CA2392997A1 - Prion protein conversion assay, prion protein intermediate and uses thereof - Google Patents

Abstract

The invention relates to a method of producing an intermediate prion protein comprising contacting a PrP c substrate with a low pH solution. The invention is useful in assays to detect prion conversion and prion diseases.

Description

TITLE: Priori Protein Conversion Assay, Priori Protein Intermediate and Uses Thereof FIELD OF THE INVENTION
The invention relates to a novel priori conversion assay involving a priori intermediate for use in diagnosis, prognosis, and therapeutics of priori diseases.
BACKGROUND OF THE INVENTION
An insoluble, ~i-sheet-rich isoform of the priori protein (herein generically designated PrPs~ is the only known component of the infectious particle associated with the priori diseases, which are a group of transmissible, fatal neurodegenerative diseases in humans and animals. A
challenge for developing diagnostic tests and therapeutics for treatment of priori diseases is detecting and obtaining prions, which are normally present at very low levels.
PrPs° is derived from its normal cellular isoform (PrPc), which is rich in a-helical structure, by a posttranslational process involving a conformational transition (~. While the primary structure of PrPc is identical to that of PrPs~, secondary and tertiary structural changes are responsible for the distinct physicochemical properties of the two isoforms. PrP~ exists as a detergent-soluble monomer and is readily degraded by protease K (PK), whereas the infectious isoform PrPs~ forms detergent-insoluble aggregates and shows high resistance to PK digestion and to phosphatidylinositol-specific phospholipase C (PI-PLC)-mediated release ("). According to the "protein only" theory of priori infectivity, PrP~ is converted to PrPS° by a template-directed process initiated by contact with PrPS~. This disease-related in vivo transition has been modeled in vitro, in which PrPc can be converted to a protease-resistant form by contact with PrPs~ (~~~, "~. Recently, it has been reported that this conversion can be promoted by protein misfolding cyclic amplification (PMCA), a process analogous to the polymerase chain reaction for nucleic acids (").
Studies using recombinant PrP (rPrP) have indicated that the structural transition of a-helix to ~i-sheet and concomitant self-association can be triggered by acidic pH combined with detergents in vitro ("', "~~, "~~~).
Although studies using rPrP have provided important information about PrP structural transition, three-dimensional structure, thermodynamic stability, and folding pathways, the absence of co-factor molecules and post-translational modifications, such as N-linked glycans and the C-terminal glycosylphosphatidylinositol (GPI) anchor, may confound attempts to model faithfully in vivo conversion events. A challenge in developing diagnostic tests and therapeutics is to obtain in vitro conversion that. accurately mimics in vivo conversion.
SUMMARY OF THE INVENTION
The present inventors have demonstrated that acid treated human brain PrP is a superior substrate for in vitro conversion than untreated PrP.
The PrP is preferably also treated with a detergent. Prior to this invention, the use of low pH-treated brain homogenates has never been recognized as an enhanced substrate for in vitro prion conversion assays.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which is demonstrated:
Figure 1: Effect of acidic pH and GdnHCI on the detergent-solubility of PrP derived from human brain tissue.
S and P represent the supernatants and pellets respectively from ultracentrifugation at 100,000 X g for 1 hour. Panel a: Immunoblotting of mock-treated and acidlGdnHCI-treated human PrP with 3F4 antibody. In mock-treated samples, PrP was predominantly found in the supernatant whereas in the acid/GdnHCI-treated sample, PrP was predominantly found in the pellet. Panel b: Immunoblotting of pH-dependent detergent solubility of human PrP with 3F4 antibody. At pH equal to or greater than 4.5, the PrP was found in the supernatants whereas at pH less than or equal to 3.5, the PrP
was found in the pellets. Panel c: Immunoblotting of human PrP treated with various concentrations of GdnHCI at pH 3.5. The blot was probed with 6H4 antibody. At GdnHCI concentration equal to or greater than 2.5 M, PrP was predominantly found in supernatants whereas at GdnHGI concentration less than or equal to 1.5 M, PrP was predominantly found in the pellets. Molecular masses are shown in kilodaltons (kDa).
Figure 2: PK-sensitivity of acidIGdnHCI-treated PrP.
Immunoblotting of human PrP treated with various PK concentrations.
The blot was probed with 6H4 antibody. Both mock-treated and acidic pH/GdnHCI-treated PrP showed PK-resistance at low concentration of PK;
however, there was no observable difference in the PK-sensitivity between the two. Molecular masses are shown in kDa.

