasm might cause neurodegeneration, indicating a possible role of this degradation pathway in the pathogenesis of prion diseases (16).

It has been reported that retrotransport of PrPC from trans-Golgi compartment toward the ER increases the production of PrPSc in persistently scrapie-infected neuroblastoma N2a cells (17). On the other hand, blocking protein export from the ER by brefeldin A inhibits de novo formation of PrPSc, suggesting that ER compartment is not competent for PrPSc formation (18).

We applied the intracellular antibody technology to specifically interfere with the intracellular trafficking of prion protein and to study the effects of PrPC re-routing on the PrPC-PrPSc conversion process. This approach is based on the ectopic expression of recombinant antibodies and their targeting to different intracellular compartments (19-21). The intrabody can mediate its effect inside the cells in multiple ways: (i) by inactivating the target protein through its binding to the functional domain (22, 23) or (ii) by relocating the antigen to a different subcellular location (24). For example, intrabodies carrying the ER retention signal, have been used to bind proteins, including GPI-anchored, in the ER, and inhibit their intracellular traffic through the secretory pathway (25-27).

Here we report the cloning and the intracellular expression of two single chain antibody fragments (scFv) directed against different epitopes of the prion protein, targeted to the secretory compartment of mammalian cells. The recombinant anti-prion scFvs, tagged with the ER retention signal KDEL, were used to specifically trap the PrPC in the ER. The stable expression of ER-tagged anti-prion scFvs in PC12 cells induces a marked surface depletion of PrPC. Relocation of PrPC to the ER also impairs its maturation, enhances its proteinase K resistance and detergent insolubility. Finally we show that anti-prion intrabodies prevent PrPSc formation in NGF-differentiated PC12 cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Engineering of the 8H4 and 8F9 scFv Fragments—The V regions of two anti-prion monoclonal antibodies (mAbs) 8H4 and 8F9 were cloned using a phage display system as previously described (28). Briefly, cDNA was synthesized from total RNA of hybridoma cell lines using CKFOR primer for VK and MOCG12FOR primer for VH. For the amplification of VK gene a mixture of eighteen 5'-primers (VKBACK mix) and a mixture of five 3'-primers (VKFOR mix) were used. For the amplification of VH gene a mixture of twenty 5'-primers (VHBACK mix) and a mixture of five 3'-primers (VHFOR mix) were used (29). The VH and VL repertoires were separately cloned into the pDAN vector for growth of phage(mid) particles (30).

Expression of scFv Fragments in Escherichia coli—Supernatants containing the scFv-expressing phages were analyzed in a phage-ELISA (31) for binding to coated recombinant human prion protein (rec-PrP23-231) and lysozyme as a negative control. Positive clones were further analyzed by fingerprinting and sequencing. Selected anti-prion scFvs and the anti-lysozyme scFv D1.3 (32) were expressed in E. coli HB2151 non-suppressor strain and purified by affinity chromatography using Ni-nitrilotriacetic acid agarose (Qiagen). ELISA assay was carried out by coating the plate with 1 µg of recombinant prion protein and 300 µg of lysozyme (Sigma). After saturation with 2% w/v PBS-BSA solution, 50 µl of purified scFv, diluted in PBST, 2% BSA, was added. ELISA and Western blotting were performed using mouse anti-V5 IgG-linked horseradish-peroxidase (Invitrogen).

DNA Constructs—For the expression in mammalian cells, phage-derived 8H4 and 8F9 scFv fragments were subcloned into the BssHII/NotI sites of the secretory (Sec) and ER-retained (KDEL) versions of the pscFvexpress vectors (33). For PCR amplification of scFvs from the bacterial pDAN-scFv vector, the following degenerate primers were designed: 5'-GCAGCAAGCGGCGCGCACTCC and 3'-TTTAGCGGCCGCTGGGATTGGTTT. The {alpha}D11 scFv, derived from pscFvexp-cyt-{alpha}D11 (34), was subcloned into the pscFvexp-KDEL vector. Plasmid encoding for human PrP for expression in mammalian cells was provided by A. Negro, University of Padova.

Cell Lines, Transfection, and Drug Treatments—HEK-293 fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were transiently transfected with Superfect reagent (Qiagen) with a DNA/Superfect ratio (w/v) of 1:5 and analyzed after 48 h. PC12 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum and 5% FBS. For generation of stable clones, cells were transfected using Lipofectamine 2000 (Invitrogen), with a DNA/lipid ratio (w/v) of 1:4. At least 50 G418-resistant clones were isolated after 3-4 weeks. 20% of resistant clones were positive for scFv expression. To induce neuronal differentiation, PC12 cells were plated on flasks coated with rat tail collagen (Sigma), in complete medium containing 100 ng/ml NGF. Medium was replaced every 2-3 days. For drug treatment, cells were cultured for 16 h in complete medium containing 5 µg/ml tunicamycin (Roche Applied Science) or 5-10 µg/ml brefeldin A (Sigma).

Immunofluorescence Staining—Immunofluorescence was carried out as described (35). For surface staining of PrPC, cells were rinsed with ice-cold PBS and incubated for 1 h at 4 °C with MAb 7A12 (36) diluted in Opti-MEM (Invitrogen) containing 0.2 mg/ml BSA. After washing with ice-cold PBS, cells were fixed in 4% paraformaldehyde for 10 min at room temperature. Affinity-purified rabbit anti-Myc (Santa Cruz Biotechnology), mouse anti-Myc IgG 9E10 (Invitrogen), mouse anti-prion 3F4 (Sigma), and goat anti-calnexin (Santa Cruz Biotechnology) were used as primary antibodies and CyTM 2-conjugated AffiniPure donkey anti-rabbit IgG, CyTM 2-conjugated AffiniPure donkey anti-mouse IgG (Jackson Immunoresearch), Texas-red goat anti-mouse IgG (Calbiochem), CyTM 2-conjugated AffiniPure rabbit anti-goat IgG (Jackson Immunoresearch) and Rhodamine RedTM-X-conjugated AffiniPure donkey anti-mouse IgG (Jackson Immunoresearch) were used as secondary antibodies. Samples were examined with DMRA Leica fluorescence microscope equipped with CCD camera. Acquired images were deconvolved using Leica Qfluoro software and processed using Adobe Photoshop.

