U.S. patent application number 11/979226 was filed with the patent office on 2008-07-17 for detection of conformationally altered proteins and prions.
This patent application is currently assigned to ADLYFE, INC.. Invention is credited to Eugene A. Davidson, Anne Grosset, Cindy Orser.
Application Number | 20080171341 11/979226 |
Document ID | / |
Family ID | 34677130 |
Filed Date | 2008-07-17 |
United States Patent
Application |
20080171341 |
Kind Code |
A1 |
Orser; Cindy ; et
al. |
July 17, 2008 |
Detection of conformationally altered proteins and prions
Abstract
The invention provides methods and kits for detecting
conformationally altered proteins and prions in a sample. In one
embodiment, the conformationally altered proteins and prions are
associated with amyloidogenic diseases.
Inventors: |
Orser; Cindy; (Lafayette,
CO) ; Grosset; Anne; (La Croix-de-Rozon, CH) ;
Davidson; Eugene A.; (Washington, DC) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
ADLYFE, INC.
|
Family ID: |
34677130 |
Appl. No.: |
11/979226 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10728246 |
Dec 4, 2003 |
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11979226 |
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10161061 |
May 30, 2002 |
7166471 |
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10728246 |
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10494906 |
Sep 7, 2004 |
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PCT/US02/17212 |
May 30, 2002 |
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10161061 |
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60295456 |
May 31, 2001 |
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60295456 |
May 31, 2001 |
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Current U.S.
Class: |
435/7.1 ;
530/300 |
Current CPC
Class: |
G01N 33/582 20130101;
G01N 33/542 20130101; G01N 33/6896 20130101; C07K 14/4711 20130101;
G01N 2800/2828 20130101 |
Class at
Publication: |
435/7.1 ;
530/300 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 16/00 20060101 C07K016/00 |
Claims
1. A peptide probe comprising an amino acid sequence corresponding
to a .beta.-sheet forming region of a target protein, wherein the
peptide probe exhibits a random coil or alpha-helix conformation in
solution, and undergoes a transition to a .beta.-sheet conformation
upon interaction with misfolded target protein exhibiting a
.beta.-sheet conformation, and wherein at least 75% of the amino
acid residues are hydrophobic amino acids.
2. The peptide probe of claim 1, wherein the peptide probe
comprises at least 10 amino acid residues.
3. The peptide probe of claim 1, wherein the amino acid sequence of
the peptide probe consists of 50 or fewer amino acid residues.
4. The peptide probe of claim 1, wherein the amino acid sequence
corresponding to a .beta.-sheet forming region of the target
protein is at least about 40%, at least about 70%, at least about
90% or 100% identical to the .beta.-sheet forming region of the
target protein.
5. The peptide probe of claim 1, wherein the target protein is
associated with a condition selected from amyloidogenic disease,
Alzheimer's Disease, Prion disease, Creutzfeld Jakob disease,
Gerstmann-Straussler-Scheinker Syndrome, chronic wasting disease,
scrapie, bovine spongiform encephalopathy, kuru, fatal familial
insomnia, transmissible spongiform encephalopathies, ALS, Pick's
disease, Parkinson's disease, Frontotemporal dementia, Diabetes
Type II, Multiple myeloma-plasma cell dyscrasias, Familial
amyloidotic polyneuropathy, Medullary carcinoma of thyroid, Chronic
renal failure, Congestive heart failure, Senile cardiac and
systemic amyloidosis, Chronic inflammation, Atherosclerosis,
Familial amyloidosis, and Huntington's disease.
6. The peptide probe of claim 1, wherein the target protein is
selected from the group consisting of APP, A.beta. peptide,
.alpha.1-antichymotrypsin, tau, non-A.beta. component, presenilin
1, presenilin 2 apoe, prion protein, SOD, neurofilament, Pickbody,
.alpha.-synuclein, amylin, IgGL-chain, transthyretin,
procalcitonin); .beta..sub.2-microglobulin, atrial natriuretic
factor, serum amyloid A, ApoA1, gelsolin, Huntingtin, low-density
lipoprotein receptor, cystic fibrosis transmembrane regulator,
insulin-related amyloid, hemoglobin, rhodopsin, crystallins, p53,
wildtype human TSE, human lung surfactant protein, cystatin C, and
human islet amyloid polypeptide.
7. The peptide probe of claim 1, wherein the peptide probe
comprises an amino acid sequence that is at least about 40%, at
least about 70%, at least about 90%, or 100% identical to an amino
acid sequence selected from the group consisting of SEQ ID NOs
1-29.
8. The peptide probe of claim 1, comprising (a) a first amino acid
sequence corresponding to a .beta.-sheet forming region of the
target protein and (b) a second amino acid sequence corresponding
to a .beta.-sheet forming region of the target protein, wherein the
first and second amino acid sequences are the same or different and
correspond to the same or different .beta.-sheet forming regions of
the target protein.
9. The peptide probe of claim 8, wherein the peptide probe
comprises: (a) a first amino acid sequence corresponding to a
.beta.-sheet forming region of the target protein oriented in the
forward direction, and (b) a second amino acid sequence
corresponding to a .beta.-sheet forming region of the target
protein oriented in the reverse direction.
10. The peptide probe of claim 9, wherein either (i) the second
amino acid sequence oriented in the reverse direction comprises the
same amino sequence, in the reverse direction, as the first amino
acid sequence oriented in the forward direction, or (ii) the first
amino acid sequence oriented in the forward direction comprises the
same amino sequence, in the reverse direction, as the second amino
acid sequence oriented in the reverse direction.
11. The peptide probe of claim 8, wherein at least one of the first
or second amino acid sequences consists of from 10 to 12 amino acid
residues.
12. The peptide probe of claim 8, further comprising a peptide
linker linking the first and second amino acid sequences.
13. The peptide probe of claim 12, wherein the amino acid sequence
of the peptide linker consists of from 1 to 10 amino acid
residues.
14. The peptide probe of claim 12, wherein the peptide linker
comprises proline.
15. The peptide probe of claim 1, labeled with a detectable
label.
16. The peptide probe of claim 15, wherein the detectable label is
selected from (i) optically detectable moieties and (ii)
radionuclides.
17. The peptide probe of claim 15, wherein the detectable label is
a chromophore.
18. The peptide probe of claim 15, wherein the detectable label is
selected from pyrene, tryoptophan, fluorescein, or rhodamine.
19. The peptide probe of claim 15, wherein both termini of the
peptide probe are labeled with a fluorophore capable of
participating in excimer formation.
20. A composition comprising a peptide probe of claim 1 bound to a
misfolded target protein.
21. A method for detecting misfolded target protein in a sample
comprising: (a) contacting a sample with a peptide probe according
to claim 1 and permitting the peptide probe to interact with any
misfolded target protein present in the sample; and (b) detecting
any interaction between the peptide probe and any misfolded target
protein present in the sample.
22. The method of claim 21, wherein the peptide probe is labeled
with a detectable label.
23. The method of claim 21, wherein (i) both termini of the peptide
probe are labeled with a fluorophore and (ii) the detecting step
comprises detecting any excimers formed upon interaction between
the fluorophore-labeled peptide probe and any target protein
present in the sample.
24. The method of claim 21, wherein the detecting step comprises
using circular dichroism to detect any interaction between the
peptide probe and any target protein present in the sample.
25. The method of claim 21, further comprising, prior to the
contacting step, subjecting the sample to a disaggregation
step.
26. The method of claim 21, wherein the sample is a biological
sample.
27. The method of claim 21, wherein the sample is selected from the
group consisting of tissue, meat, a biopsy sample, blood, a blood
fraction, plasma, serum, pharmaceutical formulations that might
contain products of animal origin, spinal fluid, saliva, urine,
bodily fluids, food products, and medical products.
28. The method of claim 21, wherein the target protein is
associated with a condition selected from amyloidogenic disease,
Alzheimer's Disease, Prion disease, Creutzfeld Jakob disease,
Gerstmann-Straussler-Scheinker Syndrome, chronic wasting disease,
scrapie, bovine spongiform encephalopathy, kuru, fatal familial
insomnia, transmissible spongiform encephalopathies, ALS, Pick's
disease, Parkinson's disease, Frontotemporal dementia, Diabetes
Type II, Multiple myeloma-plasma cell dyscrasias, Familial
amyloidotic polyneuropathy, Medullary carcinoma of thyroid, Chronic
renal failure, Congestive heart failure, Senile cardiac and
systemic amyloidosis, Chronic inflammation, Atherosclerosis,
Familial amyloidosis, and Huntington's disease.
29. The method of claim 21, wherein the target protein is selected
from the group consisting of APP, A.beta. peptide,
.alpha.1-antichymotrypsin, tau, non-A.beta. component, presenilin
1, presenilin 2 apoe, prion protein, SOD, neurofilament, Pickbody,
.alpha.-synuclein, amylin, IgGL-chain, transthyretin,
procalcitonin); .beta..sub.2-microglobulin, atrial natriuretic
factor, serum amyloid A, ApoA1, gelsolin, Huntingtin, low-density
lipoprotein receptor, cystic fibrosis transmembrane regulator,
insulin-related amyloid, hemoglobin, rhodopsin, crystallins, p53,
wildtype human TSE, human lung surfactant protein, cystatin C, and
human islet amyloid polypeptide.
30. The method of claim 21, wherein the peptide probe comprises an
amino acid sequence that is at least about 40%, at least about 70%,
at least about 90%, or 100% identical to an amino acid sequence
selected from the group consisting of SEQ ID NOs 1-29.
31. A method of diagnosing whether a subject suffers from, or is
predisposed to, a disease associated with a misfolded target
protein, comprising: (a) obtaining a sample from the subject; (b)
contacting the sample with a peptide probe according to claim 105;
and (c) detecting interaction between the peptide probe and any
misfolded target protein in the sample to assess the level of
misfolded target protein present, wherein the level of detectable
misfolded target protein correlates with a diagnosis that the
subject suffers from, or is predisposed to, a disease associated
with the misfolded target protein.
32. The method of claim 31, wherein the peptide probe is labeled
with a detectable label.
33. The method of claim 31, wherein (i) both termini of the peptide
probe are labeled with a fluorophore and (ii) the detecting step
comprises detecting any excimers formed upon interaction between
the fluorophore-labeled peptide probe and any target protein
present in the sample.
34. The method of claim 31, wherein the detecting step comprises
using circular dichroism to detect any interaction between the
peptide probe and any target protein present in the sample.
35. The method of claim 31, further comprising, prior to the
contacting step, subjecting the sample to a disaggregation
step.
36. The method of claim 31, wherein the sample is a biological
sample.
37. The method of claim 31, wherein the sample is selected from the
group consisting of tissue, meat, a biopsy sample, blood, a blood
fraction, plasma, serum, pharmaceutical formulations that might
contain products of animal origin, spinal fluid, saliva, urine,
bodily fluids, food products, and medical products.
38. The method of claim 31, wherein the target protein is
associated with a condition selected from amyloidogenic disease,
Alzheimer's Disease, Prion disease, Creutzfeld Jakob disease,
Gerstmann-Straussler-Scheinker Syndrome, chronic wasting disease,
scrapie, bovine spongiform encephalopathy, kuru, fatal familial
insomnia, transmissible spongiform encephalopathies, ALS, Pick's
disease, Parkinson's disease, Frontotemporal dementia, Diabetes
Type II, Multiple myeloma-plasma cell dyscrasias, Familial
amyloidotic polyneuropathy, Medullary carcinoma of thyroid, Chronic
renal failure, Congestive heart failure, Senile cardiac and
systemic amyloidosis, Chronic inflammation, Atherosclerosis,
Familial amyloidosis, and Huntington's disease.
39. The method of claim 31, wherein the target protein is selected
from the group consisting of APP, A.beta. peptide,
.alpha.1-antichymotrypsin, tau, non-A.beta. component, presenilin
1, presenilin 2 apoe, prion protein, SOD, neurofilament, Pickbody,
.alpha.-synuclein, amylin, IgGL-chain, transthyretin,
procalcitonin); .beta..sub.2-microglobulin, atrial natriuretic
factor, serum amyloid A, ApoA1, gelsolin, Huntingtin, low-density
lipoprotein receptor, cystic fibrosis transmembrane regulator,
insulin-related amyloid, hemoglobin, rhodopsin, crystallins, p53,
wildtype human TSE, human lung surfactant protein, cystatin C, and
human islet amyloid polypeptide.
40. The method of claim 31, wherein the peptide probe comprises an
amino acid sequence that is at least about 40%, at least about 70%,
at least about 90%, or 100% identical to an amino acid sequence
selected from the group consisting of SEQ ID NOs 1-29.
41. A kit for detecting the presence of misfolded target protein in
a sample comprising a peptide probe of claim 1 together with
instructions for use.
42. The peptide probe of claim 3, wherein the amino acid sequence
of the peptide probe consists of from 10 to 25 amino acid
residues.
43. The peptide probe of claim 15, wherein both termini of the
peptide probe is labeled with a detectable label.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/728,246 filed Dec. 4, 2003, which is a
continuation-in-part of U.S. patent application Ser. No.
10/161,061, filed May 30, 2002, which claims priority from U.S.
Provisional Patent Application Ser. No. 60/295,456, filed May 31,
2001. This application is also a continuation-in-part of U.S.
application Ser. No. 10/494,906 filed Sep. 7, 2004, which is a
National Stage of Application Serial No. PCT/US02/17212 filed May
30, 2002, which claims priority from U.S. Provisional Application
Ser. No. 60/295,456 filed May 31, 2001. The entire contents of the
aforementioned applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The invention provides methods and kits for detecting
conformationally altered proteins and prions in a sample.
[0003] In one embodiment, the conformationally altered proteins and
prions are associated with amyloidogenic diseases.
BACKGROUND OF THE INVENTION
1. Conformationally Altered Proteins and Prions and Associated
Diseases.
[0004] The conversion of normally soluble proteins into
conformationally altered insoluble proteins is thought to be a
causative process in a variety of other diseases. Structural
conformational changes are required for the conversion of a
normally soluble and functional protein into a defined, insoluble
state. Examples of such insoluble proteins include: A. beta.
peptide in amyloid plaques of Alzheimer's disease (AD) and cerebral
amyloid angiopathy (CAA); .alpha.-synuclein deposits in Lewy bodies
of Parkinson's disease, tau in neurofibrillary tangles in frontal
temporal dementia and Pick's disease; superoxide dismutase in
amylotrophic lateral sclerosis; huntingtin in Huntington's disease;
and prions in Creutzfeldt-Jakob disease (CJD): (for reviews, see
Glenner et al. (1989) J. Neurol. Sic. 94; 1-28; Haan et al. (199)
Clin. Neurol. Neurosurg. 92(4):305-310).
[0005] Often these highly insoluble proteins form aggregates
composed of nonbranching fibrils with the common characteristic of
a beta.-pleated sheet conformation. In the CNS, amyloid can be
present in cerebral and meningeal blood vessels (cerebrovascular
deposits) and in brain parenchyma (plaques). Neuropathological
studies in human and animal models indicate that cells proximal to
amyloid deposits are disturbed in their normal functions (Mandybur
(1989) Acta Neuropathol. 78:329-331; Kawai et al. (1993) Brain Res.
623:142-6; Martin et al. (1994) Am. J. Pathol. 145:1348-1381;
Kalaria et al. (1995) Neuroreport 6:477-80; Masliah et al. (1996)
J. Neurosci. 16:5795-5811). Other studies additionally indicate
that amyloid fibrils may actually initiate neurodegeneration
(Lendon et al. (1997) J. Am. Med. Assoc. 277:825-31; Yankner (1996)
Nat. Med. 2:850-2; Selkoe (1996) J. Biol. Chem. 271:18295-8; Hardy
(1997) Trends Neurosci. 20:154-9).