Figure 3: Immunoprecipitation (1P) of the treated PrP and PrPS~ with anti-PrP antibodies.
Immunoblotting of the precipitates with 3F4 antibody. Lane 1 and 3:
mock-treated samples; Lane 2 and 4: acidic pHIGdnHCI-treated samples;
Lane 5 and 7: normal brain samples; and Lane 6 and 8: CJD brain samples.
There was no significant difference in binding of 6H4 or 3F4 to the proteins from mock-treated and acidic pHlguanidine-treated samples. However, the content of PrP precipitated by the two antibodies was significantly decreased in CJD brain samples compared to normal brain samples. Molecular masses are shown in kDa.
Figure 4: In vitro conversion of the treated PrP in the absence and presence of PrPs° from human brain with CJD.
Immunoblotting of the nascent PK-resistant PrP isoforms with 3F4 antibody. Samples were incubated in 0.05% SDS, 0.5% Triton X 100 in PBS, pH 7.4 at' 37°C for 12 hours with shaking in the absence or presence of trace quantities of PrPS°. Lane 1: mock-treated PrP alone; Lane 2: acidic pH/GdnHCI-treated PrP alone; Lane 3: mock-treated PrP plus trace quantities of PrPs~, and Lane 4: acidic pHIGdnHCI-treated PrP plus trace quantities of PrPS°. All samples were PK-treated at 100 p.glml. Molecular masses are shown in kDa.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to diagnostic tests and therapeutics for prion diseases. In one aspect, the invention provides a novel intermediate prion protein and conversion assay. The intermediate species is a conformational isoform that has properties of both PrP and PrPs~. For example, the intermediate species is insoluble and aggregates, but is not protease resistant. The intermediate species is also multimeric and probably acquires more beta-sheet content. The invention also provides an in vitro prion conversion assay which faithfully mimics in vivo prion conversion. This assay _5 is useful for identifying inhibitors of priori conversion as well as compounds which stabilize the native priori state. The compounds may inhibit conversion to either the intermediate state or PrPSc.
SwissProt accession numbers for PrP proteins include the following.
Homo sapiens (Human) P04156; Mus musculus {Mouse) P04925;
Mesocricetus auratus (Golden hamster) P97895; Ovis aries (Sheep) P23907;
Capra hircus (Goat) P52113; Odocoileus virginianus (white-tailed deer) 002841; Cervus elaphus nelsoni (American elk) P79142; Bos taurus (Bovine) P10279; Bos taurus (Bovine) Q01880; Felis silvestris catus (Cat) 018754 One may also use polypeptides which have sequence identity at least about: >50%, >60%, >70%, >80% or >90% more preferably at least about >95%, >99% or >99.5%, to a PrP sequence of the invention (or a partial sequence thereof) provided that the polypeptides retain PrP identity. Identity is calculated according to methods known in the art. Sequence identity is most preferably assessed by the BLAST version 2.1 program advanced search (parameters as above). BLAST is a series of programs that are available online at http:Ilwww:ncbi.nlm.nih.gov/BLAST. The advanced blast search (http:/lwww.ncbi.nlm.nih.govlblast/blast.cgi?Jform=1) is set to default parameters. (ie Matrix BLOSUM62; Gap existence cost 11; Per residue gap cost 1; Lambda ratio 0.85 default). References to BLAST searches include:
Altschul, S.F.; Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403 4'10; Gish, W. & States, D.J. (1993) "Identification of protein coding regions by database similarity search." Nature Genet. 3:266 272; Madden, T.L., Tatusov, R.L. & Zhang, J.
(1996) "Applications of network BLAST server" Meth. Enzymol. 266:131_141;
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W.
&
Lipman, D.J. (1997) "Gapped BLAST and PSI~BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389 3402;
Zhang, J. & Madden, T.L. {1997) "PowerBLAST: A new network BLAST
application for interactive or automated sequence analysis and annotation."
Genome Res. 7:649_656. The invention includes the use of polypeptides with mutations that cause an amino acid change in a portion of the polypeptide not involved in providing activity or an amino acid change in a portion of the polypeptide involved in providing activity so that the mutation increases or decreases the activity of the polypeptide.
Therapeutic Methods The present invention comprising administering to an animal in need thereof an effective amount provides a method of treating a disease associated with a prion of an agent that inhibits prion conversion to an intermediate prion protein or PrPS~.
The term "an agent that inhibits prion conversion " as used herein means any agent that can inhibit prion conversion as compared to the level prion conversion in the same type of cell in the absence of the agent. The agent can be any type of substance including, but not limited to, nucleic acid molecules, proteins, peptides, carbohydrates, small molecules, or organic compounds. UVhether or not the prion conversion is inhibited can be readily determined by one of skill in the art using known methods described in this application.
The term "animal" as used herein includes all members of the animal kingdom. The animals are preferably human.
The term "effective amounts as used herein means an amount effective at dosages and for periods of time necessary to inhibit prion conversion.
The term "treatment or treating" as used herein means an approach for obtaining beneficial or desired results, including clinical results.
Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating" can also mean prolonging survival as compared to expected survival if not receiving treatment. The term "disease associated with a priors" means any disease or condition that is caused by the presence of a priors, including but no limited to, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, variant Creutzfeldt-Jakob disease, kuru, scrapie, bovine spongiform encephalopathy, chronic wasting disease, transmissible mink encephalopathy, and feline spongiform encephalopathy.
Screening for priors conversion inhibitors Molecules are screened to determine if they inhibit priors protein conversion. Inhibitors are preferably directed towards specific domains of priors protein. To achieve specificity, inhibitors should target the unique sequences and or conformational features of priors protein.
The present invention also includes the isolation of substances that inhibit priors protein conversion. Biological samples and commercially available libraries may be tested for substances such as proteins that bind to a priors protein. In addition, antibodies prepared to priors may be used to isolate other proteins with affinity for priors isoforms. For example, labeled antibodies may be used to probe phage displays libraries or biological samples. Once potential binding partners have been isolated, screening methods of the invention may be designed in order to determine if the substances that bind to the priors are useful in preventing priors protein conversion.
Therefore, the invention also provides methods for identifying substances which are capable of binding to priors proteins. Accordingly the invention provides a method of identifying substances which bind with a priors protein comprising the steps of:
(a) reacting a priors protein and a candidate substance, under conditions which allow for formation of a complex, and (b) assaying for complexes, for free substance, and for non-complexed protein.