Western Blot Analysis—For prion detection, cells were rinsed twice with ice-cold PBS and lysed for 30 min in ice-cold cell extraction buffer (EB)(100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 0.3 µM aprotinin). Nuclei and large debris were removed by centrifugation at 290 x g for 10 min at 4 °C. The supernatants were then precipitated with 5 volumes of MeOH at -20 °C for 2 h. After centrifugation (16,000 x g, 10 min, 4 °C), protein pellets were dissolved in 4x sample buffer (500 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 40 mM dithiothreitol, and 0.02% bromphenol blue) and heated at 95 °C for 5 min.

For scFv detection in the medium, cells were grown in Opti-MEM for 48 h, and collected supernatants were precipitated in 10% trichloroacetic acid. Pellets were dissolved in 4x sample buffer. Equal amounts of proteins were separated by SDS-PAGE in 12% acrylamide gels and transferred to polyvinylidene difluoride membranes (Amersham Biosciences) for 16 h at 30 V. Blots were probed with mAbs 7A12 and 3F4, and mouse anti-Myc 9E10 IgG (Invitrogen). Immunoreactive bands were detected with sheep anti-mouse IgG horseradish peroxidase and visualized by ECL (Amersham Biosciences).

Metabolic Labeling and Immunoprecipitation—Prior to pulse labeling, cells were washed twice in PBS and incubated in methionine/cysteine-free RPMI medium (Sigma) containing 5% dialyzed FBS for 1 h at 37 °C. Cells were metabolically labeled with 270-300 µCi/ml 35S-Trasilol (Promix, Amersham Biosciences) in methionine/cysteine-free RPMI medium containing 5% dialyzed serum for indicated times. Cells were washed and chased in complete medium containing 2 mM methionine and cysteine and 5% FBS for indicated times. At the end of the chase period, cells were harvested and lysed as described above. Clarified cell lysates were subjected to immunoprecipitation with mAb 7A12 linked to 30 µl of protein A-Sepharose beads (Amersham Biosciences) for 3 h at 4 °C. Bound proteins were washed with 50 mM Tris-HCl, pH 7.5, 500 mM NaCl and 0.1% Nonidet P-40, eluted in sample buffer and analyzed by SDS-PAGE. Gels were fixed, impregnated with Amplify (Amersham Biosciences) and exposed to autoradiographic film.

Assay of Detergent Insolubility and Proteinase K Resistance—Post-nuclear supernatants were ultracentrifuged at 100,000 x g for 1 h at 4 °C to separate detergent-soluble (S) and detergent-insoluble (P) proteins. Insoluble fractions were dissolved in 1 ml of lysis buffer (EB). After methanol precipitation, proteins from both fractions were immunoblotted. For evaluation of PK-resistance, cells were lysed in ice-cold EB in the absence of protease inhibitors and clarified by low-speed centrifugation at 290 x g. Equal amounts of proteins were digested with different concentration of PK (Roche Applied Science) (1, 15, and 20 µg/ml) for 30 or 60 min. Digestions were stopped by the addition of 0.8 mg/ml phenylmethylsulfonyl fluoride. Proteins were methanol-precipitated and immunoblotted.

Enzymatic Reactions—For enzymatic deglycosylation, clarified cell lysates were methanol-precipitated, resuspended in denaturating buffer (0.5% SDS, 1% {beta}-mercaptoethanol), and boiled for 10 min. Denaturated proteins were digested with PNGase-F (1000 units in 1% Nonidet P-40, 25 mM sodium phosphate, pH 7.5) or endoglycosidase-H (New England Biolabs) (1000 units in 50 mM sodium citrate, pH 5.5) for 3 h at 37 °C. For PI-PLC digestion cells were cultured for 16 h in Opti-MEM medium containing 0.8 units/ml PI-PLC (ICN). Supernatants containing PI-PLC-released proteins were methanol-precipitated and immunoblotted. For pulse chase experiments 0.8 units/ml of PI-PLC were added to the medium 30 min before the end of each chase point.

MTT Analysis—2 x 104 PC12 cells were grown in 96-well plates and treated with different concentration of tunicamycin or brefeldin A (as indicated in Fig. 7) for 16 h. Cell viability was quantified using MTT (Sigma) assay as described (37). The data presented represent two independent experiments performed in triplicate.



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  		FIG. 7.
MTT analysis. PC12 (dark columns) and KDEL-8H4-PC12 (white columns) cells were incubated with tunicamycin and brefeldin A for 16 h as indicated. The results shown are the average of three different experiments performed in triplicate.

