THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 5, Issue of January 30, pp. 3694 –3698, 2004 Printed in U.S.A.
Monoclonal Antibody against a Peptide of Human Prion Protein Discriminates between Creutzfeldt-Jacob’s Disease-affected and Normal Brain Tissue* Received for publication, June 11, 2003, and in revised form, October 2, 2003 Published, JBC Papers in Press, October 29, 2003, DOI 10.1074/jbc.M310868200
ˇ urin Sˇerbec‡§, Mara Bresjanac¶, Mara Popovic´储, Katrina Pretnar Hartman‡, Vladka C ˇ ernilec‡, Tanja Vranac‡, Iva Hafner**, Vesna Galvani‡, Ruth Rupreht‡, Maja C and Roman Jerala** From the ‡Blood Transfusion Center of Slovenia, Sˇlajmerjeva 6, the Institutes of ¶Pathophysiology and 储Pathology, School of Medicine, and the **Laboratory of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia
Current methods for diagnosing transmissible spongiform encephalopathies rely on the degradation of the cellular prion protein (PrPC) and the subsequent detection of the protease-resistant remnant of the pathological prion isoform PrPSc by antibodies that react with all forms of PrP. We report on a monoclonal antibody, V5B2, raised against a peptide from the C-terminal part of PrP, which recognizes an epitope specific to PrPSc. In cryostat sections from Creutzfeldt-Jacob’s disease (CJD) patients’ brains, V5B2 selectively labels various deposits of PrPSc without any pretreatment for removal of PrPC. V5B2 does not bind to non-CJD brain samples or to recombinant PrP, either in its native or denatured form. Specificity for PrP is confirmed by a sandwich enzyme-linked immunosorbent assay utilizing V5B2, which discriminates between CJD and normal samples without proteinase K treatment, and by immunoprecipitation from CJD brain homogenate. The PrPSc-specific epitope is disrupted by denaturation. We conclude that the C-terminal part of PrP in disease-associated PrPSc aggregates forms a structural epitope whose conformation is distinct from that of PrPC.
A conformational transition of the normal cellular prion protein (PrPC)1 into a pathological prion isoform (PrPSc) is believed to be responsible for the development of transmissible spongiform encephalopathies (TSE) (1, 2). PrPSc forms characteristic insoluble aggregates, is partially resistant to protease digestion, and differs in secondary structure from PrPC (1–3). The importance of TSE arose in the last decade with the epidemic of bovine spongiform encephalopathy (BSE) and the appearance of a new variant of Creutzfeldt-Jacob’s disease (vCJD), predom* This work was supported by Ministry of Education, Science and ˇ . Sˇ.) and Sport of the Republic of Slovenia Grants L3-3435 (to V. C P3-0518-0381 (to M. B. and M. P.) and the Fifth Framework Program, Human TSE, Neuropathology Network PRIONET Grant QLRT-199930837 (to M. P.). 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. § To whom correspondence should be addressed. Tel.: 386-1-5438165; E-mail: [email protected]
1 The abbreviations used are: PrP, prion protein; PrPC, cellular PrP; PrPSc, pathological PrP isoform; recPrP, recombinant PrP; BSE, bovine spongiform encephalopathy; TSE, transmissible spongiform encephalopathy; BSA, bovine serum albumin; CJD, Creutzfeldt-Jacob’s disease; sCJD, sporadic CJD; vCJD, variant of CJD; ELISA, enzyme-linked immunosorbent assay; GdnHCl, guanidine HCl; HRP, horseradish peroxidase; IHC, immunohistochemistry; KLH, keyhole limpet hemocyanin; mAb, monoclonal antibody; PBS, phosphate-buffered saline.