[0006] In both AD and CAA, the main amyloid component is the
amyloid beta protein (A. beta.). The A. beta. peptide, which is
generated from the amyloid beta precursor protein (APP) by the
action of two putative secretases, is present at low levels in the
normal CNS and blood. Two major variants, A.beta.sub.1-40 and
A.beta.sub.1-42, are produced by alternative carboxy-terminal
truncation of APP (Selkoe et al. (1988) Proc. Natl. Acad. Sci. USA
85:7341-7345; Selkoe, (1993) Trends Neurosci 16:403-409).
A.beta.sub.1-42 is the more fibrillogenic and more abundant of the
two peptides in amyloid deposits of both AD and CAA. In addition to
the amyloid deposits in AD cases described above, most AD cases are
also associated with amyloid deposition in the vascular walls
(Hardy (1997), supra; Haan et al. (1990), supra; Terry et al.,
supra; Vinters (1987), supra; Itoh et al. (1993), supra; Yamada et
al. (1993), supra; Greenberg et al. (1993), supra; Levy et al.
(1990), supra). These vascular lesions are the hallmark of CAA,
which can exist in the absence of AD.
[0007] Human transthyretin (TTR) is a normal plasma protein
composed of four identical, predominantly beta.-sheet structured
units, and serves as a transporter of the hormone thyroxin.
Abnormal self assembly of TTR into amyloid fibrils causes two forms
of human diseases, namely senile systemic amyloidosis (SSA) and
familial amyloid polyneuropathy (FAP) (Kelly (1996) Curr Opin
Struct Biol 6(1):11-7). The cause of amyloid formation in FAP is
point mutations in the TTR gene; the cause of SSA is unknown. The
clinical diagnosis is established histologically by detecting
deposits of amyloid in situ in biopsy material.
[0008] To date, little is known about the mechanism of TTR
conversion into amyloid in vivo. However, several laboratories have
demonstrated that amyloid conversion may be simulated in vitro by
partial denaturation of normal human TTR [McCutchen, Colon et al.
(1993) Biochemistry 32(45):12119-27; McCutchen and Kelly (1993)
Biochem Biophys Res Commun 197(2) 415-21]. The mechanism of
conformational transition involves a monomeric conformational
intermediate which poly_merizes into linear beta.-sheet structured
amyloid fibrils [Lai, Colon et al. (1996) Biochemistry
35(20):6470-82]. The process can be mitigated by binding with
stabilizing molecules such as thyroxin or triiodophenol (Miroy, Lai
et al. (1996) Proc Natl Acad Sci USA 93(26):15051-6).
[0009] The precise mechanisms by which neuritic plaques are formed
and the relationship of plaque formation to the disease-associated
neurodegenerative processes are not well-defined. The amyloid
fibrils in the brains of Alzheimer's and prion disease patients are
known to result in the inflammatory activation of certain cells.
For example, primary microglial cultures and the THP-1 monocytic
cell line are stimulated by fibrillar .beta.-amyloid and prion
peptides to activate identical tyrosine kinase-dependent
inflammatory signal transduction cascades. The signaling response
elicited by .beta.-amyloid and prion fibrils leads to the
production of neurotoxic products, which are in part responsible
for the neurodegeneration. C. K. Combs et al, J Neurosci 19:928-39
(1999).
2. Prions.
[0010] Prions are infectious pathogens that cause central nervous
system spongiform encephalopathies in humans and animals. Prions
are distinct from bacteria, viruses and viroids. A potential prion
precursor is a protein referred to as PrP 27-30, a 28 kdalton
hydrophobic glycoprotein that poly_merizes (aggregates) into
rod-like filaments found as plaques in infected brains. The normal
protein homologue differs from prions in that it is readily
degradable, whereas prions are highly resistant to proteases. It
has been suggested that prions may contain extremely small amounts
of highly infectious nucleic acid, undetectable by conventional
assay methods Benjamin Lewin, Genes IV (Oxford Univ. Press, New
York, 1990 at p. 1080. The predominant hypothesis at present is
that no nucleic acid component is necessary for the infectivity of
prion protein.
[0011] Complete prion protein-encoding genes have since been
cloned, sequenced and expressed in transgenic animals. PrP.sup.C is
encoded by a single-copy host gene and is normally found at the
outer surface of neurons. During a post-translational process,
PrP.sup.Sc is formed from the normal, cellular PrP isoform
(PrP.sup.C), and prion diseases result from conversion of PrP.sup.C
into a modified isoform called PrP.sup.Sc. PrP.sup.Sc is necessary
for both the transmission and pathogenesis of the transmissible
neurodegenerative diseases of animals and humans.
[0012] The normal prion protein (PrP) is a cell-surface
metallo-glycoprotein that is mostly an alpha-helix and coiled-loop
structure as shown in FIG. 8, and is usually expressed in the
central nervous and lymph systems. It is believed to serve as an
antioxidant and is thought to be associated with cellular
homeostasis. The abnormal form of PrP, however, is a confor_mer
which is resistant to proteases and is predominantly beta-sheet in
its secondary structure, as shown in FIG. 9. It is believed that
this conformational change in secondary structure leads to
aggregation and eventual neurotoxic plaque deposition in the
prion-disease process.
[0013] Prion-associated diseases include scrapie of sheep and
goats, chronic wasting disease of deer and elk, and bovine
spongiform encephalopathy (BSE) of cattle (Wilesmith, J. and Wells,
Microbiol. Immunol. 172:21-38 (1991)). Four prion diseases of
humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob
disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and
(4) fatal familial insomnia (FFI) (Gajdusek, D. C., Science
197:943-960 (1977); Medori et al., N. Engl. J. Med. 326:444-449
(1992)).
[0014] Prion diseases are transmissible and insidious. For example,
the long incubation times associated with prion diseases will not
reveal the full extent of iatrogenic CJD for decades in thousands
of people treated with cadaver-sourced HGH worldwide. The
importance of detecting prions in biological products has been
heightened by the possibility that bovine prions have been
transmitted to humans who developed new variant Creutzfeldt-Jakob
disease (nvCJD) (G. Chazot et al., Lancet 347:1181 (1996); R. G.
Will et al. Lancet 347:921-925 (1996)).
[0015] Diseases caused by prions are hard to diagnose: the disease
may be latent or subclinical (abnormal prions are detectable but
symptoms are not). Moreover, normal homologues of a
prion-associated protein exist in the brains of uninfected
organisms, further complicating detection. Ivan Roitt, et al.,
Immunology (Mosby-Year Book Europe Limited, 1993), at 15.1.
[0016] Current techniques used to detect the presence of
prion-related infections rely on gross morphological changes in the
brain and immunochemical techniques that are generally applied only
after symptoms are manifest. Many of the current detection methods
rely on antibody-based assays or affinity chromatography that use
brain tissue from dead animals, and in some cases capillary
immunoelectrophoresis of blood samples.
[0017] Brain-tissue-based assays can lead to late detection and
require slaughtering the animal to be tested. Prionic-Check also
entails slaughtering an animal to obtain a liquefied-brain tissue
sample, which is subjected to an antibody using Western Blot.
Although results are obtained in six to seven hours, the test does
not account for the six-month lag time between PrP.sup.S
accumulation in the brain and the onset of clinical symptoms.
Tonsillar biopsy sampling, and blood and cerebrospinal sampling,
while accurate, can require surgical intervention and take weeks to
obtain results. Electrospray ionization mass spectrometry (ESI-MS),
nuclear magnetic resonance NMR, circular dichroism (CD) and other
non-amplified structural techniques require large amounts of sample
and expensive equipment that is typically located a substantial
distance from the sample source.
[0018] Detection methods for conformationally altered proteins
associated with the aforementioned disorders such as Alzheimer's
disease and CAA are also inadequate in that, like the previously
mentioned prion detection techniques, they often require postmortem
tissue sampling,
[0019] Accordingly, the need exists for reliable and affordable
detection methods for conformationally altered proteins and prions.
Such methods should be applicable during the life of the subject at
issue in order to obtain rapid diagnoses and facilitate
prophylactic or remedial treatments.
SUMMARY OF THE INVENTION
[0020] The invention provides reliable, affordable, and safe
methods for the detection of conformationally altered proteins and
prions associated with a variety of diseases. Methods of the
invention can be applied to obtain rapid diagnoses and facilitate
prophylactic or remedial treatments. Significantly, the methods of
the invention use small amounts of sample and are therefore less
invasive and more readily applied than known diagnostic techniques.
Further, methods of the invention can be used to analyze samples
from a living subject and are not limited to samples obtained post
mortem; and may be utilized in a manner that ensures that
infectious material is not propagated during testing.
[0021] The invention overcomes many of the problems associated with
prior art diagnostic techniques by using catalytic propagation to
exploit conformational changes in conformationally altered protein
or prions associated with a particular disease process, such as
transmissible spongiform encephalopathy (TSE). Catalytic
propagation may be used to amplify the number of existing
conformationally altered protein fragments or prions in a sample
and causes detectable aggregates to form as follows:
[0022] Upon interaction of a sample containing conformationally
altered protein or prions with a conformational probe as defined
hereinafter, the probe undergoes a conformational change and adopts
the conformation of, and aggregates with, the conformationally
altered protein (which may be soluble or insoluble) or prions. The
resulting aggregates which exhibit .beta..beta. sheet formation,
may be readily detected using standard analytical techniques. As a
result, the invention facilitates rapid and cost-effective analysis
of small sample sizes and is widely applicable to tissues and body
fluids from a variety of sources including, but not limited to, the
brain.
[0023] The invention enables detection of small amounts of
disease-associated conformationally altered proteins such as
low-density lipoprotein receptor, cystic fibrosis transmembrane
regulator, Huntingtin, A-beta peptide, prions, insulin-related
amyloid, hemoglobin, alpha synuclein, rhodopsin, crystallins, and
p53. In a preferred embodiment, methods of the invention use
palindromic probes as otherwise described herein, preferably, for
example, a palindromic 33_mer probe containing amino acid sequences
126-104 and 109-126 of the PrP.sup.(Sc) protein to detect prions in
a sample. In a preferred embodiment, the probes are bound at each
end to moieties that are optically distinct and detectable upon
conformational conversion of the probes to a .beta.-sheet
structure.
[0024] In one embodiment, the invention provides a method for
detecting conformationally altered proteins or prions in a sample
comprising:
(a) reacting the sample with one or more .alpha.-helix or random
coil conformational probes that interact with the
.beta..beta.-sheet conformation insoluble proteins or prions in the
sample and thereby (i) undergo a conformational conversion to a
predominantly .beta..beta.-sheet conformation, and (ii) form
detectable aggregates with the .beta..beta.-sheet conformation
insoluble proteins or prions in the sample; and (b) detecting
levels of detectable aggregates, wherein levels of detectable
aggregates correlate to the levels of .beta..beta.-sheet
conformation insoluble proteins or prions in the sample and the
infectiousness of the sample.
[0025] The invention also provides kits that use these methods as
well as methods of diagnosing whether a subject suffers from, or is
predisposed to, a disease associated with conformationally altered
proteins or prions.
[0026] A kit of the instant invention comprises one or more
.alpha.-helix or random coil conformational probes that interact
with .beta..beta.-sheet conformation insoluble proteins or prions
in the sample and thereby (i) undergo a conformational conversion
predominantly to .beta..beta.-sheet conformation, and (ii) form
detectable aggregates with the .beta..beta.-sheet conformation
insoluble proteins or prions in the sample. The kit may further
include moieties that bind to, or are bound to, probe termini and
that are optically detectable upon conformational conversion of the
probe to a predominantly to .beta..beta.-sheet conformation, as
well as instructions for using the kit, and solutions for
suspending or fixing samples.
[0027] A method of diagnosing whether a subject suffers from, or is
predisposed to, a disease associated with conformationally altered
proteins or prion comprises:
(a) obtaining a sample from the subject; (b) reacting the sample
with one or more .alpha.-helix or random coil conformational probes
that interact with the .beta..beta.-sheet conformation of insoluble
proteins or prions in the sample and thereby (i) undergo a
conformational conversion preferably to a predominantly
.beta..beta.-sheet conformation, and (ii) form detectable
aggregates with the .beta..beta.-sheet conformation insoluble
proteins or prions in the sample; and (c) detecting levels of
detectable aggregates, wherein levels of detectable aggregates
correlate to the amount of the .beta..beta.-sheet conformation
insoluble proteins or prions in, and level of infectiousness of,
the sample and indicate whether the subject suffers from, or is
predisposed to, a disease associated with .beta..beta.-sheet
conformation insoluble proteins or prions.
[0028] These and other aspects of the invention are described
further in the following detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1 illustrates the alpha-helical monomer 10 and
beta-sheet dimer 12 of a TSE conformer. The normal wild-type (wt)
form of prion protein (PrP.sup.C) prefers a monomeric state, while
the abnormal, disease-causing form (PrP.sup.Sc) prefers the
multimeric state.
[0030] FIG. 2 illustrates a diagnostic analysis of a sample
containing TSE protein comprised of beta-sheets 12.
[0031] FIG. 3 illustrates a circular dichroism graph of a
diagnostic analysis that was performed in accordance with the
invention and that used a poly-L-lysine 20 micomolar (.mu.M) 52,000
molecular weight (MW) as a peptide-model.
[0032] FIG. 4 illustrates an absorbance graph of a diagnostic
analysis that was performed using poly-L-lysine, 70 micromolar
(.mu.M) 52,000 molecular weight (MW), as a peptidemodel.
[0033] FIG. 5 illustrates the results from FIG. 3, that used a
poly-L-lysine, 70 micromolar (.mu.M) 52,000 molecular weight (MW)
as a peptide model and the effect of pH and temperature on
conformational change.
[0034] FIG. 6 illustrates a spectroscopic analysis that used pyrene
as a fluorescent probe in proximal and distal locations in an alpha
helical bundle structure that underwent conformational change.
[0035] FIG. 7 illustrates energy changes associated with
conformational changes in proteinaceous material or prions.
[0036] FIG. 8 illustrates the alpha-helix and loop structure of
PrP.
[0037] FIG. 9 illustrates the predominantly beta-sheet secondary
structure of PrP.sup.Sc.
[0038] FIG. 10 illustrates a palindromic 33_mer probe (SEQ ID NO:
29) used in the methods of the instant invention and the 19_mer and
14_mer sequences disclosed as SEQ ID NOS 2-3, respectively.
[0039] FIG. 11 illustrates a circular dichroism graph of three
distinct common conformational forms that proteins and peptides can
assume (source: Woody R W (1996) In Circular Dichroism and the
Conformational Analysis of Biomolecules (Fasman, G D ed.) pp.
25-69, Plenum press NY).
[0040] FIG. 12 illustrates a circular dichroism graph of a
diagnostic analysis that was performed in aqueous conditions in
accordance with the invention and that used a palindromic 33_mer
probe and the 14_mer and the 19_mer amino acid sequences which make
it up (these three sequences are set forth in FIG. 10).
[0041] FIG. 13 illustrates a variation of the spectroscopic
analysis of FIG. 6, in which a spectrofluorometric data of a
diagnostic analysis that was performed using a palindromic 33_mer
probe (SEQ ID NO: 1, SEE FIG. 10_) that had pyrene attached to both
ends. The spectral scans in the monomer (open) conformation yielded
a strikingly fluorescent spectrum that had a maximum emission
between 370 and 385 nm, while the excited dimmer or excimer state
of the pyrene-labeled peptide has an emission max between 475 and
510 nm.