 
Scrapie Infection of NGF-treated PC12 Cells—PC12 cells were differentiated with NGF for 8 days prior to infection. Infection was performed by incubating cells with 0.5 ml serum-less RPMI medium and 0.5 ml of 0.45 micron-filtered 139A scrapie strain inoculum for 6 h at 37 °C with occasional shaking. Inoculum was obtained from 10% (w/v) PBS suspension of pooled mouse brains from clinically scrapie affected mice at terminal stages of disease. Cells were rinsed twice with serum-less medium. Some cultures were collected and stored at -80 °C. Rest cultures were maintained in the presence of NGF for 3 weeks. Cells (6 h p.i. and 3 weeks p.i.) were lysed for 10 min in 50 mM Tris/HCl pH 7.5, NaCl 150 mM, 0.5% Triton X-100, 0.5% sodium deoxycholate at 4 °C, and centrifuged at 290 x g for 10 min. Postnuclear supernatants were digested with 20 µg/ml proteinase K for 1 h at 37 °C and centrifuged at 100,000 x g for 1 h at 4 °C. Pellets were immunoblotted for PrPSc detection. Experiments were all performed in triplicates.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Phage Display Cloning of Anti-prion scFv Antibodies—We used the phage display system to clone the V regions of two well characterized anti-PrP mAbs 8H4 and 8F9. mAb 8H4 recognizes residues 175-185 of helix B and mAb 8F9 binds the linear epitope 225-231 at the C terminus of PrP (38, 39). These antibodies are IgG1 subtype, recognize all described PrPC species and are able to react with both PrPC and PrPSc isoforms (36, 38, 40).

Selected 8H4 and 8F9 anti-prion scFvs were expressed in bacteria, purified, and analyzed by ELISA assay and Western blotting. As shown in Fig. 1A the two purified scFv fragments maintain the same immunogenic properties of the original mAbs 8H4 and 8F9. Western blot of affinity-purified scFvs is shown in Fig. 1B. A major 36-kDa band, corresponding to the expected size of 8H4 (lane 1) and 8F9 (lane 2) scFvs is detected.



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  		FIG. 1.
Expression of the 8H4 and 8F9 scFv fragments in E. coli. A, ELISA to measure the binding activity of purified scFv fragments and the parental Ig. A solution of 10 µg/ml of human prion protein (dark columns) and 3 mg/ml of lysozyme (light columns) was used for coating. As a negative control, a purified anti-lysozyme D1.3 scFv fragment was used. B, Western blot analysis of the purified scFv 8H4 (lane 1) and 8F9 (lane 2) obtained by affinity chromatography. The slight difference in molecular masses between the two scFv fragments is caused by differences in the primary sequence. The migration of molecular mass markers (in kDa) is shown on the left.

 
ER-targeted Anti-prion scFv Fragments Bind PrPC in Living Cells—To test whether 8H4 and 8F9 scFv fragments are able to interact with PrPC in living cells, we subcloned them into vectors optimized for intracellular expression in the secretory pathway of mammalian cells. For retention in the ER, the ER retention sequence SEKDEL was added at the C terminus of the scFv as described (33, 41).

We used co-immunoprecipitation to determine whether the anti-prion intrabodies interact with PrPC in the transfected cells. Constructs expressing Myc-tagged 8H4, or Myc-tagged-8F9 scFvs and human PrPC were transiently co-transfected into HEK-293 cells, and cells were processed for analysis 48 h after transfection. Cellular lysates were first immunoprecipitated with anti-prion mAb 3F4, separated by SDS-PAGE, and then immunoblotted with anti-Myc mAb 9E10 to reveal the scFvs. We found an immunoreactive band, corresponding to the scFv, only in extracts derived from cells expressing the secreted 8H4 (Sec-8H4) and the ER-retained 8H4 (KDEL-8H4) scFvs (Fig. 2A, lanes 3 and 4). On the contrary, the anti-prion 8F9 (lanes 1 and 2) and the irrelevant {alpha}D11 scFvs (lane 5) did not co-immunoprecipitate with PrPC. This result suggests that the C-terminal epitope of PrPC, which is recognized by the 8F9 intrabody, is masked during PrPC biogenesis. Alternatively, the affinity of 8F9 scFv is not optimal for in vivo studies, although this antibody fragment efficiently reacts with bacterial produced recombinant human PrP (Fig. 1A). The 48-kDa band is caused by nonspecific binding of the precipitating antibody. The efficiency and the specificity of PrPC immunoprecipitation were confirmed by probing the same membrane with the anti-prion 3F4 mAb (lanes 6-10).



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  		FIG. 2.
In vivo interaction between anti-prion intrabodies and PrP in mammalian cells. A, HEK-293 cells were transiently cotransfected with DNA encoding scFv fragments and human recombinant PrP and analyzed 48 h later. Proteins from cells transfected with Sec-8F9 (lanes 1 and 6), KDEL-8F9 (lanes 2 and 7), Sec-8H4 (lanes 3 and 8), KDEL-8H4 (lanes 4 and 9) and KDEL-{alpha}D11 (lanes 5 and 10) were immunopurified with anti-prion 3F4 and then probed with anti-Myc 9E10 (lanes 1-5) and anti-prion 3F4 (lanes 6-10). The arrow points to the Sec-8H4 and KDEL 8H4 co-immunopurified scFv fragments. The slightly higher molecular mass (about 1 kDa) of the KDEL-8H4 scFv (lane 4) is caused by the presence of the retention sequence SEKDEL. The migration of molecular mass markers (in kDa) is indicated on the right and the heavy ({gamma}) and light ({kappa}) chains are indicated on the left. B, HEK-293 cells were transiently co-transfected with human recombinant PrP and KDEL-{alpha}D11 (panels A-C), KDEL-8H4 (panels D-F), and KDEL-8F9 (panels G-I) scFv fragments. The localization of the intrabodies was determined using anti-Myc antibodies (A, D, and G) and the distribution of PrP by mAb 3F4 (B, E, and H). Panels C, F, and I represent the merged images. C, HEK-293 cells were transiently transfected with KDEL-8H4-scFv and stained with anti-Myc 9E10 (panel A) and anti-calnexin antibodies (panel B). Merged image is shown in panel C.