inantly but not exclusively in the United Kingdom. A specific, rapid, and sensitive test for detecting PrPSc is required to minimize the possible risk of prion contamination. Currently available methods for immunological diagnosis of TSE rely on the use of antibodies that react with both forms of PrP (4) and, thus, depend on prior elimination of PrPC by proteinase K digestion for positive identification of the remaining PrPSc (5) as its partially digested product, PrPres. Because it has been established that the conformation of PrPSc differs from that of PrPC, the aim of our work was to identify the epitope specific for PrPSc that would enable better insight into the differences between the two isoforms and provide a tool for analyzing biological samples. An earlier study reported production of a monoclonal antibody (mAb) specific for the disease-associated form of PrP (6). It was later stated by the same authors that the availability of PrP knock-out mice and recombinant PrP (recPrP) were some of the prerequisites for successful immunization and generation of the specific antibodies (7). Our approach for generating PrPSc-selective mAbs was to immunize BALB/c mice with carefully chosen peptides based on previous investigations on the structure and mechanism of PrPSc formation. We have prepared a mAb that reacts with an epitope specific for the native PrPSc and show that this epitope could not be generated in vitro from recombinant PrP. We suggest that the C-terminal part of PrPSc folds into a distinct conformation, which may be stabilized by interaction with other molecules under disease conditions. EXPERIMENTAL PROCEDURES
Peptides—Peptides based on the human PrP sequence P1 (214 –226; CITQYERESQAYY), P2 (167–179; DEYSNQNNFVHDC) and P3 (139 – 150; CIHFGSDYEDRYY Cys was added for conjugation to KLH) were made by solid state synthesis, conjugated to keyhole limpet hemocyanin (KLH) (Bachem, Bubendorf, Switzerland), and used for immunization. Preparation of Monoclonal Antibodies—Hybridomas were prepared in BALB/c mice according to the procedures described in Ref. 16, and mAbs were selected with indirect ELISA against peptide, peptide-KLH, and KLH. Brain Tissue Sample Preparation—Human brain tissue samples (nine sCJD positive and two non-CJD) were homogenized (HT1000 Potter homogenizer) in ice-cold PBS containing 0.5% Nonidet P-40 and 0.5% deoxycholate to give a 10% (w/v) final suspension. Homogenates were centrifuged for 10 min at 5000 ⫻ g at 4 °C. Supernatants of brain homogenates were aliquoted and stored at ⫺80 °C. Dot Blot Analysis—50 l of 10⫻, 20⫻, or 40⫻ diluted supernatants of brain homogenates were loaded on 0.2-m nitrocellulose membranes using the dot blot (Bio-Rad). The membranes were blocked with 5% (w/v) nonfat dry milk (Amersham Biosciences) in Tris-buffered saline/ Tween 20 (TBS-T) at 4 °C overnight and then incubated with primary mAbs (5 g/ml V5B2 or 0.2 g/ml 3F4 in 1% milk/TBS-T) for 1 h with shaking at room temperature. Membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP)-labeled anti-mouse
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PrPSc-specific Antibodies secondary antibody (Amersham Biosciences; 1:1500 in 1% milk/TBS-T) at room temperature for 1 h. Reaction was detected using chemiluminescence detection reagents (ECL, Amersham Biosciences). Western Blot Analysis—Native or proteinase K-digested (final proteinase K concentration 5 g/ml for 30 min at 37 °C, heat inactivated) supernatants of brain homogenates, denatured with SDS-PAGE sample buffer (Novex), were loaded on SDS-Tris-glycine polyacrylamide gels (7.5% gel) and run for 60 min at 120 V with 25 mM Tris, 0.32 M glycine, and 0.16% SDS (w/v), pH 8.3. The samples were blotted on 0.2 m nitrocellulose membranes (Bio-Rad) for 60 min at 200 mA using 25 mM Tris, 192 mM glycine, and 20% methanol buffer. The membranes were blocked, and proteins were detected as described above. Sandwich ELISA with Brain Tissue Homogenates—Microtiter plates (Nunc, Roskilde, Denmark) were coated with 5 g/ml mAb 6H4 (Prionics, Zu¨ rich, Switzerland) in 50 mM carbonate/bicarbonate buffer, pH 9.6. The plates were blocked with 1% BSA and 50 l of sCJD or non-CJD brain homogenates in 1% BSA/PBS/Tween20 were applied to the wells. After 90 min of incubation at 37 °C, V5B2, conjugated with HRP and diluted 1:1000 in 1% BSA/PBS/Tween 20, was added to the wells. After 90 min incubation at 37 °C reaction was detected as an indirect ELISA. Immunoprecipitation—mAb V5B2 was covalently coupled to carboxyl-terminated magnetic beads (Sigma) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a bifunctional coupling reagent as suggested by the manufacturer. Beads were preincubated in 1% BSA/PBS at room temperature for 1 h and washed in binding buffer (3% Nonidet P-40, 3% Tween, and 250 mM NaCl in 20 mM potassium phosphate buffer, pH 7.4). 30 l of brain homogenate containing ⬃270 g of protein was added to a suspension containing 50 l of magnetic beads and 150 l of binding buffer. Suspension was shaken for 1 h at room temperature and washed three times with washing buffer (2% Nonidet P-40, 2% Tween 20, and 350 mM NaCl in 20 mM potassium phosphate buffer, pH 7.4). Bound proteins were eluted with SDS loading buffer and heated at 95 °C before application to gel electrophoresis and detection by either the 3F4 or the 6H4 antibody conjugated to horseradish peroxidase. Cryosections of Fresh Frozen Tissue Samples—Brains of clinically suspected CJD cases were collected at autopsy and, prior to whole brain formalin fixation, pieces of cerebral and cerebellar tissue were frozen rapidly in liquid nitrogen and stored at ⫺80 °C until final pathological diagnosis of CJD. Brain cryosections of two sCJD cases and one nonCJD patient (cause of death was myocardial infarction) were used in this study. Sections were cut in the cryostat at 10 m, thaw-mounted to SuperFrost Plus slides (Menzel Gla¨ ser, Germany), and labeled without prior fixation. Paraformaldehyde-fixed, Paraffin-embedded Tissue Samples—Brain tissues from 16 cases of sporadic CJD were used. Following 14 days of buffered 4% paraformaldehyde fixation, tissue blocks were immersed in 96% formic acid for 1 h and embedded in paraffin. 4-m-thick sections were either microwaved (96 °C) in EDTA buffer (pH 8) for 10 min (“mild” pretreatment) or autoclaved for 30 min (121 °C), cooled, immersed for 5 min in 96% formic acid, and washed prior to immunohistochemistry (IHC). A sample of hematoxylin-eosin stained section of a vCJD case brain was destained and labeled immunohistochemically without any pretreatment. Indirect IHC—V5B2 was tested in manual IHC on CJD and non-CJD cryostat brain sections at concentrations ranging from 0.3 to 30 g/ml. Commercially available anti-PrP antibody 3F4 (Dako, Glostrup, Denmark) was used for comparison. Manual IHC was performed on slidemounted sections using the avidin-biotin method with a peroxidase substrate as described (8). IHC on paraffin-embedded sections was performed on an automated machine (Ventana Medical Systems, Tucson, AZ) using Ventana kits and prescribed protocols at every step of the procedure. In addition to non-CJD brain tissue that served as a control for V5B2 selectivity for PrPSc, controls of IHC labeling were performed by omitting primary antibody from the IHC protocol carried out on adjacent sections as described above. Hematoxylin (Dako) was used to lightly counterstain the cell nuclei on all IHC-labeled slides. The slides were analyzed under a microscope (Eclipse E600, Nikon). Images were acquired with an attached digital camera (DXM 1200 digital camera, Nikon) linked to a PC. V5B2 conjugated with HRP (V5B2-HRP) was used following the same antigen retrieval procedure as described above for direct IHC. The concentration of V5B2-HRP conjugate was 5 g/ml. Preparation of Recombinant Proteins—The oligonucleotide (5⬘phosphate-TGCATTACCCAGTATGAACGTGAAAGCCAGGCGTATTATTATTAAATG-3⬘) encoding peptide sequence, annealed with a corresponding complementary oligonucleotide, was ligated into pET31b(⫹) expression vector (Novagen; Ref. 9) 3⬘ to the ketosteroid isomerase