_$
Any assay system or testing method that detects protein-protein interactions may be used including co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns may be used.
Biological samples and commercially available libraries may be tested for priors binding peptides. In addition, antibodies prepared to priors may be used to isolate other peptides with priors binding affinity. For example, labeled antibodies may be used to probe phage display libraries or biological samples. In. this respect peptides may be developed using a biological expression system. The use of these systems allows the production of large libraries of random peptide sequences and the screening of these libraries for peptide sequences that bind to particular proteins. Libraries may be produced by cloning synthetic DNA that encodes random peptide sequences into appropriate expression vectors. (see Christian et al 1992, J. Mol. Biol.
227:711; Devlin et al, 1990 Science 249:404; Cwiirla et al 1990, Proc. Natl.
Acad, Sci. USA, 87:6378). Libraries may also be constructed by concurrent synthesis of overlapping peptides (see U:S. Pat. No. 4,708,871). Inhibitors and stabilizers are tested in priors models.
In one embodiment, the invention includes an assay for evaluating whether a candidate compound is capable of inhibiting or stabilizing priors conversion by contacting priors protein with at least one compound whose ability to inhibit or stabilize priors protein conversion is sought to be determined and thereafter monitoring for priors protein conversion. Decreased priors protein conversion to an intermediate priors protein substrate or PrPs indicates that the candidate compound is useful for treating priors disease.
A method of determining whether a candidate compound inhibits the priors conversion (and is useful for treating priors disease) can also include:
a) contacting (i) priors protein, a fragment of priors protein or a derivative of either of the foregoing with (ii) a candidate compound in a low pH (< 7) solution; and _g _ b) determining whether prior protein conversion is decreased, thereby indicating that the compound inhibits prior conversion. Decreased prior conversion to a recruitment-efficient species indicates that the compound is useful for treating prior d iseases.
Similarly, A method of determining whether a candidate compound inhibits the prior conversion (and is useful for treating prior disease) can also include c) contacting a prior recruitment efficient prior substrate species (for example, generated by exposure of brain homogenates to low pH and denaturants) with PrPS° template and a candidate compound a candidate compound in a buffer solution supporting in vitro conversion; and d) determining whether prior protein conversion from intermediate to PrPs° is decreased, thereby indicating that the compound inhibits prior isoform conversion. Decreased prior conversion to PrPS° indicates that the compound is useful for treating prior diseases.
Similar methods may also be performed to identify compounds which stabilize the native prior state, or bind to PrPs° and block conversion of recruitable PrP isoforms.
Pharmaceutical compositions The invention includes prior protein conversion inhibitors and compounds that stabilize the native prior protein state. Compounds are preferably combined with other components, such as a carrier, in a pharmaceutical composition. These compositions may be administered to a subject, such as a human, in soluble form to prevent or treat prior disease.