[0042] FIG. 14 illustrates a spectroscopic analysis in which pyrene
was used as a fluorophor, the excitation wavelength was around 350
nm, and the observation wavelength was around 365-600 nm. The
normal emission of monomer pyrene following excitation (simple
fluorescence) was recorded as the maximum wavelength at between
about 370-385 nm.
[0043] FIG. 15 illustrates the ratio of excimer formation
((I.sub.D) to monomer formation (IM) in a diagnostic analysis that
used a palindromic 33_mer probe of sequence shown in FIG. 10 under
various conditions. We expect to see minimum solubility of a
protein when the conditions are near its isoelectric point and that
is what we observed where conditions (2) approach the isoelectric
point of the 33_mer peptide--it aggregates with itself since it has
dramatically reduced solubility under these conditions as compared
to (1) In this example, electrostatic interactions (pI=10) trigger
self-association under extremely low concentrations (10 .mu.M) at
the isoelectric point of the peptide. The following legend applies
to FIG. 15. [0044] 1. pH 6-8, KCl (100-500 mM) [0045] 2. pH 10-11,
KCl (100-500 mM)
[0046] FIG. 16 illustrates an associative curve for conformation
changes in a diagnostic analysis that used a palindromic 33_mer
probe (SEQ ID NO: 1), the 19_mer (SEQ ID NO: 2) and 14_mer (SEQ ID
NO: 3) (See FIG. 10) under various conditions to determine the
optimal parameters associated with the transformation from coiled
to .beta.-sheet.
[0047] FIG. 17 shows the results from the experiment described in
Example 6 wherein the fluorescence of a complex of prion protein
and 33_mer probe was measured as a function of time. The complex
substantially dissociated over time (1 hour-24 hours).
[0048] FIG. 18 (a)-(c) illustrate fluorescence spectra of target
peptide [520 nM] in the presence of infected brain homogenate (1),
healthy brain homogenate (2), and peptide alone (3) in TRIS:TFE
(1:1) solvent. The data were obtained for 0.01% brain homogenate
from hamster (A), sheep (B), and elk (C) (hamster [270 pg/ml],
sheep [60 pg/ml], and elk [6 pg/ml]).
[0049] FIG. 19 illustrates a preliminary calibration curve of a
fluorescent diagnostic analysis conducted in accordance with the
invention. The data illustrated in this figure evidences that the
present invention is more than two orders of magnitude more
sensitive than the validated tests in use in Europe today without
any optimization. Prion Infectivity: 1 IU=3fM=200,000 PrP
[0050] The prion protein concentration was determined using the
capillary immunoelectrophoresis method of Dr. Schmerr. See,
Schmerr, et al., J. Chromatogr. A., 853 (1-2), 207-214 (Aug. 20,
1999). The sensitivity of the diagnostics with the present
invention appears to the left of the green bar, whereas the
sensitivity of more conventional diagnostics appears to the right
of the green bar. The data are taken from FIG. 18.
DETAILED DESCRIPTION OF THE INVENTION
[0051] As used herein, the following terms have the following
respective meanings.
[0052] "Amyloidogenic diseases" are diseases in which amyloid
plaques or amyloid deposits are formed in the body. Amyloid
formation is found in a number of disorders, such as diabetes,
Alzheimer's Disease (AD), scrapie, Gerstmann-Straussler-Scheinker
(GSS) Syndrome, bovine spongiform encephalopathy (BSE),
Creutzfeldt-Jakob disease (CJD), chronic wasting disease (CWD), and
related transmissible spongiform encephalopathies (TSEs).
[0053] TSE's are fatal neurodegenerative diseases that include such
human disorders as CJD, kuru, fatal familial insomnia, and GSS.
Animal forms of TSE include scrapie in sheep, CWD in deer and elk,
and bovine spongiform encephalopathy in cattle. These diseases are
characterized by the formation and accumulation in the brain of an
abnormal proteinase K resistant isoform (PrP-res) of a normal
protease-sensitive host-encoded prion protein (PrP-sen). PrP-res is
formed from PrP-sen by a post-translational process involving
conformational changes that convert the PrP-sen into a PrP-res
molecular aggregate having a higher beta.-sheet content. The
formation of these macromolecular aggregates of PrP-res is closely
associated with TSE-mediated brain pathology in which amyloid
deposits of PrP-res are formed in the brain, which eventually
becomes "spongiform" (filled with holes).
[0054] TSE diseases appear to be transmitted by exposure to an
unusual agent, for example by ritual cannibalism in the Foret
people of New Guinea, or feeding of animal parts to cattle in
bovine spongiform encephalopathy (BSE), iatrogenic CJD has also
been caused by administration of human growth hormone derived from
cadaveric pituitaries, transplanted dura mater and corneal grafts,
as well as exposure of surgeons to affected tissue during
neurological procedures.
[0055] The presence of a native prion protein (PrP) has been shown
to be essential to pathogenesis of TSE. The cellular protein
PrP-sen is a sialoglycoprotein encoded by a gene that in humans is
located on chromosome 20. The PrP gene is expressed in neural and
non-neural tissues, with the highest concentration of its mRNA
being in neurons. The translation product of the PrP gene consists
of 253 amino acids in humans, 254 in hamsters and mice, 264 amino
acids in cows, and 256 amino acids in sheep (all of these sequences
are disclosed in U.S. Pat. No. 5,565,186, which describes methods
of making transgenic mice that express species specific PrP). In
prion protein related encephalopathies, the cellular PrP-sen is
converted into the altered PrP-res that is distinguishable from
PrP-sen in that PrP-res aggregates (Caughey and Chesebro, 1997,
Trends Cell Biol. 7, 56-62); are proteinase K resistant in that
only approximately the N-terminal 67 amino acids are removed by
proteinase K digestion under conditions in which PrP-sen is
completely degraded (Prusiner et al., 1996, Sem. Virol. 7,
159-173); and has an alteration in protein conformation in which
the amount of .alpha.-helical conformation for PrP-sen is reduced,
and the amount of .beta.-sheet conformation for PrP-res is
increased (Pan et al., 1993, Proc. Natl. Acad. Sci. USA 90,
10962-10966).
[0056] If PrP-sen is not expressed in the brain tissue of animal
recipients of scrapie-infected neurografts, no pathology occurs
outside the graft, demonstrating that PrP-res and PrP-sen are both
required for the pathology (Brander et al., Nature 379:339-343,
1996). The long latency period between infection and the appearance
of disease (months to decades depending on species) has prompted
the development of a cell-free in vitro test, in which PrP-res
induces the conversion of PrP-sen to PrP-res (Kocisko et al.,
Nature 370:471474, 1994). See also Prusiner et al., WO 97/16728
published May 9, 1997. These in vivo and in vitro observations
indicate that direct interactions between PrP-res and PrP-sen form
PrP-res and promote TSE pathogenesis.
[0057] Small synthetic peptides containing certain PrP sequences
have previously been shown to spontaneously aggregate to form
fibrils with a high degree of .beta.-sheet secondary structure of
the type seen in the insoluble deposits in TSE afflicted brains
(Gasset et al., 1992, Proc. Natl. Acad. Sci. USA 89, 10940-10944;
Come et al., 1993, Proc. Natl. Acad. Sci. USA 90, 5959-5963;
Forloni et al., 1993, Nature 362, 543-546; Hope et al., 1996),
Neurodegeneration 5, 1-11). Moreover, other synthetic PrP peptides
have been shown to interact with PrP-sen molecules to form an
aggregated complex with increased protease-resistance (Kaneko et
al., Proc. Natl. Acad. Sci. USA 92, 11160-11164, 1995; Kaneko et
al., J. Mol. Biol. 270, 574-586, 1997).
[0058] "Conformationally altered proteins" include any protein
which has a three dimensional conformation associated with a
disease. The conformationally altered protein may cause the
disease, may be a factor in a symptom of the disease, or may appear
in a sample or in vivo as a result of other factors. A
conformationally altered protein appears in another conformation
which has the same amino acid sequence. These conformationally
altered proteins are generally in the form of insoluble proteins
exhibiting .beta..beta.-sheet formation which are analyzed in the
present invention.
[0059] The following is a non-limiting list of diseases followed
parenthetically by associated insoluble proteins which assemble
into two or more different conformations wherein at least one
conformation is an example of a conformationally altered protein:
Alzheimer's Disease (APP, A.beta. peptide,
alpha.1-antichymotrypsin, tau, non-A.beta. component, presenilin 1,
presenilin 2 apoE); Prion diseases, Creutzfeld Jakob disease,
scrapie and bovine spongiform encephalopathy (PrP.sup.Sc); ALS (SOD
and neurofilament); Pick's disease (Pick body); Parkinson's disease
(alpha.-synuclein in Lewy bodies); Frontotemporal dementia (tau in
fibrils); Diabetes Type II (Amylin); Multiple myeloma--plasma cell
dyscrasias (IgGL-chain); Familial amyloidotic polyneuropathy
(Transthyretin); Medullary carcinoma of thyroid (Procalcitonin);
Chronic renal failure (beta.sub.2-microglobulin); Congestive heart
failure (Atrial natriuretic factor); Senile cardiac and systemic
amyloidosis (Transthyretin); Chronic inflammation (Serum amyloid
A); Atherosclerosis (ApoA1); Familial amyloidosis (Gelsolin);
Huntington's disease (Huntingtin).
[0060] An "insoluble protein" includes any protein associated with
an amyloidogenic disease, including but not limited to any of the
proteins identified in the preceding paragraph. Insoluble proteins
generally exhibit .beta..beta.-sheet formation in the
aggregate.
[0061] "PrP protein", "PrP" and like are used interchangeably
herein and shall mean both the infectious particle form PrP.sup.Sc
known to cause diseases (spongiform encephalopathies) in humans and
animals and the noninfectious form PrP.sup.C which, under
appropriate conditions is converted to the infectious PrP.sup.Sc
form.
[0062] The terms "prion", "prion protein", "PrP.sup.Sc protein" and
the like are used interchangeably herein to refer to the infectious
PrP.sup.Sc form of a PrP protein. "Prion" is a contraction of the
words "protein" and "infection." Particles are comprised largely,
if not exclusively, of PrP.sup.Sc molecules encoded by a PrP gene.
Prions are distinct from bacteria, viruses and viroids. Known
prions infect animals and cause scrapie, a transmissible,
degenerative disease of the nervous system of sheep and goats, as
well as bovine spongiform encephalopathy (BSE), or "mad cow
disease", and feline spongiform encephalopathy of cats. Four prion
diseases known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob
Disease (CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS),
and (4) fatal familial insomnia (FFI). As used herein "prion"
includes all forms of prions causing all or any of these diseases
or others in any animals used--and in particular in humans and
domesticated farm animals.
[0063] The term "PrP gene" is used herein to describe genetic
material which expresses proteins including known polymorphisms and
pathogenic mutations. The term "PrP gene" refers generally to any
gene of any species which encodes any form of a prion protein. The
PrP gene can be from any animal, and includes all polymorphisms and
mutations thereof, it being recognized that the terms include other
such PrP genes that are yet to be discovered. The protein expressed
by such a gene can assume either a PrP.sup.C (non-disease) or
PrP.sup.Sc (disease) form.
[0064] A "peptidomimetic" is a biomolecule that mimics the activity
of another biologically active peptide molecule.
[0065] "Protein" refers to any polymer of two or more individual
amino acids (whether or not naturally occurring) linked via a
peptide bond, and occurs when the carboxyl carbon atom of the
carboxylic acid group bonded to the alpha.-carbon of one amino acid
(or amino acid residue) becomes covalently bound to the amino
nitrogen atom of amino group bonded to the .alpha.-carbon of an
adjacent amino acid. These peptide bond linkages, and the atoms
comprising them (i.e., .alpha.-carbon atoms, carboxyl carbon atoms
(and their substituent oxygen atoms), and amino nitrogen atoms (and
their substituent hydrogen atoms)) form the "polypeptide backbone"
of the protein. In simplest terms, the polypeptide backbone shall
be understood to refer the amino nitrogen atoms, alpha.-carbon
atoms, and carboxyl carbon atoms of the protein, although two or
more of these atoms (with or without their substituent atoms) may
also be represented as a pseudoatom. Indeed, any representation of
a polypeptide backbone that can be used in a functional site
descriptor as described herein will be understood to be included
within the meaning of the term "polypeptide backbone."
[0066] The term "protein" is understood to include the terms
"polypeptide" and "peptide" (which, at times, may be used
interchangeably herein) within its meaning. In addition, proteins
comprising multiple polypeptide subunits (e.g., DNA polymerase III,
RNA polymerase II) or other components (for example, an RNA
molecule, as occurs in telomerase) will also be understood to be
included within the meaning of "protein" as used herein. Similarly,
fragments of proteins and polypeptides are also within the scope of
the invention and may be referred to herein as "proteins."
[0067] "Conformation" or "conformational constraint" refers to the
presence of a particular protein conformation, for example, an
alpha-helix, parallel and antiparallel beta. strands, leucine
zipper, zinc finger, etc. In addition, conformational constraints
can include amino acid sequence information without additional
structural information. As an example, "--C--X--X--C--" is a
conformational constraint indicating that two cysteine residues
must be separated by two other amino acid residues, the identities
of each of which are irrelevant in the context of this particular
constraint. A "conformational change" is a change from one
conformation to another.
[0068] The exact mechanism by which the sequence of a protein
encodes the proper fold is unknown. In order to achieve the native
state encoded by the fold, the protein molecule must convert to a
unique conformation selected from many alternatives. Functional
proteins are typically soluble and can adopt a variety of
structures including coils and ordered elements. Ordered elements
include the alpha helix predominant in proteins such as myoglobin
and hemoglobin. During the human aging process, in some proteins
the soluble structure (e.g. alpha helical regions) becomes
conformationally altered into beta sheet structures that undergo
aggregation associated with loss of function.
[0069] There are at least twenty proteins that are associated with
human disease when they adopt a conformationally altered state, and
some of these have been described previously. FIG. 1 illustrates
both the alpha-helical monomer 10 and the beta-sheet dimer 12 forms
of a TSE conformer. The normal wild-type (wt) form of prion protein
(PrP.sup.C) prefers a monomeric state, while the abnormal,
disease-causing form (PrP.sup.Sc) more readily takes on a
multimeric state.
[0070] Protein structures can be determined by a variety of
experimental or computational methods, several of which are
described below. Protein structure can be assessed experimentally
by any method capable of producing at least low resolution
structures. Such methods currently include X-ray crystallography
and nuclear magnetic resonance (NMR) spectroscopy. X-ray
crystallography is one method for protein structural evaluation,
and is based on the diffraction of X-ray radiation of a
characteristic wavelength by electron clouds surrounding the atomic
nuclei in the crystal. X-ray crystallography uses crystals of
purified biomolecules (but these frequently include solvent
components, co-factors, substrates, or other ligands) to determine
near atomic resolution of the atoms making up the particular
biomolecule. Techniques for crystal growth are known in the art,
and typically vary from biomolecule to biomolecule. Automated
crystal growth techniques are also known.
[0071] Nuclear magnetic resonance (NMR) currently enables
determination of the solution conformation (rather than crystal
structure) of biomolecules. Typically only small molecules, for
example proteins of less that about 100-150 amino acids, are
amenable to these techniques. However, recent advances have lead to
the experimental elucidation of the solution structures of larger
proteins, using such techniques as isotopic labeling. The advantage
of NMR spectroscopy over X-ray crystallography is that the
structure is determined in solution, rather than in a crystal
lattice, where lattice neighbor interactions can alter the protein
structure. The disadvantage of NMR spectroscopy is that the NMR
structure is not as detailed or as accurate as a crystal structure.