 
The in vivo interaction was further verified by double staining on co-transfected HEK-293 cells. Fig. 2B shows the immunofluorescence analysis performed in fixed and permeabilized cells using anti-Myc antibodies to detect Myc-tagged scFv fragments (panels A, D, and G) and mAb 3F4 to reveal human PrPC (panels B, E, and H). Merged images are shown in panels C, F, and I. This analysis revealed that both anti-prion and the irrelevant KDEL-scFvs were efficiently expressed, localized in the secretory compartment, and accumulated mostly in the ER (see panels A, D, and G). When we explored the effect of intracellular scFvs expression on PrPC localization, we found that only the expression of KDEL-8H4 scFv (panel D, green signal) resulted in a dramatic alteration in the distribution of PrPC (panel E, red signal) in all transfected cells. A strong co-localization with the anti-PrPC intrabody is evident in the merged image (panel F). On the contrary, the expression of Sec-8H4, Sec-8F9 (not shown), the irrelevant scFv fragment (panel A) or the KDEL-8F9 scFv (panel G) did not alter the intracellular distribution of PrPC (panels B, C, and H and I). Because Sec-8H4 did co-immunoprecipitate with PrPC (Fig. 2A); therefore, the redistribution of PrPC must be caused by the retention of PrPC in the ER by KDEL-8H4 scFv rather than simply binding of the scFv to PrPC.

To directly analyze the intracellular distribution of the KDEL-8H4-scFv in the endoplasmic reticulum, we co-localized the scFv (Fig. 2C, panel A) with the ER marker calnexin (panel B). As it is shown in the merged image (panel C), KDEL-8H4 scFv showed remarkably precise co-localization with calnexin.

Altered Intracellular Distribution and Glycosylation Pattern of Endogenous PrPC in KDEL-8H4-PC12 Cells—PC12 cells, originally derived from a rat pheochromocytoma, respond to the nerve growth factor (NGF) by ceasing cell division and developing into cells with morphological, electrophysiological, and biochemical properties of sympathetic neurons (42). PC12 cells express PrPC on their cell surface, and the NGF-induced differentiation does not alter the biochemical properties of the endogenous PrPC (43). Moreover, differentiated PC12 cells are susceptible to scrapie infection (44, 45).

We, therefore, generated PC12 stable cell lines expressing either KDEL-8H4 scFv (KDEL-8H4-PC12) or Sec-8H4 scFv (Sec-8H4-PC12). To determine whether the KDEL-8H4 scFv is efficiently retained in PC12 cells, we analyzed and compared, by Western blot, the quantity of scFv fragments present in the medium of Sec-8H4-PC12 and KDEL-8H4-PC12 cells. As shown in Fig. 3A, KDEL-8H4 scFv was completely retained intracellularly (lane 3) and was not detectable in the medium (lane 4). In contrast, most of Sec-8H4 scFv accumulated in the medium in 48 h (lanes 1 and 2).



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  		FIG. 3.
Characterization of PrPC in KDEL-8H4-PC12 cells. A, Western blot of cellular lysates (c) and collected medium (m) from Sec-8H4-PC12 (clone 9, lanes 1 and 2) and KDEL-8H4-PC12 cells (clone 13, lanes 3 and 4). The scFv fragments were detected by rabbit anti-Myc antibodies. B, unpermeabilized (panels A and C) and permeabilized (panels B and D) wild-type and KDEL-8H4-PC12 (clone 13) cells were immunostained for PrPC. Immunoreactivity is detected at the plasma membrane (panel A) in unpermeabilized and in the Golgi area (panel B) in permeabilized PC12 cells. In contrast, cell surface PrPC fluorescence is almost abolished in unpermeabilized KDEL-8H4-PC12 (panel C) and PrPC shows a reticular pattern (panel D) in permeabilized KDEL-8H4-PC12 cells. C, Western blot of lysates prepared from wild-type (lane 1), KDEL-{alpha}D11-PC12 (clone 24, lane 2), Sec-8H4-PC12 (clone 28, lane 3), and KDEL-8H4-PC12 (clone 13, lane 4) cells. D, Western blot of lysates prepared from wild-type PC12 (lanes 1 and 2) and KDEL-8H4-PC12 (lanes 3 and 4) cells incubated for 3 h with PNGase F (1000 units), as indicated. E, Western blot of lysates derived from wild-type (lanes 1 and 2) and KDEL-8H4-PC12 (lanes 3 and 4) cells cultivated with tunicamycin (5 µg/ml for 16 h) as indicated. F, Western blot of cellular lysates (c) and collected medium (m) from wild-type PC12 (lanes 1 and 2) and KDEL-8H4-PC12 cells (lanes 3 and 4) treated for 16 h with PI-PLC (0.8 units/ml). The asterisk highlights the 31-kDa fragment present only in KDEL-8H4-expressing cells. PrPC was detected with mAb 7A12. Molecular mass markers are given in kilodaltons. The blots shown are representative of four different experiments.

 
We used these two cell lines to distinguish whether the effects of the intrabodies are caused by the specific epitope binding and/or retention of the PrPC in the ER. A stably transfected KDEL-{alpha}D11 scFv PC12 cell line (KDEL-{alpha}D11-PC12), which expresses an irrelevant ER-retained scFv was also generated and used as control.

Immunofluorescence analysis of endogenous PrPC in control PC12 cells and KDEL-8H4-PC12 cells is shown in Fig. 3B. A much weaker PrPC surface fluorescence was detected in unpermeabilized KDEL-8H4-PC12 cells (panel C) compared with PC12 cells (panel A). When PC12 cells were fixed, permeabilized and then examined, PrPC signal was mostly concentrated in small perinuclear patches (panel B), which represent the Golgi compartment. In contrast, PrPC was not detectable in the Golgi patches of permeabilized KDEL-8H4-PC12 cells. In these cells we observed a more widespread staining pattern characteristic of typical ER distribution (panel D). PrPC localization was not altered in KDEL-{alpha}D11-PC12 cells (data not shown).