The pharmaceutical compositions can be administered to humans or animals by a variety of methods including, but not restricted to topical administration, oral administration, aerosol administration, intratracheal instillation, intraperitoneal injection, and intravenous injection. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration. Polypeptides may be introduced into cells using in vfvo delivery vehicles such as but not exclusive liposomes.
The pharmaceutical compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, such that an effective quantity of the nucleic acid molecule or polypeptide is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, 'for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA).
On this basis, the pharmaceutical compositions could include an active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and isoosmotic with the physiological fluids. The methods of combining the active molecules with the vehicles or combining them with diluents is well known to those skilled in the art. The composition could include a targeting agent for the transport of the active compound to specified sites within tissue PrP Conversion Acidic pH and GdnHCI induces a physical transition of cellular PrP
derived from normal human brain homogenates. Treated PrP is detergent insoluble, similar to PrPS~, but displays PrP~-like protease sensitivity and epitope accessibility. A small proportion of acidic pH/GdnHCI-treated human brain PrP, but not mock-treated PrP, acquires PK resistance upon further treatment with a Low concentration of SDS. Trace quantities of PrPS~ template greatly enhance conversion of acidic pHIGdnHCIISDS-treated human brain PrP to a PrPS~-like PK-resistant species.
Sequential Events in PrP Isoform Conversion Conversion of PrPc to PrPS~ progresses through two discrete stages, which can be recapitulated in vitro. Low pH and denaturants may induce the first stage of structural rearrangement, in which treated PrP becomes more "recruitable" than native PrPc. The second stage is dependent upon further rearrangement that is driven by a PrPs° template, which is fostered in vitro by low concentrations of SDS, a denaturing anionic detergent.
Low pH and denaturants can induce beta sheet conformational change and self-association in recombinant PrP from multiple species (6-8, 16), and in a prion peptide (PrP 195-213; 9). The narrow pH range (pH 3.5-4.5) in which these transitions occur is consistent with protonation of acidic amino acids (9). Molecular dynamics simulations (MDS) of Syrian hamster rPrP
reveals the importance of protonation of acidic amino acids in acid-induced structural changes of PrP species (20). At neutral pH, Asp-178 forms a charge-stabilized hydrogen bond with Tyr-128 in MDS; however, this interaction between Asp and Tyr is broken when Asp is protonated at low pH
(20). Furthermore, recombinant human PrP (hurPrP) (90-231) showed a pH-dependent exposure of hydrophobic patches on the surface of the protein molecule, as evidenced by an increase in fluorescence intensity of bound bis-ANS at pH lower than 5.5 (6). Thus, acquisition of detergent-insolubility of human brain PrP resulting from treatment with low pH and GdnHCI could be concomitant with conformational changes and associated aggregation, driven by hydrophobic interactions.
Human brain PrP treated at acidic pHIGdnHCI acquires the insolubility of PrPs', but does not display the protease resistance or epitope obscuration possessed by this isoform. Several lines of evidence indicate that ~i-structure formation in a region spanning residues ~90-120 contributes to the acquisition of PK-resistance of PrPs~ (11, 21, 22). Using anti-PrP antibody-mediated structural mapping, Peretz et al. reported epitopes in this region (residues ~90 to 120) were accessible in PrPc but largely cryptic in PrP 27-30 (11).1'he obscuration in PrPS~ of the 3F4 epitope (residues 109-112) is consistent with this hypothesis, although the decreased accessibility to the 6H4 epitope (residues 144-152), shows that the final rearranged region extends C-terminal to residues 90 to 120. If PrPS° formation is dependent upon structural changes in codons ~90-120, then structural changes in other regions of the molecule are responsible for the decreased solubility and enhanced "recruitability" of acidic pH/GdnHCI-treated human brain PrP. Precedents for structural rearrangements occurring outside residues 90 to 120 include: an acidic pH-induced a-~i transition in a C-terminal domain of hamster rPrP (121-231) (8), and the acquisition of a novel binding site for Cu(Il) in mouse rPrP (121-231) treated at pH range 3-5 (23), both of which are outside of the 90-120 region.
Acidified, but not non-acidified, human brain PrP acquires some protease resistance characteristic of PrPs° in the presence of low concentration SDS and Triton X-100. The anionic detergent SDS may impact the hydrophobicity of treated rPrP molecules (18), and induce intermolecular interactions and further structural rearrangement. The PK-resistant conversion of acidified, but not non-acidified, human brain PrP was greatly enhanced by the presence of trace amounts of native PrPs°. These data suggest that acidic pHIGdnHCI-treated PrP constitutes an acceptable substrate for the PrPc to PrPS~ recruitment process. Moreover, the acquisition of PrPs°-like properties by acidic pHIGdnHCI brain PrP is template-directed, similar to the PrP
conversion occurring in prion disease.
Subcellular Location of PrP Conversion PrP~ is synthesized in the rough endoplasmic reticulum (ER) and transits through the Golgi apparatus to the cell surface, where it is attached to the outer leaflet by a GPI-anchor in lipid rafts or caveolae (24, 25). PrP~ is internalized into endocytic compartments from which most of the molecules are recycled intact to the cell surface (26, 27). A small proportion of the endocytosed molecules are proteolytically cleaved, followed by externalization of the cleaved products (27, 28). Ultimately, PrPc is degraded in the endosomal-lysosomal pathway (27, 29), a series of progressively acidic intracellular organelles.
The finding that acidified PrP is an enhanced substrate for PrP
conversion shows that an acidic environment provides conversion in vivo. We have demonstrated that acid-induced conformational transition and aggregation is associated with protonation of the acidic amino acids aspartate (Asp) and glutamate (Glu) using a peptide (195-213) corresponding to C-terminal region of PrP ("'). PrP in acidic organelles participates in specific folding pathways, forming molecular species structurally and physicochemically distinct from PrP~. Metabolic labeling studies indicate that PrPS~ formation takes place on the cell surface or a proximate post-surface compartment (33-38), and that PrPs~ subsequently accumulates in the endosomal-lysosomal system (28, 29, 39-42). Some data suggests that conversion occurs in caveolae-like domains called "lipid rafts" (28, 42), non-acidic microdomains on the plasma membrane. However, in view of the endosomal recycling activity of PrP~, the cell surface PrPc may be composed of two populations: one directly from the Golgi and another recycled from the endosome as acidified PrP. After acidification, PrP possesses properties physically distinct from the un-acidified molecules, which are retained even when the protein is returned to physiological pH: Consistent with the notion of "recruitable" and "non-recruitable" pools of PrP~ is the finding that only ~ 5-10% PrPc has been found to be converted into PrPS~ in scrapie-infected neuroblastoma cells (35, 36).
Detection of PrPs~
In prion diseases, PrP~ is recruited to PrPs° by a template-directed process that can be mimicked in vitro (3-5). Our finding that acidic pH/GdnHCI-treated human brain PrP constitutes a superior substrate for this reaction is exploited to detect PrPS° in tissues and fluids in which the template is present in extremely low concentrations. The conversion of acidified PrP