Generally, biomolecule structures determined by NMR spectroscopy
are of moderate resolution compared relative to those determined by
crystallography.
[0072] Other techniques useful in studying biomolecule structure
include circular dichroism (CD), fluorescence, and
ultraviolet-visible absorbance spectroscopy. See, for example,
Physical Biochemistry: Applications to Biochemistry and Molecular
Biology, 2.sup.nd ed., W.H. Freeman & Co., New York, N.Y., 1982
for descriptions of these techniques.
[0073] "Equivalent" refers to amino acid sequences that are similar
in sequence to the amino acid sequence of the protein to be
analyzed but have at least one, but fewer than 5, (e.g., 3 or
fewer) differences, substitutions, additions, or deletions. Thus,
the substitution of one or more amino acid in a given sequence
which does not substantially change the basic function of that
amino acid within its use in context, is an equivalent for purposes
of describing the present invention.
[0074] "Homology", "homologs of", "homologous", or "identity" or
"similarity" refers to sequence similarity between two
polypeptides, with identity being a more strict comparison.
Homology and identity can each be determined by comparing a
position in each sequence which may be aligned for purposes of
comparison. When a position in the compared sequence is occupied by
the same amino acid, then the molecules are identical at that
position. A degree of identity of amino acid sequences is a
function of the number of identical amino acids at positions shared
by the amino acid sequences. A degree of homology or similarity of
amino acid sequences is a function of the number of amino acids,
i.e., structurally related, at positions shared by the amino acid
sequences. An "unrelated" or "non-homologous" sequence shares less
than 40% identity, though preferably less than 25% identity, with
one of the sequences used in the present invention. Related
sequences share more than 40% identity, preferably at least about
50% identity, more preferably at least about 70% identity, even
more preferably at least about 90% identity, more preferably at
least about 99% identity.
[0075] The term "percent identical" refers to sequence identity
between two amino acid sequences. Identity can each be determined
by comparing a position in each sequence which may be aligned for
purposes of comparison. When an equivalent position in the compared
sequences is occupied by the same amino acid, then the molecules
are identical at that position; when the equivalent site occupied
by the same or a similar amino acid residue (e.g., similar in
steric and/or electronic nature), then the molecules can be
referred to as homologous (similar) at that position. Expression as
a percentage of homology, similarity, or identity refers to a
function of the number of identical or similar amino acids at
positions shared by the compared sequences. Various alignment
algorithms and/or programs may be used, including FASTA, BLAST, or
ENTREZ. FASTA and BLAST are available as a part of the GCG sequence
analysis package (University of Wisconsin, Madison, Wis.), and can
be used with, e.g., default settings. ENTREZ is available through
the National Center for Biotechnology Information, National Library
of Medicine, National Institutes of Health, Bethesda, Md. In one
embodiment, the percent identity of two sequences can be determined
by the GCG program with a gap weight of 1, e.g., each amino acid
gap is weighted as if it were a single amino acid mismatch between
the two sequences. Other techniques for determining sequence
identity are well-known and described in the art.
[0076] The term "interact" as used herein is meant to include
detectable interactions (e.g., biochemical interactions) between
molecules, such as interaction between protein-protein,
protein-nucleic acid, nucleic acid-nucleic acid, and protein-small
molecule or nucleic acid-small molecule in nature.
[0077] The term "homolog of an insoluble protein" includes all
amino acid sequences that are encoded by a homolog of an insoluble
protein gene, and all amino acid sequences that are equivalent or
homologous to such sequences. Therefore, "homolog of an insoluble
protein" includes proteins that are scored as hits in the Pfam
family. To identify the presence of an "insoluble protein" domain
in a protein sequence, and make the determination that a
polypeptide or protein of interest has a particular profile, the
amino acid sequence of the protein can be searched against one of
several databases (SwissProt, PIR, for example) using various
default parameters
(http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example,
the hmmsf program, which is available as part of the HM_MER package
of search programs, is a family specific default program for
MILPAT0063 and a score of 15 is the default threshold score for
determining a hit. Alternatively, the threshold score for
determining a hit can be lowered (e.g., to 8 bits). A description
of the Pfam database can be found in Sonham_mer et al. (1997)
Proteins 28(3):405-420 and a detailed description of HMMs can be
found, for example, in Gribskov et al. (1990) Meth. Enzymol.
183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA
84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and
Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which
are incorporated herein by reference.
[0078] "Test specimen" is a sample of material to be tested. The
sample may be prepared from tissue (e.g. a portion of ground meat,
an amount of tissue obtained by a biopsy procedure) by
homogenization in a glass homogenizer. The amount of material
should be between about 1 mg and 1 gm, preferably between 10 mg and
250 mg, ideally between 20 and 100 mg. The material to be sampled
may be suspended in a suitable solvent, preferably
phosphate-buffered saline at a pH between 7.0 and 7.8. The solvent
may contain a detergent such as (Triton X-100, SDS, or sarkosyl).
Homogenization is performed for a number of excursions of the
homogenizer, preferably between 10 and 25 strokes; ideally between
15 and 20 strokes. The suspended sample is preferably centrifuged
at between 100 and 1,000 g for 5-10 minutes and the supernatant
material sampled for analysis. In some samples, it may be
preferable to treat the supernatant material with an additional
reagent such as phosphotungstic acid according to the procedure
described by Safar et al., Nature Medicine 4, pp. 1157-1165 (1998)
and as modified by Wadsworth et al. The Lancet, 358, pp.
171-180(2001).
[0079] The amount of sample to be tested is based on a
determination of the protein content of the supernatant solution as
measured by the procedure described by Bradford (1976). Preferably,
this corresponds to between 0.5 and 2 mg of protein.
[0080] In addition to the procedure described above for tissue
material, test samples may be obtained from serum, pharmaceutical
formulations that may contain products of animal origin, spinal
fluid, saliva, urine or other bodily fluids. Liquid samples may be
tested directly or may be subjected to treatment with agents such
as phosphotungstic acid as described above.
[0081] "Conformational probes" are preferably peptides that have
amino acid sequences that are similar to, and more preferably
identical to, some of those in the target protein and that also
have the potential to undergo conformational alteration to produce
.beta..beta.-sheet formation when complexed with the target protein
(insoluble protein). Such alteration typically leads to a .beta.
sheet structure not normally evidenced by the probe. Ideally, a
probe has a palindromic structure with two amino acid sequences
derived from the target protein. Preferred .alpha.-helix or random
coil conformational probes (i.e., probes that exhibit .alpha.-helix
or random coil conformation in solution) useful in the instant
invention include the following:
a palindromic 33_mer comprising amino acid sequences that are
identical to amino acids 122-104 and 109-0122 of the PrPSC protein
(SEQ ID NO: 13 and 14) (Swiss-Prot PO4156 (Pfam ID Prion Pf00377
& 03991)
TABLE-US-00001 SEQ ID NO: 29 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV
(murine) SEQ ID NO: 1 VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV
(human);
a palindromic 33_mer comprising amino acid sequences that are
equivalent to amino acids 122-104 and 109-0122 of the PrPSC protein
(SEQ ID NO: 13 and 14) (Swiss-Prot PO4156 (Pfam ID Prion Pf00377
& 03991)
TABLE-US-00002 SEQ ID NO: 29 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV
(murine) SEQ ID NO: 1 VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV
(human);
a palindromic 33_mer comprising amino acid sequences that are
between about 70% to about 90% identical to amino acids 122-104 and
109-122 of the PrPSC protein SEQ ID NO: 13 and 14) (Swiss-Prot
PO4156 (Pfam ID Prion Pf00377 & 03991)
TABLE-US-00003 SEQ ID NO: 29 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV
(murine) SEQ ID NO: 1 VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV
(human);
a probe comprising amino acid sequences that are identical to amino
acids 1-40 of the Abeta peptide (Nref 00111747 (human))
TABLE-US-00004 (SEQ ID NO: 4)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV;
a probe comprising amino acid sequences that are equivalent to
amino acids 1-40 of the Abeta peptide (Nref 00111747 (human))
TABLE-US-00005 (SEQ ID NO: 4)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV;
a probe comprising amino acid sequences that are between about 70%
to about 90% identical to amino acids 1-40 of the Abeta peptide
(Nref 00111747 (human))
TABLE-US-00006 (SEQ ID NO: 4)
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV;
a probe comprising amino acid sequences that are identical to amino
acids 11-34 of the Abeta peptides (Nref 00111747 (human))
TABLE-US-00007 EVHHQKLVFFAEDVGSNKGAIIGL; (SEQ ID NO: 5)
a probe comprising amino acid sequences that are identical to amino
acids 11-34 of the Abeta peptides (Nref 00111747 (human)) but with
residue H13 substituted with R to reduce metal ion interactions and
to increase the solubility of the peptide
TABLE-US-00008 EVRHQKLVFFAEDVGSNKGAIIGL; (SEQ ID NO: 6)
a probe comprising amino acid sequences that are identical to amino
acids 25-35 of the Abeta peptides (Nref 00111747 (human))
TABLE-US-00009 GSNKGAIIGLM; (SEQ ID NO: 7)
a probe that has a helix-loop-helix conformation found in
polylysine and an amino acid sequence that is at least 10 amino
acid residues in length and is equivalent or homologous to SEQ ID
NO:8 KKKKKKKKKKKKKKKKKKKKKKKKKKK (27_mer); a probe that has a
conformation found in polyglutamine and an amino acid sequence that
is equivalent or homologous to SEQ ID NO:9 QQQQQQQQQQQQQQQQQQQQQQQ;
a probe comprising amino acid sequences that are homologous to
amino acids 104-122 of wild-type (wt) TSE (Human NREF 00130350)
TABLE-US-00010 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO:10)
a probe comprising amino acid sequences that are equivalent to
amino acids 104-122 of wild-type (wt) TSE (Human NREF 00130350)
TABLE-US-00011 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO:10)
a probe comprising amino acid sequences that are between about 70%
to about 90% identical to amino acid sequences 104-122 of wild-type
(wt) TSE (Human NREF 00130350)
TABLE-US-00012 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO:10)
a probe that comprise an amino acid sequence that: (a) is a
selectively mutated TSE sequence; (b) is destabilized and
noninfectious; and (c) has an amino acid sequence that is
homologous to amino acid sequences 104-122 of wild-type (wt) TSE
(Human NREF 00130350)
TABLE-US-00013 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO:10)
a probe that comprise an amino acid sequence that: (a) is a
selectively mutated TSE sequence; (b) is destabilized and
noninfectious; and (c) has an amino acid sequence that is
equivalent to amino acid sequences 104-122 of wild-type (wt) TSE
(Human NREF 00130350)
TABLE-US-00014 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO:10)
a probe that comprise an amino acid sequence that: (a) is a
selectively mutated TSE sequence; (b) is destabilized and
noninfectious; and (c) has an amino acid sequence that is between
about 70% to about 90% identical to amino acid sequences 104-122 of
wild-type (wt) TSE (Human NREF 00130350)
TABLE-US-00015 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO:10)
a probe comprising amino acid sequences that are identical to amino
acids 1-38 of the human islet amyloid polypeptide precursor
(amylin) protein (Accession # NP.sub.--000406 (human) implicated in
human diabetes
TABLE-US-00016 (SEQ ID NO: 11)
MGILKLQVFLIVLSVALNHLKATPIESHQVEKRKCNTA;
a probe comprising amino acid sequences that are identical to at
least 10 contiguous amino acid residues within the sequence
corresponding to amino acids 1-38 of the human islet amyloid
polypeptide precursor (amylin) protein (Accession # NP.sub.--000406
(human) implicated in human diabetes
TABLE-US-00017 (SEQ ID NO: 11)
MGILKLQVFLIVLSVALNHLKATPIESHQVEKRKCNTA;
a probe comprising amino acid sequences that are identical to amino
acids 1-25 of the human lung surfactant protein (NCBI Accession #
AAH32785 (human) implicated in human infant SIDS
TABLE-US-00018 MAESHLLQWLLLLPTLCGPGTAAW (SEQ ID NO: 12)
a probe comprising amino acid sequences which include at least 10
contiguous amino acid residues of amino acids 104-122 of the human
or amino acids 103-121 of the murine PrP.sup.Sc protein (SEQ ID NO:
13 and 14) (Swiss-Prot PO4156 (Pfam ID Prion Pf00377 &
03991)
TABLE-US-00019 Human prion protein Accession : PO4156 (SEQ ID NO:
13) 10 20 30 40 50 60 | | | | | | MANLGCWMLV LFVATWSDLG LCKKRPKPGG
WNTGGSRYPG QGSPGGNRYP PQGGGGWGQP 70 80 90 100 110 120 | | | | | |
HGGGWGQPHG GGWGQPHGGG WGQPHGGGWG QGGGTHSQWN KPSKPKTNMK HMAGAAAAGA
130 140 150 160 170 180 | | | | | | VVGGLGGYML GSAMSRPIIH
FGSDYEDRYY RENMHRYPNQ VYYRPMDEYS NQNNFVHDCV 190 200 210 220 230 240
| | | | | | NITIKQHTVT TTTKGENFTE TDVKMMERVV EQMCITQYER ESQAYYQRGS
SMVLFSSPPV 250 | ILLISFLIFL IVG #2 Mouse prion protein Accession :
PO4925 (SEQ ID NO: 14) 10 20 30 40 50 60 | | | | | | manlgywlla
lfvtmwtdvg lckkrpkpgg wntggsrypg qgspggnryp pqggtwgqph 70 80 90 100
110 120 | | | | | | gggwgqphgg swgqphggsw gqphgggwgq gggthnqwnk
pskpktnlkh vagaaaagav 130 140 150 160 170 180 | | | | | |
vgglggymlg samsrpmihf gndwedryyr enmyrypnqv yyrpvdqysn qnnfvhdcvn
190 200 210 220 230 240 | | | | | | itikqhtvtt ttkgenftet
dvkmmervve qmcvtqyqke sqayydgrrs sstvlfsspp 250 | villisflif
livg;
a probe comprising amino acid sequences which include at least 10
contiguous amino acid residues of amino acids 235-269 (emphasized
below) of the human plasma gelsolin (SEQ ID NO: 15) (PO6396),
Maury, et al. FEBS Lett., 260(1), pp. 85-87 (1990);
TABLE-US-00020 (SEQ ID NO: 16); 1 maphrpapal lcalslalca lslpvraata
srgasqagap qgrvpearpn smvvehpefl 61 kagkepglqi wrvekfdlvp
vptnlygdff tgdayvilkt vqlrngnlqy dlhywlgnec 121 sqdesgaaai
ftvqlddyln gravqhrevq gfesatflgy fksglkykkg gvasgfkhvv 181
pnevvvqrlf qvkgrrvvra tevpvswesf nngdcfildl gnnihqwcgs nsnryerlka
241 tqvskgirdn ersgrarvhv seeqtepeam lqvlgpkpal pagtedtake
daanrklakl 301 ykvsngagtm svslvadenp faqgalksed cfildhgkdg
kifvwkgkqa nteerkaalk 361 tasdfitkmd ypkqtqvsvl peggetplfk
qffknwrdpd qtdglglsyl sshianverv 421 pfdaatlhts tamaaqhgmd
ddgtgqkqiw riegsnkvpv dpatygqfyg gdsyiilyny 481 rhggrqgqii
ynwqgaqstq devaasailt aqldeelggt pvqsrvvqgk epahlmslfg 541
gkpmiiykgg tsreggqtap astrlfqvra nsagatrave vlpkagalns ndafvlktps
601 aaylwvgtga seaektgaqe llrvlraqpv qvaegsepdg fwealggkaa
yrtsprlkdk 661 kmdahpprlf acsnkigrfv ieevpgelmq edlatddvml
ldtwdqvfvw vgkdsqeeek 721 tealtsakry ietdpanrdr rtpitvvkqg
feppsfvgwf lgwdddywsv dpldramael 781 aa
YERLKATQVSKGIRDNERSGRARVHVSEEGTEPEAM;
a probe comprising amino acid sequences which include at least 10
contiguous amino acid residues of amino acids 27-146 (emphasized
below) of the cytastain C protein sequence depicted below (SEQ ID
NO: 17) (P01034), Levy, et al. J. Exp. Med., 169(5), pp. 1771-8
(1989). The amyloid forming version of the peptide is 120 amino
acids corresponding to amino acid residues 27-146 below. An
appropriate probe is any portion thereof of at least 10 amino
acids, numerous probes can be posited accordingly;
TABLE-US-00021 1 magplrapll llailavala vspaagsspg kpprlvggpm
dasveeegvr raldfavgey 61 nkasndmyhs ralqvvrark qivagvnyfl
dvelgrttct ktqpnldncp fhdqphlkrk 121 afcsfqiyav pwqgtmtlsk
stcqda
Palindromic probe of cystatin C protein (from amino acids 39-47 of
the above sequence with a four unit proline linker)
TABLE-US-00022 EEEVSADMPPPPMDASVEEE ((SEQ ID NO: 18)
a probe comprising amino acid sequences which include at least 10
and up to 23 contiguous glutamine amino acid residues oligo or
polyglutamine (from residues 18-40) of the Huntingtin (Huntington's
Disease Protein) protein sequence depicted below (SEQ ID NO: 19)
(P42858) [gi:1170192]:
TABLE-US-00023 1 matleklmka feslksfqqq qqqqqqqqqq qqqqqqqqqq
pppppppppp pqlpqpppqa 61 qpllpqpqpp ppppppppgp avaeeplhrp
kkelsatkkd rvnhcltice nivaqsvrns 121 pefqkllgia melfllcsdd
aesdvrmvad eclnkvikal mdsnlprlql elykeikkng 181 aprslraalw
rfaelahlvr pqkcrpylvn llpcltrtsk rpeesvqetl aaavpkimas 241
fgnfandnei kvllkafian lksssptirr taagsavsic qhsrrtqyfy swllnvllgl
301 lvpvedehst llilg..... ....