FIG. 1. Comparison of reactivity of V5B2 and 3F4 with CJD positive and non-CJD human brain homogenates. Dot blots of 10⫻, 20⫻, and 40⫻ diluted sCJD-positive (sCJD) and -negative (neg.) human brain homogenates show specific reactivity of V5B2 with PrPSc under native conditions (a) contrasted with the non-discriminatory reactivity of 3F4 (b). gene. Fusion protein was overproduced in Escherichia coli in the form of inclusion bodies. Inclusion bodies were washed, solubilized in 6 M GdnHCl, pH 8, dialyzed against deionized water, and the precipitate (⬎95% pure) was used in assays. Recombinant human PrP (amino acids 23–230 and 90 –230) forms were expressed in E. coli in form of inclusion bodies and purified (10). Preparation of Different Structural Forms of Prion Protein—Several structural forms of human recPrP (23–230) (Prionics) were produced to test the reactivity of V5B2 toward them. Human recPrP (0.2 mg/ml) was unfolded by overnight incubation at room temperature in 4.8 M GdnHCl, 100 mM dithiothreitol, and 16 mM Tris/HCl, pH 8. Buffer was exchanged to 50 mM sodium acetate pH 4, to maintain the cysteine residues in a reduced state. Thermal denaturation was performed by heating the protein solution for 6 min at 95 °C. Oligomers of PrP with a high content of ␤-sheet structure were formed by the following reaction conditions: 1 or 3.5 M urea, 0.15 M NaCl, 50 mM sodium acetate pH 4 or 8 (11); 1 M GdnHCl, 50 mM sodium acetate, pH 4 (12); or reduction in 4 M urea, pH 4 (13), and reduced PrP in 0.1% or trifluoroacetic acid, pH 2 (34). RESULTS
Monoclonal Antibody V5B2 Prepared against a Synthetic Peptide in BALB/c Mice—Of the three peptides based on the sequence of human PrP used for immunization (see “Experimental Procedures”), the best results were obtained with a 13-residue synthetic peptide from the C-terminal part of human PrP (amino acids 214 –226). We have selected this region of PrP because it is part of the protease-resistant structure as determined by reactivity with mAb raised against the peptide 218 –232 (14). This part of the molecule has already been shown to be promising for its immunogenicity and specificity (6, 7, 15). BLAST search showed that the sequence of our immunizing peptide is unique to prion proteins, and the most similar segment among the human non-prion sequences differed at 6 of 13 residues. Polyclonal antibodies against a slightly larger peptide from this region of the bovine molecule reacted with PrPC but not with PrPSc (15). Three epitopes, described as specific for bovine PrPSc, have been defined, one of which is located in the same region of the protein (6, 7). Prnp0/0 mice and the recombinant bovine prion protein were a prerequisite for developing an mAb with this specificity. Additionally, the presence of glutamate and multiple tyrosines (two and three of each, respectively, in our peptide) has been reported to be favorable in stimulating production of conformationally specific antibodies (16). Immunization with this peptide resulted in a strong immune response in all the injected BALB/c mice. Over 2000 hybridomas were prepared from three fusions and, of the 44 clones that produced antibodies reactive against P1, three were found to distinguish between PrPSc and PrPC. ELISA, dot blot, Western blot, and IHC were used to select mAb V5B2 from a number of mAbs of different specificity for PrPC or PrPSc. PrPSc Selectivity of V5B2—V5B2 was found to distinguish reproducibly between normal and CJD brain homogenates in dot blot analysis (Fig. 1a). This is in sharp distinction to mAb 3F4 (Senetec, Maryland Heights, MO, and Dako), the widely accepted reference antibody for prion detection (17), which
FIG. 2. Sandwich ELISA discriminates between CJD-positive and -negative tissue. sCJD-positive (sCJD) and -negative (neg.) human brain homogenates tested with sandwich ELISA composed of 6H4 as the capture and the V5B2-HRP conjugate as the reporter antibody, showing the specific reactivity of V5B2 with PrPSc in sCJD-positive samples without treatment with proteinase K.