into a PK-resistant form more closely resembles PrPs~ propagation in vivo, compared with two other in vifro PrP conversion systems previously reported (3, 4} in which a 50-fold or a 10-fold molar excess of PrPS~ are required for the.
conversion to occur. Saborio et al (5) have also reported an in vitro conversion system in which trace concentrations of hamster brain PrPS~ can be detected by a process called PMCA utilizing sequential incubation-sonication to enhance conversion. However, the hamster brain PMCA system optimized by Saborio et al (5) does not appear to support significant amplification of human PrPS~ (Zou, V11. Q. and Cashman, N. R., not shown).
AcidicIGdnHCI-treated PrPc is a superior substrate to untreated PrP~ in amplification-detection of human PrPs~, and also obviates the requirement for lengthly incubation-sonication cycles. Acidic pH/GdnHCI-treated human brain PrP is useful to determine the conformational events of underlying prion protein conversion in disease, the molecular cofactors and post-translational modifications critical in conversion; and pharmaceutical agents which might prevent PrPs~ formation in vitro and in vivo.
Accordingly, in one embodiment, the invention includes a diagnostic test for the presence of PrPSc comprising the steps of (a) contacting a PrP-intermediate substrate with detergent;
(b} incubating the substrate with a subject sample;
(c) digesting the mixture with proteinase, preferably Proteinase K;
(d) identifying the presence of PrPSc in the sample, preferably by PK-resistance.
In a preferred embodiment, the subject is selected from the group consisting of human, sheep, cow, goat, cervid, mouse and hamster and the sample is selected from the group consisting of brain, lymphoid tissues, ocular tissues, blood or blood fractions, urine, saliva and spinal fluid and preferably comprises at feast 15ng/ml of prion protein. Most preferably, the subject is suspected of having a prion disease. Examples of prion disease include -~ 5 -Creutzfeldt-Jakob disease, Gerstmann-Straussler-~Scheinker syndrome, fatal familial insomnia, variant Creutzfeldt-Jakob disease, kuru, scrapie, bovine spongiform encephalopathy, chronic wasting disease, transmissible mink encephalopathy, and feline spongiform encephalopathy.
Diagnosis of PrPsc containing samples can also be done using a plaque lift. PrPc in normal brain homogenates is converted to an "efficiently recruitable" species by treatment at low pH in the presence of denaturants, followed by precipitation in methanol and ultracentrifugation. This material is resuspended in conversion buffer (containing low concentration SDS and non-denaturing detergents) mixed with fow percentage (preferably 0.5-0.75 °l°) "top" agar and plated on a conventional agar plate (preferably 1.5%) prepared with PBS or conversion buffer. The recruitable PrP intermediate in brain homogenate is permitted to permeate the gel in a short incubation at room temperature to form a "lawn" of convertible PrP species. A test sample is diluted in conversion buffer (as above) and applied to the conversion lawn.
The plate is incubated at an optimal temperature for a set time, both of which are to be determined. Alternatively, the "convertible substrate" and the "misfolded template" are mixed together in the "top" agar and plated simultaneously and incubated as described. A "plaque lift" is prepared by applying a filter membrane (nitrocellulose, PVDF, or other composition to be determined) to the surface of the agarose plate. A UV cross-linking step may be used on the membrane to immobilize adsorbed proteins. The membrane is then subjected to digestion with proteinase K or other proteases, to be determined and optimized. The membrane is then washed and probed with anti-PrP antibodies and secondary antibodies possessing a chromogen detection label. In vifro conversion on the plate is identified by spots of protease-resistant PrP. The number of converted spots is correlated with the number of infectious prions in the test sample. Accordingly, in another embodiment, the invention includes a diagnostic test for detection of prion protein comprising contacting the intermediate prion substrate with a lawn of brain PrP and detecting Protease-resistant PrP, detected with an anti-PrP
antibody.