exemplary probe:
TABLE-US-00024 QQQQQQQQQQQQQQQQQ; (SEQ ID NO: 20)
a probe comprising amino acid sequences which include at least 6
contiguous amino acid residues of amino acid residues 12-17 and
15-20 (emphasized below) of the (8-20) domain of the human islet
amyloid polypeptide involved in fibrillogenesis, sequence depicted
below (SEQ ID NO: 21) NP.sub.--000406 [gi:4557655] Scrocchi, et
al., J. Struct. Biol., 141(3), pp. 218-27 (2003).
TABLE-US-00025 1 mgilklqvfl ivlsvalnhl katpieshqv ekrkcntatc
atqrlanfiv hssnnfgail 61 sstnvgsnty gkrnavevlk replnylpl
Exemplary probes contain the following sequences which are minimal
sequences within the sequence 8-20 of the above peptide sequence,
which may be used without modification or may be used to form
palindromic probes of the present invention:
TABLE-US-00026 (SEQ ID NO: 22) LANFV; (SEQ ID NO: 23)
VFNALPPPPLANFV (palindromic probe); (SEQ ID NO: 24) FLVHSS; (SEQ ID
NO: 25) SSHVLFPPPFLVHSS (palindromic probe);
a probe comprising amino acid sequences which include at least 5
contiguous amino acid residues of amino acid residues 10-19
(emphasized below) of the peptide fragment of transthyretin,
sequence depicted below (SEQ ID NO: 26) AAH20791 [gi: 18089145]
MacPhee and Dobson, J. Mol. Biol., 297(5), pp. 1203-15 (2000)
TABLE-US-00027 1 mashrllllc laglvfvsea gptgtgeskc plmvkvldav
rgspainvav hvfrkaaddt 61 wepfasqkts esgelhgltt eeefvegiyk
veidtksywk algispfheh aevvftands 121 gprrytiaal lspysystta
vvtnpke;
a palindromic probe based upon the above referenced sequence (amino
acid residues 10-19):
TABLE-US-00028 ESVFVLGALPPPPLAGLVFVSE. (SEQ ID NO: 27)
[0082] Numerous other probes may be readily produced without undue
experimentation using standard laboratory techniques and peptide
and related chemical syntheses.
[0083] The native conformation of the probe is determined by one or
more spectroscopic methods such as circular dichroism, Fourier
transform infra-red, ultra-violet, nuclear magnetic resonance, or
fluorescence, among others. This conformation in a solvent as
described below should correspond to that of an alpha-helix or
random coil (in circular dichroism, for example, the nature of the
spectrum is indicative of the conformation).
[0084] The probe is modified to contain substituents that are
detectable by optical means. Such substituents may include
tryptophan (an amino acid), pyrene or similar fluorophores, all
attached at or near the terminal positions of the peptide probes.
Attachment of such fluorophores proceeds according to conventional
chemical methods which are well-known in the art, preferably, but
not necessarily through covalent attachment of the fluorophore to
the probe. Ideally, the substituents have the capability to
interact in such a manner as to produce a species known as an
excimer. An excimer represents the interaction of two fluorophores
that, upon excitation with light of a specific wavelength, emits
light at a different wavelength which is also different in
magnitude from that emitted by either fluorophore acting alone.
Thus, structural alterations of the conformational probe that allow
for the formation of such excimers can be detected by a change in
optical properties. Such changes can be measured by known
fluorimetric techniques, including UV, IR, CD, NMR, or
fluorescence, among numerous others, depending upon the fluorophore
attached to the probe. The magnitude of these changes is related to
the degree to which the probe has undergone the conformational
alteration.
[0085] In another embodiment, the probe may be substituted with a
radioactive material. Ideally, this should be positron emission of
a sufficient energy to be detected by machines currently employed
for this purpose. Such an entity would preferably contain oxygen-15
(an isotope of oxygen that decays by positron emission) or other
radionuclide. In this embodiment, the radiolabeled probe may be
injected into a patient and the binding of the probe to the protein
target monitored externally.
[0086] A probe may comprise a peptide or peptidomimetic of at least
five, preferably about ten or more amino acid residues that
exhibits a random coil or alpha-helical conformation in solution. A
peptide or peptidomimetic probe solvent may be aqueous and have a
pH of between about 4 and about 10, preferably between about 5 and
about 8, and may have an ionic strength of between about 0.05 and
about 0.5 (when typically prepared with a chloride salt such as
sodium chloride or potassium chloride). The solvent may also
contain a percentage of a water-miscible organic material such as
trifluoroethanol in amounts between about 30 to about 70% by
volume, preferably between about 45 to about 60%. The solvent may
be prepared with a suitable buffering system such as acetate/acetic
acid, Tris, or phosphate.
[0087] The sequence of probe amino acids is determined from the
nature of the target protein to be analyzed and usually comprises a
region of the target that is known to undergo a structural
transition from either an alpha-helix or coil to a beta-sheet. This
latter structure is associated with the pathogenic form of the
target protein. The conformational probe sequence ideally contains
two repeats of the target sequence of interest, preferably between
about 10 and 25 amino acids in length; more preferably between
about 14 and 20 amino acids in length. These are arranged
preferably in the probe to form a palindrome as illustrated in FIG.
10.
[0088] Preferred probes used in methods and kits of the invention
have amino acid sequences corresponding to .beta.-sheet regions of
the protein to be analyzed. These probes are preferably at least 5
amino acids units in length and can be about 300-400 amino acid
units in length (_mer) or more, although, preferably these are
about 10 amino acids to about 50 amino acids in length. In certain
aspects of the invention, preferred probes which correspond to the
.beta.-sheet region are about 15 to about 100_mer, in others
preferred probes may range from about 20 to about 50_mer. The
preferred length of a given probe will be a function of the probes
ability to complex and produce .beta..beta.-sheet formation with
the target protein.
[0089] Probes for use in the present invention are readily
determined from existing information in sequence databases already
in existence or alternatively, may be readily determined
experimentally. Thus, the probe will generally correspond to a
minimum number of amino acids, preferably at least 10, and more
preferably about 10 to 25 amino acids, which correspond to at least
a portion of a peptide sequence of a target protein which
undergoing a conformational transition from alpha-helix or random
coil to .beta..beta.-sheet formation in the insoluble protein.
[0090] Noted that within the experimental information which will
guide the presentation and synthesis of an appropriate probe, there
are some constraints which can guide the practitioner in making use
of the present invention. Because there are only a few kcal
difference separating a population in the initial conformation
state from a population predominantly in the transformed
conformational state (in complex). This transformation is provided
by the driving force due either to the Kd of association between
the probe molecule and its natural associate to form
.beta..beta.-sheet complex, or due to changes in the electrostatic
interactions between the molecules (for example, by lowering the
ionic strength of the solution. If metal ions such as Al are
involved, or the binding of another ligand, other electrostatic or
steric effects could contribute. The size of the probe peptide can
vary, but should be of sufficient length to have "reasonably"
well-defined secondary structure under detection conditions and to
have sufficient recognitional specificity for the prion selected.
The probe peptide should also accommodate single-site mutations in
order to be generally applicable to mutated strains, recognizing
that these changes and/or heterogeneities affect the thermodynamic
stability of the molecule. Moreover, the probe must be
non-contagious to the patient population, whether that population
is a human patient population, a domesticated animal population or
other mammalian population.
[0091] Once a peptide sequence is established for a probe (which
corresponds to at least a portion of a target protein responsible
for .beta.-sheet formation as described above), the peptide
sequence may be endcapped (at one, but preferably both ends of the
peptide) with a moiety or chemical entity which can facilitate
analysis of the peptide probe. Preferably, this moiety is a
fluorophore, such as pyrene, but may vary widely, depending upon
the analytical technique to be used for analysis. The moiety or
chemical entity may be complexed or covalently bonded at or near
the amino or carboxy end of the peptide, which is preferably
endcapped with a short, hydrophobic peptide sequence. In preferred
aspects of the present invention, both the amino and carboxy ends
of the probe peptides are endcapped with small hydrophobic peptides
ranging in size from about 1 to about 5 amino acids. These may be
natural or synthetic, but are preferably natural (i.e., derived
from a .beta.-sheet formation region of a target protein. The
fluorophore are preferably attached at or near the amino and/or
carboxy end of the probe (preferably both) and may be, for example,
pyrene, tryptophan, flurescein, rhodamine, among numerous others
and is preferably pyrene. It is preferable that the fluorophores
form excimers when in the correct geometric orientation.
[0092] Conformational probes according to the present invention are
preferably palindromic in nature. This refers to the organization
of a given conformational probe sequence such that it will contain
first and second peptide sequences corresponding to a portion of
the target protein responsible for .beta.-sheet formation, but
which peptide sequences are presented in a palindromic manner,
i.e., from the carboxy end to the amino end (or amino end to
carboxy end) in the first peptide sequence, and from the amino end
to the carboxy end (or carboxy end to amino end) in the second
peptide sequence. The first and second peptide sequence in the
palindromic conformational probe do not have to be identical in
length, although this may be preferred in certain embodiments, but
should be at least roughly equivalent (the two peptide sequences
{"arms" of the probe} should not be more than 15, preferably no
more than 10 and even more preferably no more than 5 amino acids
different in length). Preferably, the first and second peptide
sequences within a palindromic probe sequence are separated by a
linker comprising between 1 and 5 amino acids, preferably between 1
and 3 amino acids, which preferably contain at least one proline
amino acid and more preferably comprise primarily proline amino
acids. FIG. 10 presents an exemplary palindromic 33_mer
conformation probe useful in the present invention.
[0093] Preferably, conformational probes according to the present
invention contain a hydrophobic amino acid sequence which is
preferably derived from the relevant peptide sequence of the target
protein (i.e., the peptide sequence responsible for .beta.-sheet
formation), and which may vary in length from 1 amino acid to 20 or
more amino acids, preferably about 2-10 amino acids in length and
appears at or near one of the two ends of the conformation probe.
In the case of palindromic conformation probes, these hydrophobic
amino acid sequences appear at the ends of the two peptide arms of
the probe. Optionally, the probe also may contain a synthetic
hydrophobic amino acid sequence (i.e., not natural to the peptide
sequence of the target protein responsible for .beta.-sheet
formation) at least one end of the probe and in the case of
palindromic probes, at or near each end of the probe, which may
vary in length from as few as one amino acid to 20 or more amino
acids, preferably about 3-10 amino acids in length.
[0094] By way of example and without limitation, if a desired
peptide sequence in a target protein contains the sequence, reading
from amino end to carboxy end, QRSTVVURLKAAAV (SEQ ID NO: 30)
(where AAAV (SEQ ID NO: 31) is a hydrophobic amino acid sequence)
then the palindrome would contain a first peptide sequence which is
VAAAKLRUVVTSRQ (SEQ ID NO: 32) and a second peptide sequence which
is QRSTVVURLKAAAV (SEQ ID NO: 30) (or a close variation to that
sequence), with the two sequences separated by a linker comprising
from 1 to 5 amino acids, with at least one of those amino acids,
and preferable most, if not all, of those amino acids, being
proline amino acids. The probe would therefore be:
TABLE-US-00029 VAAAKLRUVVTSRQPPPPQRSTVVURLKAAAV SEQ ID NO: 28
(hypothetical palindromic probe)
[0095] Preferably, the palindromic probe would contain a
hydrophobic amino acid sequence obtained from the relevant sequence
of the target protein. Conformational probes according to the
present invention may be readily obtained.
[0096] The following rules may be used to guide the formation of an
appropriate preferred conformational probe according to the present
invention. These rules apply generally to conformational probes
according to the present invention without limitation, but are more
specifically used in context to produce the preferred palindromic
conformational probes according to the present invention.
[0097] The following rules may be applied to the instant invention
to produce preferred conformational peptide probes: [0098] 1. Each
"arm" of the peptide palindrome should have a minimum of five, and
preferably at least 10-12 amino acids and, ideally, not more than
about 25 amino acids. [0099] 2. The amino acid sequence is selected
from a region of a larger protein that is known to undergo a
conformational transition from alpha-helix or random coil to beta
sheet. [0100] 3. One or more of the following additional criteria:
[0101] a) A high proportion of hydrophobic amino acids--generally
not less than about 75% (based upon the number of amino acids),
ideally 80% or greater [0102] b) Amino acid repeats of at least 20
and preferably 25 (such as is present in huntingtin) [0103] c)
Clustered charges of opposite sign (as described in Zhang, S.,
Altman, M. and Rich, A. in Conformational Disease, A Compendium,
Solomon, Taraboulos and Katchalski-Katzir, eds. The Center for the
Study of Emerging Diseases, 2001. [0104] d) A linker sequence
between each of the peptide arms that has 1 or more amino acids,
preferably less than five and that contains one or more proline
residues Test criteria for peptide probe: [0105] 1. The
conformation of the palindrome peptide probe should be that of an
alpha helix or random coil but not a beta sheet. [0106] 2.
Determination of the conformation of the peptide is ideally
accomplished by circular dichroism measurements that can identify
solution conformations. These are performed using a CD spectrometer
in one or more solvents that can include aqueous buffers and/or
organic agents such as trifluoroethanol--see FIG. 11.
[0107] Applying the general rules obtained above and using readily
available methods in the art, one of ordinary skill can produce
large numbers of conformational peptide probes having favorable
characteristics to be useful in the present invention.