displayed strong reactivity with non-CJD as well as pathological brain homogenate (Fig. 1b). Sandwich ELISA, employing commercially available mAb 6H4 (6) as the capture antibody and V5B2 as the detection antibody, gave a strong signal with the brain homogenate from CJD-affected brain, unlike that from non-CJD brain without proteinase K digestion (Fig. 2), demonstrating that the target of V5B2 in tissue is indeed PrPSc characteristic for the prion disease and is not found in normal brain. V5B2 gave no reaction on Western blot analysis with extracts from CJD and non-CJD brain subjected to denaturing SDS-PAGE (Fig. 3), as expected for a conformational antibody. mAb 3F4 was used as a positive control. V5B2 conjugated to magnetic beads precipitated PrP from CJD-affected, but not normal brain homogenate (Fig. 4), confirming the specificity of V5B2. Additional confirmation of the selectivity of V5B2 for PrPSc and a demonstration of its usefulness come from its strong and selective labeling of PrPSc aggregates in unfixed, thawmounted cryosections of human brain tissue from CJD cases (Fig. 5a). No labeling occurred with normal human brain tissue (Fig. 5b) or with brain tissue from other neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease (not shown). In contrast, strong, non-selective labeling of tissue components in parallel tissue cryosections of both CJD (Fig. 5c) and non-CJD (Fig. 5d) brain was obtained with mAb 3F4. The conformational epitope was still recognized by V5B2 after paraformaldehyde fixation and could be detected in sections from paraffin-embedded fixed brains (Fig. 5e), even when a direct method of immunodetection was used in which V5B2 was directly conjugated with HRP without additional amplification of the signal through the secondary antibody and the avidin-biotin complex (Fig. 5f). Inclusion of a formic acid incubation step in the antigen retrieval pre-treatment protocol further enhanced V5B2 immunoreactivity with the CJD brain sections, permitting detection of fine, “synaptic” type prion deposits (Fig. 5g). V5B2 was also tested on fixed, paraffinembedded tissue sections from vCJD, where it displayed strong immunoreactivity to prion aggregates (Fig. 5h). We have also found that mAb V5B2 can be used to diagnose BSE and scrapie in the brain tissue of infected cattle and sheep (data not shown). mAb V5B2 Does Not React with Any Form of Recombinant PrP—V5B2 did not bind to human or bovine recPrP in either dot blot or ELISA despite the presence of the identical peptide sequence at the C terminus, although it bound to the immunogenic peptide and its conjugate to KLH. To test whether the antibody would recognize the aggregated protein, we prepared
FIG. 3. Western blot of sCJD-positive (sCJD) and negative (neg.) human brain homogenates under denaturing conditions. Samples were treated (⫹) or untreated (⫺) with proteinase K. Immunodetection was performed with antibodies 3F4 and V5B2.
FIG. 4. V5B2 selectively immunoprecipitates PrPSc. CJD⫺ and CJD⫹, homogenates from nonaffected and CJD-affected brain, respectively, loaded to SDS-PAGE (positive control); IP-CJD⫹ and IP-CJD⫺, immunoprecipitates from CJD-positive and CJD-negative brain homogenates by V5B2 immobilized to magnetic beads (V5B2 mag. beads). Homogenates contained comparable total amounts of protein in controls (CJD⫹ and CJD⫺) as well as in homogenates used in immunoprecipitation. V5B2 magnetic beads were a negative control without brain homogenate. PrP was detected by direct immunoblotting with 3F4.