The misfolded PrP species created by treatment of brain homogenates at low pH and denaturants is reactive with antibodies raised against a PrPS~-specific determinant (Cashman et al 2001), indicating that this intermediate PrP species shares some (but not all) physiochemical properties of PrPS~. The fact that PrP treated in this manner is immunoreactive with anti-PrPS°-specific antibodies also raises the possibility that it may serve as a non-infectious positive control in PrPs' detection assays based on accessibility of this epitope, and perhaps other epitopes, exposed upon conversion of PrP~ to PrPs~ (Cashman, N. R., Paramithiotis, E., Pinard, M., Lawton, T., LaBossiere, S., Leathers, V., Zou, W. Q., Estey, L., Kondejewski, L., Haghighat, A., Spatz, S. J., Tonelli, Q., Ledebur, H. C., and Ghakrabartty, A. (2001) 31th Annual Meefing of Neuroscience, San Diego (USA), p:338.) The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Examale 1 Detergent Solubility of Acidic pHlGdnHCI Treated Human Brain PrP
Recombinant mouse PrP (23-231 ) undergoes an a-helix to ~i-sheet conformational transition upon treatment with 1.5 M ~dnHCl in PBS at pH 3.5, as indicated by circular dichroism spectroscopy (Zou WQ and Cashman NR, unpublished data). The effect of these conditions on the physicochemical properties of cellular PrP derived from normal human brain tissue was shown.
After removal of acidic buffer and detergent from normal human brain homogenates, the detergent- soluble (S) and insoluble fractions (P) of the samples in lysis buffer were separated by ultracentrifugation. The distribution of PrP in the supernatants and in the pellets was determined by immunoblotting. As shown in panel a of Fig. 1, while PrPc in the mock-treated samples was predominantly found in the detergent-soluble fraction, PrP in the treated brain homogenates was recovered mostly in the detergent-insoluble fraction. Therefore, acidic pH and GdnHCI can not only induce a conformational transition of the recombinant protein, but can also induce a physical conversion from detergent-soluble PrP~ into a detergent-insoluble PrPs°-like species in native, properly post-translationally modified PrP from brain tissue. Interestingly, treated protein retained the property of insolubility even when returned to physiological pH for at least 7 days (Fig. 1, panel a, and not shown).
To further characterize the effect of pH and GdnHCI on the solubility of PrP in non-denaturing detergents, titrations of these two treatments on human brain homogenates were performed. Panel b of Fig. 1 demonstrates the pH-dependent insolubility of PrP in the presence of low concentrations of GdnHCI. At pH less than or equal to 3.5, PrP from human brain became insoluble, while at pH equal to or greater than 4.5 PrP was soluble. This pH
range corresponds to the pKa of the side chains of the Asp and Glu, showing that the pH-dependent change in solubility of PrP could be associated with the protonation of these acidic residues. To determine the effect of GdnHCI on the solubility of PrP at low pH, brain homogenates were incubated with various concentrations of GdnHCI at pH 3.5. As shown in panel c of Fig. 1, when the concentration of GdnHCI was increased to 2.5 M or higher, most of the brain PrP became soluble. However, PrP still remained insoluble at the concentration of GdnHCi less than and equal to 1.5 M. Therefore, acidic pH-treated PrP may possess a unique structure at 1.5 M GdnHCI.
Example 2 PK Sensitivity of Acidic pHlGdnHCI Treated' Human Brain PrP
Partial protease resistance is a hallmark of the PrPs~ isoform, presumably resulting from structural conversion of the protein. To determine if brain PrP treated with acid/GdnHCI possesses this property, samples were incubated with PK at various concentrations at 37°C for 1 hour. As shown in Fig. 2, both mock-treated and acidic pH/GdnHCI-treated PrP display an intrinsic PK-resistance at low concentrations of PK (equal to or less than 1 pg/ml PK), which is consistent with the observations of Buschmann et al (");
however, there was no difference in PK-sensitivity between the two, showing that despite acquiring PrPS°-like detergent-insolubility, the new species of PrP
is not identical to PrPs~.
Example 3 Epitope Accessibility of Acidic pHlGdnHCI Treated Human Brain PrP
Monoclonal antibodies to diverse epitopes of PrP have been used to probe conformational rearrangement in PrP structural isoforms ("~,"", "'~~, "~", ~', ""'). Immunoprecipitations with 3F4 antibody (against residues 109-112) and 6H4 antibody (against residues 144-152) were used to identify differences in epitope accessibility between mock-treated and acidIGdnHCI-treated brain proteins. In lysis buffer containing low concentrations of non-denaturing detergents (0.5 % NP-40 and 0.5% DOC), 6H4 and 3F4 antibodies precipitated PrP equally well from both mock-treated and acid/GdnHCI-treated brain homogenates (Fig. 3, panel a), showing that these two epitopes are not obscured in the structural changes induced by the treatment conditions. In contrast, the amount of PrP precipitated from CJD brain homogenates by the two antibodies was much less than that from normal brain homogenates (Fig.
3, panel b). Since 6H4 recognizes only native PrPc and not native PrPS
under these immunoprecipitation conditions ("""), and 3F4 is likewise poorly accessible in native PrPs~ (11), the small amounts of PrP detected in the CJD
brain homogenates could simply be residual PrP~. These data confirm that residues 109-112 and 144-152 are cryptic in PrPS~ molecules (11, 17) and suggest that the content of PrP~ may be decreased in the prion disease-affected brain (14). Moreover, acidic pH/GdnHCI-treated human brain PrP
does not share the epitope obscuration properties of native PrPS~.
ExamJ~le 4 Conversion In vitro of Acidic pHlGdnHCI-Treated Human Brain PrP
Recently, SDS has been found to induce structural conversion of a-helices to (3-sheets and aggregation of hamster rPrP (23-231) ~""", "'°'). Also, a low concentration of SDS is present in the PMCA protocol pioneered by Saborio et of (5). To determine if acid/GdnHCI-treated protein can undergo further structural rearrangement upon treatment with SDS, treated and mock-treated brain samples were incubated in 0.05% SDS, 0.5% Triton X 100 in PBS, pH
7.4 at 37°C for 12 hours with shaking. As shown in Fig. 4, small amounts of a PK-resistant fragment were found in the acid/GdnHCi-treated PrP sample (Fig. 4, lane 2) while none was observed in mock-treated sample (Fig. 4, lane 1), showing that SDS and/or Triton X 100 may induce conformational change in treated brain PrP. Although SDS has been shown to induce aggregation and structural transition of recombinant hamster PrP, these preparations do not acquire PK resistance at pH 6.5 {18), which is consistent with our data generated with mock-treated PrP~ from brain tissue.
Remarkably, the formation of PK-resistant PrP from the treated PrP
was greatly enhanced by incubation with trace quantities of PrPS° from CJD
brain homogenate (Fig. 4, lane 4). Enhanced conversion of treated human brain PrP in the presence of a PrPS~ template was confirmed in four independent experiments. No PK-resistant PrP was derived from similar experiments with mock-treated brain PrP (the very faint PK-resistant fragments seen in lane 3 of Fig. 4 are protease-resistant fragments of the exogenous PrPs~ template): Notably, this conversion reaction system contained only 0.6 p.1 of 10% CJD brain homogenate in 79.4 p,1 of 10% normal brain homogenate, and each sample in lane 3 and 4 of Fig. 4 contained only 65 n1 of CJD brain homogenate.
Materials and Methods Reagents and Antibodies Phenylmethylsulfonyl fluoride (PMSF) and protease K were purchased from Sigma Chemical Co. (St. Louis, MO, USA): Magnetic beads (Dynabeads M-280 Tosylactivated) were from Dynal Co. (Dynal AS, Oslo, Norway). Mouse monoclonal antibody 6H4 from Prionics Co. (Zurich, Switzerland) recognizes the sequence DYEDRYYRE in the prion protein (human PrP residues 144-152). Mouse monoclonal antibody 3F4 from Signet Laboratories, lnc.
(Dedham, MA, USA) recognizes an epitope of human PrP residues 109 -112 including residues MKHV. Horseradish peroxidase (HRP)-conjugated sheep anti-mouse antibody was purchased from Amersham Pharmacia Biotech, Inc.
(Piscataway, NJ, USA).
Brain Tissues and Homogenate Preparation Necropsied human brain tissue was collected within 24 hours of death.
The normal human brain was obtained from an individual determined by histology to be free of neurological disorders and a prion-infected brain was from an individual with Creutzfeldt-Jakob disease (CJD) confirmed by histological examination and western blot analysis to show the presence of PrPs°. Both samples were homozygous for Met at codon 129. All tissues were frozen immediately after collection and stored at -80 °C. 10% (w/v) brain homogenates were prepared in lysis buffer (100 mM NaCI, 10 mM EDTA, 0.5% Nonidet P 40 (NP-40), 0.5% sodium deoxycholate (DOC), 10 mM
TrisHCl, pH 7.5). After homogenization on ice, samples were centrifuged at 1,000 X g for 10 min to remove cellular debris.
Immunoblotting Samples were mixed with an equal volume of 2 X electrophoresis loading buffer (6% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, 4 mM EDTA, 20% glycerol, 125 mM TrisHCl, pH 6.8) and boiled for 10 min Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred onto polyvinylidene difluoride (PVDF) membranes at 25 V for 2 h. The membranes were blocked with 5% non-fat milk in TBST (10 mM Tris-HCI, pH 7.6, 150 mM
NaCI, 0.05% Tween 20) overnight at 4°C or 1 hour at 37°C prior to incubation with antibodies. Membrane-bound proteins were probed with 6H4 antibody at 1:5,000 or with 3F4 antibody at 1:50,000. After washing with TBST, the blot was incubated with HRP-conjugated sheep anti-mouse antibody at 1:3,000.
After washing with TBST, the proteins were visualized by enhanced chemiluminescence + plus (ECL + Plus, Amersharn Pharmacia Biotech, Inc., Piscataway, NJ, USA).
Preparation of Acid/GdnHCt-Treated PrP
100 p.1 of 10% brain homogenate was mixed with an equal volume of 3.0 M GdnHCI (final concentration 1.5 M) in PBS at pH 7.4 or pH 3.5 adjusted with 1 N HCI, followed by incubation at room temperature with shaking. After 5 h, samples were mixed with 5 volumes of pre-chilled methanol and incubated at -20°C for 2 h to precipitate the proteins. The samples were subjected to centrifugation at 16,000 X g for 20 min at 4°C to remove the acidic buffer and GdnHCI, and pellets were resuspended in 100 p1 of lysis buffer or in 100 p,1 of 0.05% SDS, 0.5% Triton X-100 in PBS pH 7.4, according to the experimental design. The samples treated at pH 7.4 were designated mock-treated samples.
Assay of Detergent Insolubility and Protease K Resistance 100 w1 of the treated or mock-treated sample in lysis buffer was centrifuged at 100,000 X g (Beckman, TL-100 Ultracentrifuge) at 4°C for 1 h.
Supernatants (containing detergent-soluble PrP) were transferred to clean tubes and pellets (containing detergent-insoluble PrP) were resuspended in an equal volume of lysis buffer. The distribution of detergent-soluble PrP or detergent-insoluble PrP was determined by immunblotting. To determine the PK-resistance of the treated PrP, 20 p,1 of sample was incubated with PK at 50 p.g/ml for 1 h at 37°C and the digestion reaction was terminated by addition of PMSF to 2 mM final concentration. The sample was mixed with equal volumes of loading buffer, boiled for 10 min, and subjected to SDS-PAGE and immunoblotting.