We next investigated whether the expression of the intrabody alters the steady state synthesis of PrPC in PC12 cells. Immunoblots were probed with mAb 7A12, which is specific to PrPC residues 143-155. Immunoreaction of PC12 and KDEL-{alpha}D11-PC12 cell lysates showed three major species of PrPC comprising the unglycosylated form at 29 kDa, the intermediate forms of 32-35 kDa, and the highly glycosylated forms of 43-45 kDa (Fig. 3C, lanes 1 and 2). In contrast, KDEL-8H4-PC12 cells displayed a distinctive pattern of PrPC immunoreactivity (lane 4), which was characterized by an increase of the unglycosylated and intermediate species, and a decrease of the highly glycosylated species. Moreover, an additional 31-kDa band (see asterisk in lane 4) is evident. Interestingly, the profile of the PrPC in Sec-8H4-PC12 cells was not changed (lane 3). These results suggest that retention of PrPC in the ER drastically alters the post-translational modifications of PrPC.

We used in vitro deglycosylation with PNGase-F to determine the contribution of N-linked glycans to the complexity of PrPC species in PC12 and KDEL-8H4-PC12 cells. Removal of the N-linked glycans resulted in the appearance of a major band of 29 kDa, which is the full-length, protein backbone of PrPC, and a minor species migrating at 21 kDa. This N-terminal truncated PrPC fragment is present in both cell types (Fig. 3D, lanes 2 and 4). Similar results were also obtained by the inhibition of N-linked glycosylation in vivo with tunicamycin (Fig. 3E, lanes 2 and 4). Since deglycosylation of PrPC led to the total disappearance of the 31-kDa band (see asterisks), we concluded that this PrPC species is not an unprocessed PrPC, with either the leader peptide sequence at the N terminus or the GPI anchor signal at the C terminus, but instead a partially glycosylated PrPC species. Densitometric analysis revealed that the total amounts of PrPC did not significantly differ between PC12 cells and KDEL-8H4-PC12 cells; the percentage of various glycoforms however differed significantly.

We next compared the quantity of membrane-bound PrPC in PC12 and KDEL-8H4-PC12 cells. Cells were treated with PI-PLC to release the cell surface PrPC. Seventy percent of total PrPC is released by PI-PLC in PC12 cells (Fig. 3F, lane 2). In contrast, only about 30% of total PrPC was recovered into the medium in similarly treated KDEL-8H4-PC12 cells (lane 4). In these cells, the bulk of the PrPC is present intracellularly, including the 31-kDa form, which is not translocated to the cell surface. Moreover, biotinylation of cell surface proteins confirms the down-regulation of membrane-associated PrPC (not shown). This finding excludes the possibility that intrabody-PrPC interaction may lead to an aberrant association of the PrPC with the plasma membrane, resulting in resistance to PI-PLC digestion.

These experiments revealed that retention of PrPC in the ER of KDEL-8H4-PC12 cells has a profound effect on post-translational modifications and cellular trafficking of PrPC, resulting in a decrease in the matured PrPC isoforms, and the accumulation of an additional 31-kDa glycoform.

The 31-kDa PrPC Species in KDEL-8H4-PC12 Cells Is Susceptible to Digestion by Endoglycosidase H—Endo-H is an enzyme that cleaves high mannose and hybrid structures forming in the ER compartment; consequentially, proteins with complex N-linked oligosaccharides, present in the mid-Golgi are resistant to endo-H. The expression of KDEL-tagged anti-prion scFv fragments should trap the PrPC in the ER. Therefore, these trapped PrPC species may be sensitive to endo-H treatment. To assess whether this is indeed the case, PC12 and KDEL-8H4 PC12 cell lysates were first treated with endo-H, and then immunoblotted with mAb 7A12 as described (Fig. 4A). As expected, no change in PrPC pattern was observed after endo-H digestion in PC12 cells (lanes 1 and 2). In contrast, endo-H treatment of KDEL-8H4-PC12 cells produced a slight decrease in the highly and intermediately glycosylated PrPC species and the complete disappearance of the 31-kDa species (lanes 3 and 4). This finding indicated that the 31-kDa band is most likely a high mannose core glycosylated PrPC species, which has not yet transited beyond the mid-Golgi.



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  		FIG. 4.
The 31-kDa PrPC species in KDEL-8H4-PC12 cells is Endo-H-sensitive. A, Western blot of lysates prepared from wild-type PC12 (lanes 1 and 2) and KDEL-8H4-PC12 cells (lanes 3 and 4) incubated for 3 h with Endo-H (1000 units). The asterisk points to the 31-kDa glycoform in KDEL-8H4-PC12 cells. B, Western blot of wild-type PC12 cells incubated with brefeldin A, as indicated. Arrow indicates the 31-kDa species, which appears in BFA-treated PC12 cells. PrPC was detected with mAb 7A12. Molecular mass markers are shown on the right.

 
To gain additional support for this interpretation, we treated wt-PC12 cells with BFA, a fungal metabolite, which causes fusion of the ER with cis/medial Golgi and inhibits the export of the proteins from these compartments (46). Indeed, treatment of PC12 cells with different concentrations of BFA (Fig. 4B, lanes 2 and 3) resulted in a profile strikingly similar to that observed in KDEL-8H4-PC12 cells (see Fig. 3C, lane 4): decrease in the mature PrPC glycoforms, accumulation of the intermediate and unglycosylated forms and appearance of the 31-kDa species.