[0108] "Circular dichroism" ("CD") is observed when optically
active matter absorbs L and R hand circular polarized light
slightly differently, as measured by a CD spectropolarimeter.
Differences are very small and represent fractions of degrees in
ellipticity. FIG. 11 depicts an associative CD curve representative
of the three distinct common conformational forms that proteins and
peptides can assume. CD spectra for distinct types of secondary
structure present in peptides and proteins are distinct. Measuring
and comparing CD curves of complexed vs uncomplexed protein
represents an accurate measuring means of practicing the instant
invention.
[0109] Unexpectedly, we have determined that under near
physiological conditions, the palindrome, 33_mer (SEQ ID NO: 1 or
29), which covalently connects two peptides-the 14_mer (SEQ ID NO:
3 and the 19_mer (SEQ ID NO:2) exhibits a largely coil conformation
despite the proximity of two hydrophobic chains resembling the
14_mer structure, as illustrated in FIG. 12. The addition of a
pyrene at each end of the palindromic 33_mer peptide allows for
spectral observation of the conformational change, as illustrated
in FIG. 13. The spectral scans for pyrene attached to the ends of
the 33_mer in the monomer (open) conformation gives a strikingly
different fluorescent spectrum, having a maximum emission between
370 and 385 nm, while the excited dimer or excimer state of the
pyrene-labeled peptide has an emission max between 475 and 510
nm.
[0110] Although it is possible to follow conformational changes by
any of the several optical methods described above, a preferred
embodiment of the invention utilizes fluorescence spectroscopy
since that technique provides sensitivity, rapidity and simplicity
of operation. The probe is modified by attachment at both termini
of a fluorophore that has specific optical properties. It is
preferred that these include the ability to fluoresce upon
irradiation with light of a specific wavelength (defined by the
absorption and emission spectra of the chromophore itself). Thus,
irradiation with light of a wavelength near that of the absorption
maximum and emission of light at a sufficiently higher wavelength
so as to be distinguished from the excitation wavelength--this
measurement is well known to those versed in the art. Examples of
such fluorophores include, but are not limited to, pyrene,
tryptophan, fluorescein, rhodamine. It is also preferred that the
attached fluorophores have the capacity to form excimers when in
the correct geometric orientation.
[0111] An "excimer" is an adduct that is not necessarily covalent
and that is formed between a molecular entity that has been excited
by a photon and an identical unexcited molecular entity. The adduct
is transient in nature and exists until it fluoresces by emission
of a photon. It is possible to recognize an excimer (or the
formation of an excimer) by the production of a new fluorescent
band at a wavelength that is longer than that of the usual emission
spectrum. An excimer can be distinguished from fluorescence
resonance energy transfer since the excitation spectrum is
identical to that of the monomer.
[0112] The formation of the excimer is dependent on the geometric
alignment of the fluorophores and is heavily influenced by the
distance between them. In a preferred embodiment, fluorophores are
present at each probe terminus and excimer formation between
fluorophores is negligible as long as the overall probe
conformation is alpha-helix or random coil. This is readily
determined by measurement of the fluorescent behavior of the probe
in the solvent to be used for analysis in the absence of the target
protein to be measured.
[0113] Preferred conformational transition following interaction
with an analyte target is achieved by measuring fluorescence
spectra under conditions where excimer formation can be analyzed.
Typically, using pyrene as an exemplary fluorophor, the excitation
wavelength would be about 350 nm and the observation wavelength
365-600 nm. The normal emission of monomer pyrene following
excitation (simple fluorescence) is recorded as the maximum
wavelength between about 370-385 nm. Representative data is shown
in FIG. 14.
[0114] As shown in FIG. 14, the excimer or excited dimer state is
recorded at a maximum of between 475-510 nm. The formation of the
excited dimer state can also be encouraged through the addition of
high salt and by conducting measurements at pH approaching the pI
of the peptide (e.g., in the illustrated case, a pH of around
10).
[0115] Therefore, in a preferred method of the invention,
interaction of the probe with the specific protein to be analyzed
causes a conformational change in the probe such that excimer
formation occurs. This is readily measured by the procedures
described herein. Conversion of the probe structure from that
exhibited in the absence of analyte (alpha-helix or random coil) to
a beta-sheet structure enables fluorophores attached to the probe
to form excimers that can be readily identified. Further, the
magnitude of excimer formation is directly related to the amount of
protein analyte present.
[0116] Proteins or prions may be detected in aggregated form or in
the presence of other cellular constituents such as lipids, other
proteins or carbohydrates. A sample preparation for analysis is
preferably homogenized or subjected to a similar disruption of
tissue or aggregate structures, and cellular debris is preferably
removed by centrifugation. This process is ideally performed in the
presence of a buffered salt solution and may utilize one of several
detergents such as SDS, Triton X-100, or sarkosyl. Further
concentration of the sample may be achieved by treatment with any
of several agents; one preferred agent is phosphotungstate, which
is employed according to the method of Safar et al Nature Medicine
4:1157-1165 (1998).
[0117] In a preferred embodiment of the invention, peptide probes
are selected in order for addition to an unknown or test sample.
The peptide probes are preferably proteins or peptide sequences
that have secondary structures of predominately alpha-helix or
random coil, but which are preferably, but not necessarily derived
from portions of a target peptide responsible for .beta.-sheet
formation. In a particularly preferred embodiment, the peptide
probes are peptide fragments consisting of a helix-loop-helix
structure found in polylysine. In another particularly preferred
embodiment, the peptide probes can be made of a peptide sequence
chosen from wild-type (wt) TSE, from a desired species-specific TSE
peptide sequence, or even from a selectively mutated TSE sequence
that has been mutated in such a manner as to render it destabilized
and noninfectious. Additionally, extrinsic fluors such as pyrene
can be added or designed into the peptide probe to allow detection
of anticipated conformational changes using common fluorescence
detection techniques.
[0118] Once a peptide probe is selected, it is added to a test
sample. Prior to the addition of the peptide probe, however, it is
preferred to have the sample subjected to disaggregation techniques
commonly known in the art, such as sonication. The disaggregation
step allows any potentially aggregated sample material to break
apart so that these disaggregated sample materials are free to
combine with the newly introduced peptide probe, thereby
facilitating the anticipated catalytic propagation.
[0119] After the test sample or disaggregated test sample is
allowed to interact with the peptide probes, the resulting mixture
is then subjected to analytical methods commonly known in the art
for the detection of aggregates and to fluorescence measurements in
cases where fluorescent peptide probes are used. Unknown or test
samples containing any dominant beta-sheet formation characteristic
of abnormally folded or disease-causing proteins result in an
increase in beta-sheet formation and consequently aggregate
formation in the final mixture containing both the test sample and
the peptide probes. Conversely, unknown or test samples which lack
a predominantly beta-sheet secondary structure will neither
catalyze a transition to beta-sheet structure nor will propagate
the formation of aggregates.
[0120] The initial conformational change can be triggered in the
test samples in a number of ways. Without intending to be bound by
any theory, the binding of a metal ligand could direct a change in
the protein conformation and favor aggregation. The expression or
cleavage of different peptide sequences can promote advanced
aggregation leading to fibril and plaque formation. Genetic point
mutations can also alter the relative energy levels required of the
two distinct conformations, resulting in midpoint shifts in
structural transitions. Furthermore, an increase in concentration
levels could be sufficient to favor the conformational transition.
Regardless of the initial trigger mechanism, however, the disease
process in many of the abnormal protein conformations such as in
prion-related diseases involves the catalytic propagation of the
abnormal conformation, resulting in structural transformation of
the previously normal protein.
[0121] Optical detection techniques useful in the instant invention
include but are not limited to light scattering, or hydrophobicity
detection using extrinsic fluors such as 1-anilino-8-napthalene
sulfonate (ANS) or Congo Red stain, fluorescence resonance energy
transfer (FRET) and quenching of intrinsic tryptophan fluorescence
through either conformational change of monomer or binding at an
interface in an alpha-beta heterodimer.
[0122] Other structural techniques include equilibrium
ultracentrifugation or size-exclusion chromatography.
[0123] The instant invention uses propagated conformational change
to correlate directly levels of abnormal proteins or prions with
levels of infectivity. For this reason, it is preferable to utilize
the methods of the invention in a manner in which there is no
increase in infectious products a; a result of the propagation.
This can be achieved by placing a "break" in the links between the
chain of infection, transmission, and propagation of the abnormal
form. Such a "break" must occur at the transitional stage between
the dimer and multimer forms of the aggregate. The physical
formation of the multimer form (an be blocked by simply impeding
the step which leads to its formation. This may be achieved by
using a probe in which the sequence of interest is attached to a
non-relevant peptide, or by a neutral "blocker" segment, with the
understanding that probes on linkers or "tethers" are more likely
to encounter each other and thus result in amplifying the
signal.
[0124] The invention is described further in the following
examples, which are illustrative and in no way limiting.
EXAMPLE 1
Materials and Methods
[0125] A sample may be obtained for testing and diagnosis through
use of the instant invention as follows. A sample may be prepared
from tissue (e.g. a portion of ground meat, or an amount of tissue
obtained by a biopsy procedure) by homogenization in a glass
homogenizer or by mortar and pestle in the presence of liquid
nitrogen. The amount of material should be between about 1 mg and 1
gm, preferably between 10 mg and 250 mg, ideally between 20 and 100
mg. The material to be sampled may be suspended in a suitable
solvent, preferably phosphate-buffered saline at a pH between 7.0
and 7.8. The addition of Rnase inhibitors is preferred. The solvent
may contain a detergent (e.g., Triton X-100, SDS, or sarkosyl).
Homogenization is performed for a number of excursions of the
homogenizer, preferably between 10 and 25 strokes; ideally between
15 and 20 strokes. The suspended sample is preferably centrifuged
at between 100 and 1,000 g for 5-10 minutes and the supernatant
material sampled for analysis. In some samples, it may be
preferable to treat the supernatant material with an additional
reagent such as phosphotungstic acid according to the procedure
described by Safar et al., Nature Medicine, 4, 1157-1165 (1998) and
as modified by Wadsworth, The Lancet, 358, 171-180 (2001). Eight
prion strains have PrP.sup.Sc molecules with different
conformations. See, Safar, et al. and Wadsworth, ibid. Tissue
distribution of protease resistant prion protein in variant
Creutzfeldt Jakob disease has been reported using a highly
sensitive immunoblotting assay as described in Wadsworth, et al.,
ibid.
[0126] The amount of sample to be tested is based on a
determination of the protein content of the supernatant solution as
measured by the procedure described by Bradford, Anal. Biochem.
72:248-254 (1976). A rapid and sensitive method for determining
microgram quantities of protein utilizes the principle of
protein-dye binding. Preferably, this corresponds to between about
0.5 and 2 mg of protein.
[0127] In addition to the procedure described above for tissue
material, test samples may be obtained from serum, pharmaceutical
formulations that may contain products of animal origin, spinal
fluid, saliva, urine or other bodily fluids. Liquid samples may be
tested directly or may be subjected to treatment with agents such
as phosphotungstic acid as described above.
Illustrative Analysis
[0128] A sample containing TSE may be analyzed in accordance with
the invention as follows. Referring to FIG. 2, the top row of the
schematic illustrates an unknown sample of TSE protein represented
as containing beta-sheets 12. The beta-sheets are disaggregated by
sonication. Labeled peptide probes 14 are added and are allowed to
bind to the sample 12. The beta-sheet conformation in sample 12
induces the peptide probes to conform to beta-sheet conformation
16. Beta-sheet propagation among the peptide probes 14 forms
aggregates 18. The resulting transition to a predominately
beta-sheet form and amplified aggregate formation is detected by
techniques such as light scattering and circular dichroism (CD). In
a particularly preferred embodiment, the peptide probe is
fluorescently labeled and fluorescence detection is used.
[0129] The bottom row of FIG. 2 shows an alternative example in
which the unknown sample of TSE protein is represented in its
normal alpha-helical form 10. For consistency, the sample is
subjected to the same disaggregation process described above. Upon
addition of the labeled peptide probes 14, neither a transition to
beta-sheet form nor binding to the unknown samples occurs. As a
result, there is no aggregate fluorescence signal in the case of a
labeled peptide probe and there is no detection of aggregate
formation by other analytical tools. Based on this schematic,
unknown samples can be tested for the presence or absence of such
abnormal protein conformations or sequences.
EXAMPLE 2
[0130] Poly-lysine was used as a model peptide. Experiments were
performed using model systems to illustrate the conformational
changes involved in the transition from a predominately alpha-helix
to a beta-rich form. The model system chosen used non-neurotoxic
polyamino acid polylysine. The polyamino acid was chosen because of
availability and safety; and normally evidences random coil
conformation at pH values between 5 and 9.
[0131] FIG. 3 depicts a CD graph of an experiment in which
poly-L-lysine 20 micro Molar (.mu..mu.M) 52,000 molecular weight
(MW) was used as a peptide model.
[0132] As also illustrated in FIG. 3:
Sample 24, which was maintained at pH 7, 25.degree. C., exhibited a
minimum at approximately 205 nanometers (nm), indicating a random
coil structure; Sample 26 which was maintained at pH 11 (near the
isoelectric point), at 50.degree. C., resulted in a minimum at
approximately 216 nanometers (nm) indicating a .beta.-sheet
structure (see FIG. 11 for exemplary CD spectra of protein
conformations); Sample 28, which was a 1:1 combination of samples
maintained at pH7, 25.degree. C. and at pH11, 50.degree. C.,
resulted in a minimum at approximately 216 namometers (nm)
indicating .beta.-sheet structure, Sample 30, which was a 1:1
combination of samples maintained at pH 7, 50.degree. C. and at pH
11, 50.degree. C., resulted in a minimum at approximately 216
namometers (nm), indicating .beta.-sheet structure.
EXAMPLE 3
[0133] FIG. 4 illustrates general CD results of experiments that
were conducted: (1) using poly-L-lysine; and (2) at varying
temperatures and pH, to observe the effect of random coil to
beta-sheet conformational changes under varying environmental
conditions. The results indicate that both temperature and pH play
an important role in the transition. The results also indicate that
the addition of a relatively small amount of .beta.-sheet peptide
to a random coil sample can result in a shift towards a .beta.-rich
conformation and that such changes can be accelerated depending on
the temperature and pH environment of the samples.
[0134] More specifically, FIG. 4 illustrates an absorbance graph
generated using a poly-L-lysine of 52,000 molecular weight (MW) at
70 micromolar (.mu.M) as a peptide probe in accordance with the
experimental technique described in Examples 1-3. FIG. 4
illustrates that:
Sample 32 (pH 11, 25.degree. C.) evidenced a plateau at
approximately 0.12, indicating a pre-dominantly .alpha.-helical
structure; Sample 34 (maintained at pH 7, 50.degree. C.) evidenced
a plateau at approximately 0.22, which indicated a predominantly
random coil structure; Sample 36 (a 10:1 combination of samples
maintained at pH 7, 50.degree. C. and at pH 11, 50.degree. C.)
resulted in a steeper incline from approximately 0.22 to 0.33,
indicating an accelerated transition from random coil to
.beta.-sheet structure; Sample 38 (a 10:1 combination of samples
maintained at pH 7, 25.degree. C. and at pH 11, 50.degree. C.)
resulted in a gradual incline from approximately 0.22 to 0.26,
indicating a transition from random coil to .beta.-sheet
structure.
[0135] The observations based on all of the experiments described
above show that the addition of a relatively small amount of
.beta.-sheet peptide to random coil sample can result in a shift
towards a beta-rich conformation and that such changes can be
accelerated depending on the temperature and pH environment of the
samples.