human recPrP in the form of insoluble inclusion bodies, but there was no reaction with V5B2 although, as a control, there was strong reaction with 3F4 (Fig. 6). Additionally, we have fused the peptide to the C terminus of ketosteroid-isomerase to test whether the non-reactivity against PrP inclusion bodies might be due to its inaccessibility in the aggregated form. This fusion protein also formed inclusion bodies but, in contrast, reacted strongly with V5B2. These results indicate that some of the residues of the peptide that react with V5B2 are buried in the native conformation of the recPrP and PrPC as well as in denatured aggregated PrP. These results indicate that the conformation of the peptide in the form that reacts with V5B2 differs from that in recPrP and PrPC as well as in denatured aggregated PrP. Circular dichroism and NMR spectra and the prediction of the ␣-helical content of the peptide by AGADIR (18) (not shown) indicate that the short immunizing peptide itself does not possess an ␣-helical structure but is disordered in solution whereas, when it is part of the protein it is stabilized in a defined conformation by long range interactions. recPrP has been subjected to a variety of denaturing conditions that were shown previously to cause a transition in vitro to a conformation with a high content of ␤-structure with subsequent aggregation (11, 13, 19). These conditions included intermediate or high concentrations of urea and GdnHCl, low pH, and the presence of salt or reducing agents (see “Experimental Procedures”). None of these conditions resulted in any reactivity with V5B2 (not shown).
FIG. 6. Comparison of reactivity of recPrP inclusion bodies with V5B2 and 3F4. Dot blot of human recPrP using antibodies 3F4 and V5B2. a and b, recPrP (amino acids 23–230); c and d, recPrP (amino acids 90 –230).
FIG. 5. mAb V5B2 selectively labels pathological aggregates of PrPSc in fixed and unfixed tissue samples of CJD patients. a– d, cryosections (10 m) of fresh frozen cerebellum samples from a case of sCJD with primitive PrPSc plaques in the cerebellum (a and c) and a non-CJD autopsy case (b and d) labeled with V5B2 (a and b) or 3F4 mAb (c and d). Although morphology is suboptimal in all unfixed cryosections, even the fine pathological aggregates can be discerned in V5B2labeled CJD cerebellum (a) without detectable labeling of tissue components in either the CJD-afflicted (a) or normal cerebellum (b). In contrast, 3F4 labels most tissue components in both CJD (c) and nonCJD cerebellum (d). Magnified 200⫻. e– h, standard fixation does not abolish the epitope for V5B2. Paraformaldehyde-fixed, formic acidtreated, paraffin-embedded cerebellar tissue sections reveal PrPSc when labeled with V5B2. e, plaques and small aggregates are labeled by the indirect peroxidase method on a mildly pretreated section (see “Experimental Procedures”). f, adjacent section of the same cerebellum shows plaques and fine aggregates of PrPSc by direct peroxidase IHC following the same mild antigen-retrieval procedures, illustrating the potency of V5B2. g, paraffin section from the brain of another sporadic CJD case displays a fine, synaptic pattern of PrPSc deposits following hydrated autoclaving and formic acid pre-treatment (see “Experimental Procedures”). Magnified 200⫻. h, prion plaques in a vCJD brain section visualized with V5B2. Magnified 400⫻. DISCUSSION
Particularly for the transplantation and transfusion services there is an urgent need for fast, preferably pre mortem diagnostics that require antibodies that react specifically with the PrPSc without any pretreatment. Sandwich ELISA, as described in the present paper and which uses the PrPSc-specific antibody V5B2, distinguishes well between samples from normal and CJD-affected patients and is a useful tool for fast diagnosis. In contrast to other assays such as the conformation-
dependent assay (20), where the content of PrPSc is estimated from the difference in reactivity between the native and denatured sample or by degradation of PrPC by proteinase K, our assay estimates directly the content of PrPSc in the presence of PrPC. The sequence of the immunizing peptide partially overlaps with the conformational epitope of the previously reported PrPSc-specific mAb, 15B3 (6, 7). However, the latter mAb has never been available for either research or diagnostic purposes and has not been shown to work in immunohistochemical detection of PrPSc. While our manuscript was under revision, another paper reporting PrPSc-specific antibodies appeared (21). Paramithiotis et al. report that antibodies against the Tyr-Tyr-Arg motif are selective for the misfolded conformation of PrP. A similar sequence is present at the C terminus of our peptide, suggesting that both antibodies may recognize the