Immunoprecipitation Anti-PrP monoclonal antibodies (6H4 and 3F4) at 30 p.glml were coupled to magnetic Dyna beads in PBS at 37 °C for 20 h and washed twice with washing buffer (0.1 % BSAIPBS). The antibody-conjugated beads were incubated with 0.1 % BSA, 0.2 M TrisHCl, pH 8.5 at 37°C for 4 h to block non-specific binding sites, and then washed twice with 0.1 % BSAIPBS. The antibody-conjugated beads could then be stored in PBS at 4°C. For immunoprecipitation of PrP, 50 ~;1 of antibody-conjugated beads was incubated with 945 ~.I of lysis buffer in the presence of 5 ~,l of 10% (w/v) brain homogenate (mock-treated, acidic pHIGdnHCI-treated, or CJD brain) at room temperature for 3 h. The immune complex-containing beads were washed three times with washing buffer (2% NP-4.0, 2% Tween-20, PBS pH 7.4). After the last wash, all liquids were removed and 30 ~.! of loading buffer was added (without reducing agents such as dithiothreitol and ~i-mercaptoethanol to prevent antibody fragments from eluting off the beads). The samples were heated at 95°C for 5 min and then centrifuged at 3,000 rpm for 3 min.
The supernatants were subjected to SDS-PAGE and immunoblotting.
In vitro Conversion of AcidIGdnHCI-Treated PrP
To perform in vitro conversion of PrP, proteins precipitated by pre-chilled methanol were resuspended in equal volumes of 0.05% SDS, 0.5%
Triton X-100 in PBS pH 7.4, rather than in lysis buffer. This buffer has been used in PMCA of PrPs° (5). Conversion in vifro was performed in an 80 ~,f volume of the appropriate test substrate material (79.4 p1 of sample and 0.6 p,1 of 10% CJD brain homogenate for the template-directed experiments), and was incubated in a thermomixer at 37°C for 12 h with shaking. After PK-digestion at 37°C for 1 hour and boiling in loading buffer, the samples were subjected to SDS-PAGE and immunoblotting.
While the present invention has been described with reference to what are presently considered to be the preferred examptes, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims (40)

1. A method of producing an intermediate prion protein comprising contacting PrP c with a solution having a pH less than 7Ø

2. The method of claim 1 wherein the PrP c comprises native PrP.

3. The method of claims 1 or 2 wherein the PrP c comprises post-translational modifications.

4. The method of claim 3 wherein the post-translational modifications comprise two N-linked glycans and a glycosylphsophatidylinositol anchor.