EXAMPLE 4
[0136] The experiment that led to the results illustrated in FIG.
15 involved use of the 33_mer target peptide (SEQ ID NO: 1 and 29)
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human) alone, and probing
peptide association through the observation of excimer formation.
The 33_mer target peptide (SEQ ID NO: 1 or 29) used was a murine
amino acid sequence which differed from a corresponding human
sequence in the substitution of methionine for valine and leucine
at positions_M11V_, M14L_, _M20L_, and M23V_, as illustrated in
FIG. 10B. We compared the results we observed using CD (in which
peptides were unlabeled) and spectrofluorometric studies (using
pyrene-labeled peptides). No homogenate was used. The experiment
that lead to the results illustrated in FIG. 15 was a detailed
study undertaken to understand what triggered the 33_mer target
peptide (SEQ ID NO: 1 or 29) to conformationally change from
predominately monomeric to dimeric (excimeric) and become
aggregated. Conditions were found that encouraged 33_mer
labeled-peptide association in the .mu.M-range.
[0137] Conditions that screened the electrostatic interactions of
the 33 mer target peptide and thereby minimized its solubility
(pI=10) triggered self-association of the peptide under extremely
low concentrations (10 .mu.M). This self association is evident in
the formation of dimers or excimers and the concomitant far red
shift in fluorescence by virtue of the pyrene fluorophor on the
ends of the peptides. As an example, Curve 1 of FIG. 15 represents
the conditions of pH 6-8, KCl (100-500 .mu.M) where the predominant
peptide conformer is monomeric; while Curve 2 of FIG. 15 represents
the conditions of pH 10-11, KCl (100-500 .mu.M), where at very low
concentrations of peptide, we observed strong excimer formation
(aggregation of the monomers).
EXAMPLE 5
[0138] The experiment that led to the results illustrated in FIG.
16 involved use of various individual peptides, and the 33_mer
probe (comprising 19_mer and 14_mer) target peptide (SEQ ID NO:1,
29, 2 or 3) VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human) The assay conditions were
changed to observe the effect on conformation as monitored by CD.
The goal was to determine what thermodynamic conditions result in
one step transition from monomeric random coil to aggregated .beta.
sheet and avoid the associative `X` state that is probably micelle
formation of the peptides.
[0139] In the experiment that lead to the results illustrated in
FIG. 16, a specific .lamda. (205 nm) wavelength was used to monitor
peptide association by CD to obtain detailed conformational
information over a range of solvent conditions and across a range
of peptide concentrations (peptide concentrations are presented in
log scale and also refer to the standard diagram for CD--FIG.
11).
[0140] The associative curve (.theta..sub.205) recovered for the
target peptides showed two conformational transitions at the 50
.mu.M and 3 mM range, respectively, moving from a coil through to
`X` state and to .beta.-sheet.
[0141] Referring to FIG. 16, for solvent conditions above 50% (far
left dashed line), the 33_mer target peptide (SEQ ID NO: 1 and 29)
VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human) transitioned from the
coil state to a .beta.-sheet state at 3 .mu.M, while the component
19_mer or 14_mer were able to transition, but at nearly 10-fold
higher peptide concentration (middle line). Under aqueous
conditions, (thick line) none of the peptides were able to self
associate into a .beta. sheet structure.
[0142] The 33_mer palindromic peptide target peptide (SEQ ID NO: 1
and 29) exhibited unique properties at very low concentrations (ie.
1 .mu.M) under 50% solvent (acetonitrile or trifluoroethanol)
conditions in that it avoided the "dead-end" associative state (as
exhibited by the plateauing effect under aqueous conditions).
[0143] FIG. 16 shows that a variation in solvent and temperature
does not significantly affect the associative behavior of target
peptides and that all of the peptides follow the same curve,
indicating that sequence specificity is not an important feature in
this kind of molecular assembling.
EXAMPLE 6
[0144] The experiment that led to the results illustrated in FIG.
17 was conducted as follows.
[0145] One gram of scrapie infected (strain 293) hamster brain
material was homogenized in liquid nitrogen in sterile phosphate
buffered saline. Ten-fold serial dilutions were made into sterile
PBS. The concentration of protease resistant prion protein
(PrP.sup.Sc) in the brain homogenates was determined by capillary
electrophoresis antibody-capture. Brain homogenate equivalent to 10
ng of protease resistant prion protein (PrP.sup.Sc) was mixed with
1.5 .mu.M of 33_mer target peptide in 50% TFE (trifluoroethanol)
and incubated for 1 hour at room temperature prior to excitation at
350 nm in a dual chronometer spectrofluorometer and emission from
350 to 600 nm recorded, the excitation and emission scan was
repeated at 5 hours and 24 hours. The 33_mer peptide alone was used
as a control.
[0146] Addition of the infectious prion protein led to the
significant increase in the fluorescence of the 33-_mer target
peptide, which was found to be in near .beta.-sheet conformation by
CD data under conditions of 50% Tris:50% TFE. This increase of
fluorescence indicated the formation of 33_meraggregates. The
33_mer aggregates were found to be unstable and dissociated
irreversibly with time.
[0147] Following the emission of fluorescence for the complex
versus the peptide over time illustrated that the complex
dissociated with time, while the peptide fluorescence remained
stable monitoring at two different wavelengths, 377 nm (triangle)
and 475 nm (square).
EXAMPLE 7
[0148] The experiment that led to the results illustrated in FIG.
18 was conducted as follows.
[0149] One gram of scrapie infected and healthy hamster brain,
sheep brain and elk brain were homogenized in liquid nitrogen in
sterile phosphate buffered saline. Ten-fold serial dilutions were
made into sterile PBS. The concentration of protease resistant
prion protein (PrP.sup.Sc) in the brain homogenates was determined
by capillary electrophoresis antibody-capture. Brain homogenates,
infected and healthy, were mixed with 0.52 .mu.M of 33_mer target
peptide in 50% TFE (trifluoroethanol):50% TRIS and incubated for 1
hour at room temperature prior to excitation at 350 nm in a dual
chromometer spectrofluorometer and emission at 350 to 600 nm
recorded. The 33_mer peptide alone in 50% TFE:50% TRIS was used as
an additional control.
[0150] Fluorescence spectra of the target peptide [520 nM] in the
presence of infected brain homogenate (graph line 1-), healthy
brain homogenate (graph line 2-), and peptide alone (graph line 3-)
in TRIS:TFE (1:1) solvent are shown in FIG. 18. The data are for
0.01% brain homogenate from hamster (panel. A), sheep (panel B),
and elk (panel C). hamster [270 pg/ml], sheep [60 pg/ml], and elk
[6 pg/ml].
Sequence CWU 1
1
61133PRTHomo sapiens 1Val Val Ala Gly Ala Ala Ala Ala Gly Ala Met
His Lys Met Asn Thr1 5 10 15Lys Pro Lys Met Lys His Met Ala Gly Ala
Ala Ala Ala Gly Ala Val 20 25 30Val219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Lys
Pro Lys Thr Asn Leu Lys His Val Ala Gly Ala Ala Ala Ala Gly1 5 10
15Ala Val Val314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 3Leu Lys His Val Ala Gly Ala Ala Ala Ala
Gly Ala Val Val1 5 10440PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 4Asp Ala Glu Phe Arg His Asp
Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu
Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly
Gly Val Val 35 40524PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Glu Val His His Gln Lys Leu Val Phe Phe
Ala Glu Asp Val Gly Ser1 5 10 15Asn Lys Gly Ala Ile Ile Gly Leu
20624PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Glu Val Arg His Gln Lys Leu Val Phe Phe Ala Glu
Asp Val Gly Ser1 5 10 15Asn Lys Gly Ala Ile Ile Gly Leu
20711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met1 5
10827PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
20 25923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln1 5 10 15Gln Gln Gln Gln Gln Gln Gln
201019PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Lys Pro Lys Thr Asn Leu Lys His Val Ala Gly Ala
Ala Ala Ala Gly1 5 10 15Ala Val Val1138PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Met
Gly Ile Leu Lys Leu Gln Val Phe Leu Ile Val Leu Ser Val Ala1 5 10
15Leu Asn His Leu Lys Ala Thr Pro Ile Glu Ser His Gln Val Glu Lys
20 25 30Arg Lys Cys Asn Thr Ala 351225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Met
Ala Glu Ser His Leu Leu Gln Trp Leu Leu Leu Leu Leu Pro Thr1 5 10
15Leu Cys Gly Pro Gly Thr Ala Ala Trp 20 2513253PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
13Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp1
5 10 15Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp
Asn 20 25 30Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly
Asn Arg 35 40 45Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His
Gly Gly Gly 50 55 60Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro
His Gly Gly Gly65 70 75 80Trp Gly Gln Pro His Gly Gly Gly Trp Gly
Gln Gly Gly Gly Thr His 85 90 95Ser Gln Trp Asn Lys Pro Ser Lys Pro
Lys Thr Asn Met Lys His Met 100 105 110Ala Gly Ala Ala Ala Ala Gly
Ala Val Val Gly Gly Leu Gly Gly Tyr 115 120 125Met Leu Gly Ser Ala
Met Ser Arg Pro Ile Ile His Phe Gly Ser Asp 130 135 140Tyr Glu Asp
Arg Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln145 150 155
160Val Tyr Tyr Arg Pro Met Asp Glu Tyr Ser Asn Gln Asn Asn Phe Val
165 170 175His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr
Thr Thr 180 185 190Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys
Met Met Glu Arg 195 200 205Val Val Glu Gln Met Cys Ile Thr Gln Tyr
Glu Arg Glu Ser Gln Ala 210 215 220Tyr Tyr Gln Arg Gly Ser Ser Met
Val Leu Phe Ser Ser Pro Pro Val225 230 235 240Ile Leu Leu Ile Ser
Phe Leu Ile Phe Leu Ile Val Gly 245 25014254PRTMus sp. 14Met Ala
Asn Leu Gly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp1 5 10 15Thr
Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 25
30Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg
35 40 45Tyr Pro Pro Gln Gly Gly Thr Trp Gly Gln Pro His Gly Gly Gly
Trp 50 55 60Gly Gln Pro His Gly Gly Ser Trp Gly Gln Pro His Gly Gly
Ser Trp65 70 75 80Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly
Gly Thr His Asn 85 90 95Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn
Leu Lys His Val Ala 100 105 110Gly Ala Ala Ala Ala Gly Ala Val Val
Gly Gly Leu Gly Gly Tyr Met 115 120 125Leu Gly Ser Ala Met Ser Arg
Pro Met Ile His Phe Gly Asn Asp Trp 130 135 140Glu Asp Arg Tyr Tyr
Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val145 150 155 160Tyr Tyr
Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val His 165 170
175Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr Thr
180 185 190Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu
Arg Val 195 200 205Val Glu Gln Met Cys Val Thr Gln Tyr Gln Lys Glu
Ser Gln Ala Tyr 210 215 220Tyr Asp Gly Arg Arg Ser Ser Ser Thr Val
Leu Phe Ser Ser Pro Pro225 230 235 240Val Ile Leu Leu Ile Ser Phe
Leu Ile Phe Leu Ile Val Gly 245 25015782PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Met Ala Pro His Arg Pro Ala Pro Ala Leu Leu Cys Ala Leu Ser Leu1
5 10 15Ala Leu Cys Ala Leu Ser Leu Pro Val Arg Ala Ala Thr Ala Ser
Arg 20 25 30Gly Ala Ser Gln Ala Gly Ala Pro Gln Gly Arg Val Pro Glu
Ala Arg 35 40 45Pro Asn Ser Met Val Val Glu His Pro Glu Phe Leu Lys
Ala Gly Lys 50 55 60Glu Pro Gly Leu Gln Ile Trp Arg Val Glu Lys Phe
Asp Leu Val Pro65 70 75 80Val Pro Thr Asn Leu Tyr Gly Asp Phe Phe
Thr Gly Asp Ala Tyr Val 85 90 95Ile Leu Lys Thr Val Gln Leu Arg Asn
Gly Asn Leu Gln Tyr Asp Leu 100 105 110His Tyr Trp Leu Gly Asn Glu
Cys Ser Gln Asp Glu Ser Gly Ala Ala 115 120 125Ala Ile Phe Thr Val
Gln Leu Asp Asp Tyr Leu Asn Gly Arg Ala Val 130 135 140Gln His Arg
Glu Val Gln Gly Phe Glu Ser Ala Thr Phe Leu Gly Tyr145 150 155
160Phe Lys Ser Gly Leu Lys Tyr Lys Lys Gly Gly Val Ala Ser Gly Phe
165 170 175Lys His Val Val Pro Asn Glu Val Val Val Gln Arg Leu Phe
Gln Val 180 185 190Lys Gly Arg Arg Val Val Arg Ala Thr Glu Val Pro
Val Ser Trp Glu 195 200 205Ser Phe Asn Asn Gly Asp Cys Phe Ile Leu
Asp Leu Gly Asn Asn Ile 210 215 220His Gln Trp Cys Gly Ser Asn Ser
Asn Arg Tyr Glu Arg Leu Lys Ala225 230 235 240Thr Gln Val Ser Lys