Intrabody-mediated ER Trapping Affects PrPC Maturation—To better understand the complexity and kinetic of PrPC biogenesis, we performed metabolic labeling experiments. After increasing periods of chase, total cell-associated radiolabeled PrPC species were immunoprecipitated with mAb 7A12 and analyzed by SDS-PAGE. In PC12 cells, after 20-min pulse, three major PrPC species were detected (Fig. 5A, lane 1). The upper diffuse band (at around 43 kDa) was barely visible. This was the fully matured PrPC that had undergone terminal processing (complex type glycans). This PrPC species increases during chase and becomes the predominant form within 70 min (lane 4). Noticeably, in KDEL-8H4-PC12 cells the 43-kDa complex glycosylated form was missing, or only barely detectable at the latest chase time (lanes 5-8). In addition, in PC12 cells there was a faster migrating 31-kDa band (arrow), which corresponds to the endo-H-sensitive PrPC species (see Fig. 4A) with high mannose in KDEL-8H4-PC12 cells. This species is completely converted to complex structures within 40 min of chase in PC12 cells (lane 3). On the contrary, in KDEL-8H4-PC12 cells the 31-kDa species (asterisk) was stable within 70 min of chase (lanes 5-8). It is worth noting that the 29-kDa unglycosylated form of PrPC was almost undetectable in the KDEL-8H4-PC12 cells. While the maturation of PrPC in control PC12 cells is rapid and efficient, on the contrary the N-linked glycans of PrPC in KDEL-8H4-PC12 cells are not processed into complex structures, and the overall PrPC maturation is significantly disrupted.



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  		FIG. 5.
Kinetics of PrPC biosynthesis in wild-type PC12 and KDEL-8H4-PC12 cells. A, exponentially growing wild-type (lanes 1-4) and KDEL-8H4-PC12 (lanes 5-8) cells were metabolically labeled for 20 min with [35S]Trasylol and incubated in chase medium for increasing times, as indicated. Total cell-associated-labeled PrPC species were immunoprecipitated with mAb 7A12. B, following 30-min pulse, cell surface (PI-PLC released) (lanes 1-3) and intracellular (after PI PLC treatment) (lanes 4-7) PrPC, derived from wild-type PC12 cells, were separately immunoprecipitated at increasing chase times, as indicated. C, same protocol was applied to KDEL-8H4-PC12 cells to study the kinetic of cell surface (lanes 1-3) and intracellular (after PI-PLC treatment) (lanes 4-7) PrPC. The asterisks point to the 31-kDa species in KDEL-8H4-PC12 cells. Arrows indicate the 31-kDa glycoform in wild-type PC12 cells. The results shown are representative of three similar experiments. Molecular mass markers are shown on the left.

 
To further investigate the processing, turnover and transport of cell-surface PrPC, we treated the metabolically labeled PC12 cells and KDEL-8H4-PC12 cells with PI-PLC. The surface PrPC (PI-PLC-released) and the "intracellular" PrPC (after PI-PLC treatment) were then analyzed separately. As shown in Fig. 5B, 50% of matured PrPC reached the cell surface and was releasable by PI-PLC treatment in PC12 cells after a 90-min chase (lane 1) and 70-80% after 180 min (lanes 2 and 3). On the contrary, in KDEL-8H4-PC12 cells, 80-90% of PrPC was still retained intracellularly within 180 min of chase (Fig. 5C, compare lanes 6 and 2). Interestingly, the small amount of PrPC that reached the cell surface within the chase period did not include the 31-kDa form. Therefore, this species never reached the cell surface (lanes 1-3). Moreover, degradation of the membrane-associated PrPC is also impaired in KDEL-8H4-PC12 cells. Thus, while in PC12 cells the membrane PrPC started to be degraded within 390 min chase (Fig. 5B, lane 3), at this time the PrPC was still accumulating in KDEL-8H4-PC12 cells (Fig. 5C, lane 3), probably indicating either a strong delay in the translocation process or an alteration in its catabolism.

Finally, we performed densitometric analyses of PrPC in pulse chase experiments and found that the amount of immunoprecipitable PrPC in PC12 cells is 40% higher than in KDEL-8H4-PC12 cells. Since there is no difference in total label incorporation, the lower amount of soluble PrPC in KDEL-8H4-PC12 cells is related to misfolding and/or aggregation of the ER-retained PrPC.

Trapping PrPC in the ER Induces Detergent Insolubility and Partial Protease Resistance—The forced retention of PrPC in the ER of KDEL-8H4-PC12 cells allowed us to investigate whether the intrabody-mediated impairment of maturation provokes an accumulation of misfolded PrPC in the ER at steady state, finally resulting in its aggregation.

To explore PrPC solubility in non-denaturing detergents, deoxycholate-Nonidet P-40 cell lysates derived from wt-PC12 cells or KDEL-8H4-PC12 cells were ultracentrifuged to prepare a detergent-soluble supernatant (S) and a detergent-insoluble pellet (P). After separation by SDS-PAGE, each fraction was immunoblotted with mAb 7A12 (Fig. 6A). As expected, in PC12 cells nearly all the PrPC is detected in the detergent-soluble fraction (S). In contrast, in KDEL-8H4-PC12 cells, ~40% of total PrPC is found in the detergent-insoluble pellet (P).



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  		FIG. 6.
ER-retained PrPC is detergent-insoluble and partially protease-resistant. A, postnuclear supernatants from wild-type PC12 and KDEL-8H4-PC12 cells were ultracentrifuged to separate detergent soluble (S) and insoluble (P) proteins. B, postnuclear supernatants from wild-type PC12 and KDEL-8H4-PC12 cells were digested with increasing concentration of PK, as indicated. PrPC was detected by Western blotting using mAb 7A12. Asterisks (in A and B) denote the 31-kDa species in KDEL-8H4-PC12 cells. Blots are the mean of three independent experiments.