5. The method of any one of claims 1 to 4 wherein the PrP c interacts with other endogenous molecules.

6. The method of claim 1 further comprising contacting the PrP c with a denaturing agent.

7. The method of claim 6, wherein the denaturing agent comprises at least one compound selected from the group consisting of a chaotropic ion, urea, an organic denaturant, an acidic amino acid and GdnHCl.

8. The method of any of claims 1-7, further comprising isolating the intermediate prion protein.

9. The method of claim 8, wherein the intermediate prion protein is isolated from the detergent-insoluble portion of the solution.

10. A method according to claim 1 wherein the PrP c protein is in a mammalian brain homogenate.

11. The method of claim 10, wherein the mammalian brain homogenate is obtained from a mammal selected from the group consisting of human, sheep, cow, goat, cervid, mouse, and hamster.

12. The method of claim 1, further comprising identifying and/or quantifying the intermediate prion protein.

13. The method of claim 12, wherein the intermediate prion protein is identified and/or quantified by immunoblotting, ELISA, an immunoassay or a detection system.

14. The method of any of claims 1-13, comprising the step of isolating the intermediate prion protein.

15. The method of claim 14, wherein the intermediate prion protein is isolated from the detergent-insoluble portion of the solution.

16. A method of producing a PrP Sc prion protein comprising the steps of treating a PrP-intermediate substrate with detergent

17. A method according to claim 16 further comprising the step of incubating the treated protein with PrP Sc template.

18. A method according to claims 16 or 17 further comprising the step of determining PK-resistance of the PrP Sc prion protein.

19. A method according to any one of claims 16 to 18 wherein the PrP-intermediate prion protein is obtained in accordance with the method of claim 1.

20. A method according to any one of claims 16 to 19 wherein the detergent comprises SDS or triton X-100.

21. An in vitro prion conversion assay for the production of an intermediate PrP Sc prion protein comprising treating a PrP-intermediate protein with detergent.

22. An assay according to claim 21, further comprising the step of incubating the treated protein with PrP Sc template.

23. An assay according to claims 21 or 22 further comprising the step of determining PK-resistance.

24. An assay according to claim 21 or 22 wherein the PrP-intermediate prion protein is obtained in accordance with the method of claim 1.

25. A method according to any one of claims 21 to 24 wherein the detergent comprises SDS or triton X-100.

26. A diagnostic test for detecting the presence of PrP Sc in a subject sample comprising the steps of {a) contacting a PrP-intermediate protein with detergent;
(b) incubating the PrP-intermediate protein with the subject sample;
(c) digesting the protein with proteinase, preferably Proteinase K;
{d) identifying the presence of PrP Sc in the sample, preferably by PK-resistance.

27. A diagnostic test according to claim 26 wherein the PrP-intermediate prion protein is obtained in accordance with the method of claim 1.

28. A diagnostic test according to claims 26 or 27 wherein the subject is a mammal selected from the group consisting of a human, sheep, cow, goat, cervid, deer, elk, mouse, and hamster.

29. A diagnostic test according to any one of claims 26 to 28 wherein the subject sample is selected from the group consisting of brain, lymphoid tissues, ocular tissues, blood or blood fractions, urine, saliva, and spinal fluid and preferably comprises at least 15ng/mL of prion protein.

30. A diagnostic test according to any one of claims 26 to 29 wherein the subject is suspected of having a prion disease.

31. The diagnostic test according to claim 26-30 wherein the prion disease is selected from the group consisting of Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, variant Creutzfeldt-Jakob disease, kuru, scrapie, bovine spongiform encephalopathy, chronic wasting disease, transmissible mink encephalopathy, and feline spongiform encephalopathy.

32. A diagnostic test for detection of prion protein comprising contacting the intermediate prion protein obtained according to the method of claim 1 with a lawn of brain PrP and detecting protease-resistant PrP.

33. The test of claim 32, wherein after the contacting step, plaque is lifted with a membrane and the membrane-bound material is digested, preferably with PK.

34. The test of claim 33, wherein the membrane comprises nitrocellulose or a PVDF filter.

35. The test of claim 32, wherein protease-resistant PrP is detected with an anti-PrP antibody.

36. A method of determining whether a candidate compound is an inhibitor of conversion of PrP c into a misfolded prion protein comprising, contacting the candidate compound with a PrP c substrate in a low pH solution and determining whether the candidate compound inhibits formation of an intermediate prion protein.

37. A method of identifying an inhibitor of conversion of PrP c into a recruitable PrP isoform comprising, contacting a candidate compound with a PrP c protein in a low pH solution and determining whether the candidate compound inhibits formation of an intermediate prion protein.

38. The method of claim 36 or 37, further comprising determining whether the compound inhibits formation of PrP Sc.

39. A method of determining whether a candidate compound is an inhibitor of conversion of an intermediate prion protein into PrP Sc comprising, contacting the candidate compound with the intermediate prion protein and determining whether the candidate compound inhibits formation of PrP Sc.

40. A method for using an intermediate prion protein as a positive control in an anti-PrP Sc antibody assay for detection of PrP Sc, comprising contacting the intermediate prion protein with the anti-PrP Sc antibody to determine whether the protein binds to the anti-PrP Sc antibody, wherein binding to intermediate prion protein indicates that the antibody is capable of binding PrP Sc and that the assay is operative.