Note: Descriptions are shown in the official language in which they were submitted.
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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
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(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.
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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.
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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
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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
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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
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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.
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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
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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.
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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
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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
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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
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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
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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
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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.
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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
CA 02392997 2002-07-11
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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
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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
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(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
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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
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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.
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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
CA 02392997 2002-07-11
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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.