 
We next tested the protease resistance of the PrPC species from PC12 and KDEL-8H4-PC12 cells by subjecting them to treatment with different concentrations of PK for 30 min at 37 °C. As shown in Fig. 6B, PrPC species from PC12 cells were completely digested with 15 µg/ml of PK. Under identical conditions, PrPC species from KDEL-8H4-PC12 cells were significantly more resistant to PK, yielding three major protease-resistant bands, at 20, 28, and a prominent band of 23 kDa. A complete digestion of PrPC was obtained at 20 µg/ml PK treatment for 1 h. From these studies, we concluded that, in our cell model, the endoplasmic reticulum is the site where PrPC becomes detergent-insoluble and acquires partial PK resistance.

The accumulation of misfolded, detergent-insoluble, and PK-resistant PrPC molecules in KDEL-8H4 PC12 cells did not alter cellular viability and proliferation rate under normal conditions. However, since accumulation of misfolded proteins in the ER can sensitize the cells to ER-stress stimuli (47), we investigated whether specific ER stress responses are activated in KDEL-8H4-PC12 cells. In order to study the vulnerability to treatments with ER stress inducers, we treated the cells with different concentrations of brefeldin A and tunicamycin and assayed the cell viability by MTT analysis. As it can be seen in Fig. 7, the response to these drugs was similar in wild-type PC12 and KDEL-8H4-PC12 cells.

Down-regulation of Cell Surface PrPC Prevents PrPSc Accumulation—Finally, we addressed the question whether intrabody expression prevents PrPSc accumulation in NGF-differentiated PC12 cells. It has been shown previously that these cells support 139A scrapie strain replication beginning from 3 weeks postinfection as determined by bioassays (44, 45). Thus, we infected NGF-differentiated PC12 and KDEL-8H4-PC12 cells with 139A scrapie strain (Fig. 8, lane 1). At 6 h or 21 days after infection, the presence of PK resistant PrPSc in control non-infected cells or infected cells were analyzed by immunoblots with mAb 7A12. As expected, no PK-resistant PrPC species is detected in non-infected wild-type PC12 cells or KDEL-8H4-PC12 cells (lanes 2 and 5). At 6 h postinfection, equal amount of PK resistant PrPSc were detected in wild-type PC12 cells and KDEL-8H4-PC12 cells (lanes 3 and 6). These PK-resistant PrPSc likely represent the cell-bound inoculum, which has not been removed after washing. Therefore, both PC12 and KDEL-8H4-PC12 cells are able to take up comparable amounts of PrPSc. In sharp contrast, at 21 days postinfection, PK-resistant PrPSc were detected only in wild-type PC12 cells (lane 4), but not in KDEL-8H4-PC12 cells (lane 7). These results suggest that KDEL-8H4-PC12 cells are unable to support de novo PrPSc synthesis. In vivo bioassays to determine whether in KDEL-8H4-PC12 cells scrapie infectivity is also prevented are ongoing.



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  		FIG. 8.
PrPSc accumulation in NGF-differentiated PC12 cells. Detection of PrPSc in NGF-differentiated wild-type (lanes 2-4) and KDEL-8H4-PC12 (lanes 5-7) cells incubated with 139A scrapie strain (lane 1). Postnuclear supernatants derived from noninfected cells (ni), cells incubated with the scrapie agent for 6 h (6h) or cells cultured for 21 days (21d) after infection were digested with 20 µg/ml PK for 1 h at 37 °C before ultracentrifugation and running on SDS-PAGE. PrPSc was detected using mAb 7A12. The blot shown is representative of three different experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented here demonstrate, for the first time, that anti-prion intrabodies targeted to the lumen of the ER provide a simple and effective means to inhibit the transport of PrPC to the cell surface. The availability of an effective tool to interfere selectively with the intracellular location of PrPC without affecting other membrane proteins allowed to study the biochemical features of ER-retained PrPC molecules and the role of the plasma membrane-associated PrPC in the process of PrPSc formation.

An absolute requirement for intracellular antibodies to function properly is their ability to interact with the endogenous antigen in vivo, in the specific compartment where they are targeted to (20). Our studies and others have highlighted the particular efficiency of this approach to interfere with proteins in the secretory compartment, because of the higher stability and solubility of single chain antibodies in this environment (20, 48). Both anti-prion 8H4- and 8F9-scFvs, described in this report, are efficiently expressed in mammalian cells and localize in the ER. Furthermore, there is no toxicity associated with the expression of these scFv fragments.

During its biosynthesis, PrPC undergoes a series of post-translational modifications, which includes cleavage at both the N and C termini to remove the signal peptide and the GPI anchor signal peptide, addition of N-linked glycans, formation of a disulfide bond, and attachment of a GPI anchor. The KDEL-8H4 anti-prion intrabody expression leads to the accumulation of PrPC in the ER and to the impairment of this maturation process. We found, in particular, aberrant glycosylation of PrPC, with the appearance of a new 31-kDa species. This aberrant PrPC species is not an unprocessed N- or C-terminal product. In fact, it migrates as the unglycosylated 29-kDa species in SDS gels following treatment by either tunicamycin in vivo or PNGase-F in vitro. The 31-kDa species is most likely produced because of a delay in its exit from the ER. This interpretation is supported by several pieces of evidences. First, this species is sensitive to endo-H digestion, a hallmark of glycoproteins that have not yet transit the mid-Golgi compartment. Second, blocking protein export from the ER by brefeldin A in control PC12 cells results in a striking similar pattern of PrPC glycosylation, including the appearance of the 31-kDa isoform. Third, we found a marked alteration in the kinetic of PrPC maturation. The 31-kDa aberrant isoform remains, ER-trapped, stable within 3 h of chase and is never found in the PI-PLC released pool.

mAb 8H4 binds the epitope between residues 175 and 185 of helix B, which contains one of the two potential N-glycosylation sites (Asn-180). However, glycosylation does not impede the binding of mAb 8H4 to native cellular PrPC in vitro (38). We found a profound alteration in PrPC glycosylation pattern only in PC12 cells expressing the KDEL-retained version of the 8H4 scFv but not in cells expressing the secretory version of the same intrabody. Hence, we conclude that the altered processing of PrPC is caused by the inhibition of its transit from the ER to the mid-Golgi.