same region of the protein. Although the conformational transition in PrP is believed to occur mainly in the N-terminal part of the structured domain, the region of PrP from which the peptide of this study originates has attracted attention before. The largest differences in the conformation of the structured domains of PrPC (amino acids 121–231) from different species are observed at the end of the C-terminal helix, which is less structured in murine than in human and bovine PrP (22, 23). NMR investigations of the full-length PrP have indicated that the disordered N-terminal tail probably interacts with the residues at the end of the third helix (22). Our results provide evidence that the conformation at the C terminus of PrPSc differs from the ␣-helical conformation of PrPC. Analysis of inter-helical contacts in PrPC indicate that any structural transformation of helix 2, which is believed to be frustrated, would affect the third helix, particularly its C terminus (24). Hydrogen exchange experiments have shown that even unfolded PrP retains an extremely stable core of ⬃10 residues around the disulfide bond, of which most are part of the peptide of this study (25). It is likely, therefore, that this region nucleates the rapid refolding on removal of denaturants, which explains the masking of the epitope for V5B2. The region C-terminal to our peptide (amino acids 225–231) was shown to be accessible in both PrPSc and PrPC (26), which indicates that only part of the helix 3 is in different conformation or, alternatively, that an insufficient number of hybridomas were screened. Several mAbs have been characterized that are reactive against both PrPC and denatured PrPSc, indicating that denaturation disrupts the specific metastable conforma-
tion of PrPSc and that refolding takes place, on removal of denaturant, to a PrPC-like conformation (26). V5B2 is, in this respect, directly opposite to those antibodies, recognizing PrPSc but not PrPC or denatured PrPSc. The conformation of PrPSc can be disrupted with strong denaturants that inactivate its infectivity irreversibly (27). We have observed that the specific conformation of PrPSc was disrupted and the reactivity with V5B2 was eliminated by treatment with SDS and NaOH but not by boiling (data not shown), conditions which have been shown to decrease or destroy the infectivity of PrPSc (2). It has been suggested that the high content of the ␤-structure and the ability to aggregate are not sufficient for the onset of the disease. Binding of additional components present in tissue, such as a putative factor X, may be necessary for keeping the C-terminal part of PrP in the conformation recognized by V5B2. Involvement of this region in the species barrier and interaction with a putative factor X has been suggested previously (28, 29). Interestingly, quinacrine, proposed for treatment of CJD, also binds to the Tyr-Tyr motif at the end of P1 (30). Finally, the alleles Q218K in mouse and E219K in human, which are located in the immunogenic peptide, protect against occurrence of the disease (31, 32). Peptides from the C terminus and antibodies against this region have been shown to prevent formation of PrP aggregates in vivo (33), and, in view of the strong immunogenicity of the peptide reported in this study, it warrants evaluation as a potential vaccine. We show that mAb V5B2 recognizes specifically the conformation of the C-terminal region of the PrPSc isoform, which differs from the PrPC. V5B2 does not react with recombinant PrP, regardless of treatment. The potent PrPSc-specific mAb V5B2 permits easy detection of PrPSc by standard immunohistochemical procedures on tissue from CJD patients’ in dot blot assays or ELISA. Its production does not require the use of knock-out mice (6, 7), and, being sub-class IgG1, it is suitable for improved immunodiagnostic procedures and possibly novel treatment options for TSE in humans as well as in animals. Finally, the results show that V5B2 is a potentially valuable tool for TSE research and may help to identify and manipulate the region of PrP, which experiences a change in conformation during the transition to the pathological form. Acknowledgments—We thank Prof. Kurt Wu¨ thrich (Eidgeno¨ ssische Technische Hochschule, Zu¨ rich, Switzerland) for the generous gift of vectors for bacterial expression of human PrP, Dr. James Ironside from the CJD Unit, Edinburgh, UK, for providing HE-stained paraffin sections of vCJD brain tissue, Robert Bremsˇ ak, Marjana Sˇ prohar, and Melita Gracar for technical assistance, and Prof. Roger Pain for his careful reading of the manuscript and valuable suggestions. We thank Dr. Bozˇ idar Voljcˇ for his support and Prof. Mirko Jung for valuable discussions.
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