A number of human diseases have been attributed to defects in the export of mutated secretory proteins (49). In the simplest case, the aberrant protein misfolds and fails to reach its final location resulting in the loss of normal physiological function. Misfolded proteins may also form aggregates in the ER or in the cytoplasm, known as Russel bodies and aggresomes (50). These products may gain "toxic functions" as well as stimulating the ER-stress responses to further compromise the functionality and viability of affected cells (47). On the other hand, the quality control machinery of secretory proteins itself can also harbor defects, as it occurs in congenital bleeding disorder or in congenital disorders of glycosylation (CDG) diseases, which are caused, respectively, by mutation of the transmembrane lectin ERGIC 53 (51) and by mutation in the enzymes catalyzing glycosylation of proteins (52).

We demonstrate here that PrPC molecules, when significantly delayed in their transit along the early pathway of the secretory compartment, aggregate, become partially resistant to PK and detergent-insoluble soon after the synthesis. These biochemical characteristics are reminiscent of those described in cell models of genetic prion diseases (11, 53, 54) in which PrPC molecules carrying mutations are delayed in their maturation, accumulate in the ER, tend to misfold and aggregate (55, 56). In our model we find that ER-trapped wild type PrPC can acquire the same properties of inherited prion mutants but in the absence of mutations. Therefore, the endoplasmic reticulum appears to be a competent compartment for the acquisition of detergent insolubility and protease resistance of endogenous PrPC. These results led us to speculate that accumulation of aberrant PrPC molecules might arise from a defect in the ER quality control machinery, such as an impairment of glycosylation as in CDG disorders. Altogether these findings have important implications in the understanding of sporadic forms of prion diseases in which specific mutations of PrPC are not found.

Although KDEL-8H4-PC12 cells accumulate misfolded PrPC molecules in the ER, there is no discernible effect on their viability, proliferation or susceptibility to ER-stress inducers. In line with other in vitro cell models of genetic prion diseases (11), this may be related to the ER-associated degradation system of PC12 cells. Misfolded proteins, accumulating in the ER lumen, are retrotranslocated and targeted for degradation by the ER-associated degradation (ERAD) mechanism. Several studies have shown that this pathway is involved in the degradation of pathogenic PrPC mutants. Whether the misfolded ER-retained PrPC molecules are retrotranslocated and proteasome degraded in our model system is not known.

The locale of PrPC to PrPSc conversion is not completely understood. Some studies in cell models have highlighted the role of the cell surface, while other studies suggested that conversion mainly occurs in the endosomal/lysosomal vescicles (8-10). We demonstrate here that in contrast to PC12 cells, KDEL-8H4-PC12 cells exposed to 139A scrapie agent do not accumulate detectable levels of PrPSc. This suggests that either the initial phase of PrPC-PrPSc conversion and/or the following PrPSc propagation process have been inhibited. The mechanism by which KDEL-8H4 scFv hampers PrPSc formation is not known. Since the amount of cell associated PrPSc at the end of the infection period (6 h) is similar in both cell types, the inability of KDEL-8H4-PC12 cells to produce PrPSc is not simply due to the impairment of PrPSc present in the inoculum to bind cell membrane. It is possible that KDEL-8H4-PC12 cells miss the critical number of cell membrane PrPC molecules needed to start the initial phase of PrPC-PrPSc conversion, as shown by pulse chase experiments where only about 10% of fully mature PrPC molecules reach the cell surface within 6 h. Consistent with a model where a specific diverting of PrPC by intracellular antibodies prevents the formation of PrPSc, down-regulation and re-routing of surface proteins by a chemical compound, suramin, has recently been reported to prevent scrapie propagation in vitro and significantly delayed the onset of prion disease in vivo (57).

Alternatively, direct intracellular interaction between the KDEL-8H4 scFv and PrPSc or PrPC may interfere with the PrPC-PrPSc conversion process. In vivo injection of mAb 8H4 but not 8F9 also delayed the onset of prion diseases in PrPSc-infected mice (58).

In conclusion, the intracellular antibody strategy represents a novel approach not only for studying the biogenesis, the cellular trafficking and the degradation of the prion protein but holds the promise to help in elucidating the molecular events in the pathogenesis of prion diseases, including the site of conversion and the cellular control mechanisms counteracting the formation of PrPSc. Finally, these in vitro studies set the groundwork for developing promising and specific anti-prion strategies useful for therapy.


   FOOTNOTES
 
* This work was supported by Fondo per gli Investimenti della Ricerca di Base (FIRB) Grant RBNE01ZK8F_002 (to S. B.). 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

|| To whom correspondence should be addressed. Tel.: 39-06-7259-6428; Fax: 39-06-7259-6407; E-mail: biocca{at}med.uniroma2.it.

1 The abbreviations used are: PrPC, cellular isoform of prion protein; PrPSc, scrapie isoform of PrP; GPI, glycosylphosphatidylinositol; scFv, single-chain variable fragment; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin; NGF, nerve growth factor; PK, proteinase K; ER, endoplasmic reticulum; PNGase, peptide-N-glycosidase; Endo-H, endoglycosidase H; PI-PLC, phosphatidylinositol-specific phospholipase C; MTT, 3,(4,5-di-methylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide; BFA, brefeldin A; FBS, fetal bovine serum. Back


   ACKNOWLEDGMENTS
 
We thank S. Mattei for skillful help, A. Negro for the human recombinant PrP and vector for expression of PrP in mammalian cells, and D. Mercanti for providing purified nerve growth factor.



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 DISCUSSION
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