Gly Ile Arg Asp Asn Glu Arg Ser Gly Arg Ala 245 250 255Arg Val His
Val Ser Glu Glu Gly Thr Glu Pro Glu Ala Met Leu Gln 260 265 270Val
Leu Gly Pro Lys Pro Ala Leu Pro Ala Gly Thr Glu Asp Thr Ala 275 280
285Lys Glu Asp Ala Ala Asn Arg Lys Leu Ala Lys Leu Tyr Lys Val Ser
290 295 300Asn Gly Ala Gly Thr Met Ser Val Ser Leu Val Ala Asp Glu
Asn Pro305 310 315 320Phe Ala Gln Gly Ala Leu Lys Ser Glu Asp Cys
Phe Ile Leu Asp His 325 330 335Gly Lys Asp Gly Lys Ile Phe Val Trp
Lys Gly Lys Gln Ala Asn Thr 340 345 350Glu Glu Arg Lys Ala Ala Leu
Lys Thr Ala Ser Asp Phe Ile Thr Lys 355 360 365Met Asp Tyr Pro Lys
Gln Thr Gln Val Ser Val Leu Pro Glu Gly Gly 370 375 380Glu Thr Pro
Leu Phe Lys Gln Phe Phe Lys Asn Trp Arg Asp Pro Asp385 390 395
400Gln Thr Asp Gly Leu Gly Leu Ser Tyr Leu Ser Ser His Ile Ala Asn
405 410 415Val Glu Arg Val Pro Phe Asp Ala Ala Thr Leu His Thr Ser
Thr Ala 420 425 430Met Ala Ala Gln His Gly Met Asp Asp Asp Gly Thr
Gly Gln Lys Gln 435 440 445Ile Trp Arg Ile Glu Gly Ser Asn Lys Val
Pro Val Asp Pro Ala Thr 450 455 460Tyr Gly Gln Phe Tyr Gly Gly Asp
Ser Tyr Ile Ile Leu Tyr Asn Tyr465 470 475 480Arg His Gly Gly Arg
Gln Gly Gln Ile Ile Tyr Asn Trp Gln Gly Ala 485 490 495Gln Ser Thr
Gln Asp Glu Val Ala Ala Ser Ala Ile Leu Thr Ala Gln 500 505 510Leu
Asp Glu Glu Leu Gly Gly Thr Pro Val Gln Ser Arg Val Val Gln 515 520
525Gly Lys Glu Pro Ala His Leu Met Ser Leu Phe Gly Gly Lys Pro Met
530 535 540Ile Ile Tyr Lys Gly Gly Thr Ser Arg Glu Gly Gly Gln Thr
Ala Pro545 550 555 560Ala Ser Thr Arg Leu Phe Gln Val Arg Ala Asn
Ser Ala Gly Ala Thr 565 570 575Arg Ala Val Glu Val Leu Pro Lys Ala
Gly Ala Leu Asn Ser Asn Asp 580 585 590Ala Phe Val Leu Lys Thr Pro
Ser Ala Ala Tyr Leu Trp Val Gly Thr 595 600 605Gly Ala Ser Glu Ala
Glu Lys Thr Gly Ala Gln Glu Leu Leu Arg Val 610 615 620Leu Arg Ala
Gln Pro Val Gln Val Ala Glu Gly Ser Glu Pro Asp Gly625 630 635
640Phe Trp Glu Ala Leu Gly Gly Lys Ala Ala Tyr Arg Thr Ser Pro Arg
645 650 655Leu Lys Asp Lys Lys Met Asp Ala His Pro Pro Arg Leu Phe
Ala Cys 660 665 670Ser Asn Lys Ile Gly Arg Phe Val Ile Glu Glu Val
Pro Gly Glu Leu 675 680 685Met Gln Glu Asp Leu Ala Thr Asp Asp Val
Met Leu Leu Asp Thr Trp 690 695 700Asp Gln Val Phe Val Trp Val Gly
Lys Asp Ser Gln Glu Glu Glu Lys705 710 715 720Thr Glu Ala Leu Thr
Ser Ala Lys Arg Tyr Ile Glu Thr Asp Pro Ala 725 730 735Asn Arg Asp
Arg Arg Thr Pro Ile Thr Val Val Lys Gln Gly Phe Glu 740 745 750Pro
Pro Ser Phe Val Gly Trp Phe Leu Gly Trp Asp Asp Asp Tyr Trp 755 760
765Ser Val Asp Pro Leu Asp Arg Ala Met Ala Glu Leu Ala Ala 770 775
7801636PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Tyr Glu Arg Leu Lys Ala Thr Gln Val Ser Lys Gly
Ile Arg Asp Asn1 5 10 15Glu Arg Ser Gly Arg Ala Arg Val His Val Ser
Glu Glu Gly Thr Glu 20 25 30Pro Glu Ala Met 3517146PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
17Met Ala Gly Pro Leu Arg Ala Pro Leu Leu Leu Leu Ala Ile Leu Ala1
5 10 15Val Ala Leu Ala Val Ser Pro Ala Ala Gly Ser Ser Pro Gly Lys
Pro 20 25 30Pro Arg Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu Glu
Glu Gly 35 40 45Val Arg Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn
Lys Ala Ser 50 55 60Asn Asp Met Tyr His Ser Arg Ala Leu Gln Val Val
Arg Ala Arg Lys65 70 75 80Gln Ile Val Ala Gly Val Asn Tyr Phe Leu
Asp Val Glu Leu Gly Arg 85 90 95Thr Thr Cys Thr Lys Thr Gln Pro Asn
Leu Asp Asn Cys Pro Phe His 100 105 110Asp Gln Pro His Leu Lys Arg
Lys Ala Phe Cys Ser Phe Gln Ile Tyr 115 120 125Ala Val Pro Trp Gln
Gly Thr Met Thr Leu Ser Lys Ser Thr Cys Gln 130 135 140Asp
Ala1451820PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Glu Glu Glu Val Ser Ala Asp Met Pro Pro Pro Pro
Met Asp Ala Ser1 5 10 15Val Glu Glu Glu 2019315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Met Ala Thr Leu Glu Lys Leu Met Lys Ala Phe Glu Ser Leu Lys Ser1
5 10 15Phe Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln 20 25 30Gln Gln Gln Gln Gln Gln Gln Gln Pro Pro Pro Pro Pro Pro
Pro Pro 35 40 45Pro Pro Pro Gln Leu Pro Gln Pro Pro Pro Gln Ala Gln
Pro Leu Leu 50 55 60Pro Gln Pro Gln Pro Pro Pro Pro Pro Pro Pro Pro
Pro Pro Gly Pro65 70 75 80Ala Val Ala Glu Glu Pro Leu His Arg Pro
Lys Lys Glu Leu Ser Ala 85 90 95Thr Lys Lys Asp Arg Val Asn His Cys
Leu Thr Ile Cys Glu Asn Ile 100 105 110Val Ala Gln Ser Val Arg Asn
Ser Pro Glu Phe Gln Lys Leu Leu Gly 115 120 125Ile Ala Met Glu Leu
Phe Leu Leu Cys Ser Asp Asp Ala Glu Ser Asp 130 135 140Val Arg Met
Val Ala Asp Glu Cys Leu Asn Lys Val Ile Lys Ala Leu145 150 155
160Met Asp Ser Asn Leu Pro Arg Leu Gln Leu Glu Leu Tyr Lys Glu Ile
165 170 175Lys Lys Asn Gly Ala Pro Arg Ser Leu Arg Ala Ala Leu Trp
Arg Phe 180 185 190Ala Glu Leu Ala His Leu Val Arg Pro Gln Lys Cys
Arg Pro Tyr Leu 195 200 205Val Asn Leu Leu Pro Cys Leu Thr Arg Thr
Ser Lys Arg Pro Glu Glu 210 215 220Ser Val Gln Glu Thr Leu Ala Ala
Ala Val Pro Lys Ile Met Ala Ser225 230 235 240Phe Gly Asn Phe Ala
Asn Asp Asn Glu Ile Lys Val Leu Leu Lys Ala 245 250 255Phe Ile Ala
Asn Leu Lys Ser Ser Ser Pro Thr Ile Arg Arg Thr Ala 260 265 270Ala
Gly Ser Ala Val Ser Ile Cys Gln His Ser Arg Arg Thr Gln Tyr 275 280
285Phe Tyr Ser Trp Leu Leu Asn Val Leu Leu Gly Leu Leu Val Pro Val
290 295 300Glu Asp Glu His Ser Thr Leu Leu Ile Leu Gly305 310
3152017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln1 5 10 15Gln2189PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Met Gly Ile Leu Lys Leu Gln
Val Phe Leu Ile Val Leu Ser Val Ala1 5 10 15Leu Asn His Leu Lys Ala
Thr Pro Ile Glu Ser His Gln Val Glu Lys 20 25 30Arg Lys Cys Asn Thr
Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe 35 40 45Leu Val His Ser
Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn 50 55 60Val Gly Ser
Asn Thr Tyr Gly Lys Arg Asn Ala Val Glu Val Leu Lys65 70 75 80Arg
Glu Pro Leu Asn Tyr Leu Pro Leu 85225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Leu
Ala Asn Phe Val1 52314PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 23Val Phe Asn Ala Leu Pro Pro
Pro Pro Leu Ala Asn Phe Val1 5
10246PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Phe Leu Val His Ser Ser1 52515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Ser
Ser His Val Leu Phe Pro Pro Pro Phe Leu Val His Ser Ser1 5 10
1526147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 26Met Ala Ser His Arg Leu Leu Leu Leu Cys Leu
Ala Gly Leu Val Phe1 5 10 15Val Ser Glu Ala Gly Pro Thr Gly Thr Gly
Glu Ser Lys Cys Pro Leu 20 25 30Met Val Lys Val Leu Asp Ala Val Arg
Gly Ser Pro Ala Ile Asn Val 35 40 45Ala Val His Val Phe Arg Lys Ala
Ala Asp Asp Thr Trp Glu Pro Phe 50 55 60Ala Ser Gly Lys Thr Ser Glu
Ser Gly Glu Leu His Gly Leu Thr Thr65 70 75 80Glu Glu Glu Phe Val
Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys 85 90 95Ser Tyr Trp Lys
Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu 100 105 110Val Val
Phe Thr Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala 115 120
125Ala Leu Leu Ser Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn
130 135 140Pro Lys Glu1452722PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 27Glu Ser Val Phe Val Leu Gly
Ala Leu Pro Pro Pro Pro Leu Ala Gly1 5 10 15Leu Val Phe Val Ser Glu
202832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Val Ala Ala Ala Lys Leu Arg Xaa Val Val Thr Ser
Arg Gln Pro Pro1 5 10 15Pro Pro Gln Arg Ser Thr Val Val Xaa Arg Leu
Lys Ala Ala Ala Val 20 25 302933PRTMus sp. 29Val Val Ala Gly Ala
Ala Ala Ala Gly Ala Val His Lys Leu Asn Thr1 5 10 15Lys Pro Lys Leu
Lys His Val Ala Gly Ala Ala Ala Ala Gly Ala Val 20 25
30Val3014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Gln Arg Ser Thr Val Val Xaa Arg Leu Lys Ala Ala
Ala Val1 5 10314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 31Ala Ala Ala Val13214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Val
Ala Ala Ala Lys Leu Arg Xaa Val Val Thr Ser Arg Gln1 5
103333PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Val Val Ala Gly Ala Ala Ala Ala Gly Ala Met His
Lys Met Lys Pro1 5 10 15Lys Thr Asn Met Lys His Met Ala Gly Ala Ala
Ala Ala Gly Ala Val 20 25 30Val3419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Val
Val Ala Gly Ala Ala Ala Ala Gly Ala Val His Lys Leu Asn Thr1 5 10
15Lys Pro Lys3514PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 35Val Val Ala Gly Ala Ala Ala Ala Gly
Ala Val His Lys Leu1 5 103640PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 36Val Val Gly Gly Val Met Leu
Gly Ile Ile Ala Gly Lys Asn Ser Gly1 5 10 15Val Asp Glu Ala Phe Phe
Val Leu Lys Gln His His Val Glu Tyr Gly 20 25 30Ser Asp His Arg Phe
Glu Ala Asp 35 403724PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 37Leu Gly Ile Ile Ala Gly Lys
Asn Ser Gly Val Asp Glu Ala Phe Phe1 5 10 15Val Leu Lys Gln His His
Val Glu 203824PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 38Leu Gly Ile Ile Ala Gly Lys Asn Ser
Gly Val Asp Glu Ala Phe Phe1 5 10 15Val Leu Lys Gln His Arg Val Glu
203911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Met Leu Gly Ile Ile Ala Gly Lys Asn Ser Gly1 5
104027PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
20 254123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln1 5 10 15Gln Gln Gln Gln Gln Gln Gln
204219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Val Val Ala Gly Ala Ala Ala Ala Gly Ala Val His
Lys Leu Asn Thr1 5 10 15Lys Pro Lys4338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Ala
Thr Asn Cys Lys Arg Lys Glu Val Gln His Ser Glu Ile Pro Thr1 5 10
15Ala Lys Leu His Asn Leu Ala Val Ser Leu Val Ile Leu Phe Val Gln
20 25 30Leu Lys Leu Ile Gly Met 354425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Trp
Ala Ala Thr Gly Pro Gly Cys Leu Thr Pro Leu Leu Leu Leu Leu1 5 10
15Trp Gln Leu Leu His Ser Glu Ala Met 20 2545253PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
45Gly Val Ile Leu Phe Ile Leu Phe Ser Ile Leu Leu Ile Val Pro Pro1
5 10 15Ser Ser Phe Leu Val Met Ser Ser Gly Arg Gln Tyr Tyr Ala Gln
Ser 20 25 30Glu Arg Glu Tyr Gln Thr Ile Cys Met Gln Glu Val Val Arg
Glu Met 35 40 45Met Lys Val Asp Thr Glu Thr Phe Asn Glu Gly Lys Thr
Thr Thr Thr 50 55 60Val Thr His Gln Lys Ile Thr Ile Asn Val Cys Asp
His Val Phe Asn65 70 75 80Asn Gln Asn Ser Tyr Glu Asp Met Pro Arg
Tyr Tyr Val Gln Asn Pro 85 90 95Tyr Arg His Met Asn Glu Arg Tyr Tyr
Arg Asp Glu Tyr Asp Ser Gly 100 105 110Phe His Ile Ile Pro Arg Ser
Met Ala Ser Gly Leu Met Tyr Gly Gly 115 120 125Leu Gly Gly Val Val
Ala Gly Ala Ala Ala Ala Gly Ala Met His Lys 130 135 140Met Asn Thr
Lys Pro Lys Ser Pro Lys Asn Trp Gln Ser His Thr Gly145 150 155
160Gly Gly Gln Gly Trp Gly Gly Gly His Pro Gln Gly Trp Gly Gly Gly
165 170 175His Pro Gln Gly Trp Gly Gly Gly His Pro Gln Gly Trp Gly
Gly Gly 180 185 190His Pro Gln Gly Trp Gly Gly Gly Gly Gln Pro Pro
Tyr Arg Asn Gly 195 200 205Gly Pro Ser Gly Gln Gly Pro Tyr Arg Ser
Gly Gly Thr Asn Trp Gly 210 215 220Gly Pro Lys Pro Arg Lys Lys Cys
Leu Gly Leu Asp Ser Trp Thr Ala225 230 235 240Val Phe Leu Val Leu
Met Trp Cys Gly Leu Asn Ala Met 245 25046254PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
46Gly Val Ile Leu Phe Ile Leu Phe Ser Ile Leu Leu Ile Val Pro Pro1
5 10 15Ser Ser Phe Leu Val Thr Ser Ser Ser Arg Arg Gly Asp Tyr Tyr
Ala 20 25 30Gln Ser Glu Lys Gln Tyr Gln Thr Val Cys Met Gln Glu Val
Val Arg 35 40 45Glu Met Met Lys Val Asp Thr Glu Thr Phe Asn Glu Gly
Lys Thr Thr 50 55 60Thr Thr Val Thr His Gln Lys Ile Thr Ile Asn Val
Cys Asp His Val65 70 75 80Phe Asn Asn Gln Asn Ser Tyr Gln Asp Val
Pro Arg Tyr Tyr Val Gln 85 90 95Asn Pro Tyr Arg Tyr Met Asn Glu Arg
Tyr Tyr Arg Asp Glu Trp Asp 100 105 110Asn Gly Phe His Ile Met Pro
Arg Ser Met Ala Ser Gly Leu Met Tyr 115 120 125Gly Gly Leu Gly Gly
Val Val Ala Gly Ala Ala Ala Ala Gly Ala Val 130 135 140His Lys Leu
Asn Thr Lys Pro Lys Ser Pro Lys Asn Trp Gln Asn His145 150 155
160Thr Gly Gly Gly Gln Gly Trp Gly Gly Gly His Pro Gln Gly Trp Ser
165 170 175Gly Gly His Pro Gln Gly Trp Ser Gly Gly His Pro Gln Gly
Trp Gly 180 185 190Gly Gly His Pro Gln Gly Trp Thr Gly Gly Gln Pro
Pro Tyr Arg Asn 195 200 205Gly Gly Pro Ser Gly Gln Gly Pro Tyr Arg
Ser Gly Gly Thr Asn Trp 210 215 220Gly Gly Pro Lys Pro Arg Lys Lys
Cys Leu Gly Val Asp Thr Trp Met225 230 235 240Thr Val Phe Leu Ala
Leu Leu Trp Tyr Gly Leu Asn Ala Met 245 25047782PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
47Ala Ala Leu Glu Ala Met Ala Arg Asp Leu Pro Asp Val Ser Trp Tyr1
5 10 15Asp Asp Asp Trp Gly Leu Phe Trp Gly Val Phe Ser Pro Pro Glu
Phe 20 25 30Gly Gln Lys Val Val Thr Ile Pro Thr Arg Arg Asp Arg Asn
Ala Pro 35 40 45 Asp Thr Glu Ile Tyr Arg Lys Ala Ser Thr Leu Ala
Glu Thr Lys Glu 50 55 60Glu Glu Gln Ser Asp Lys Gly Val Trp Val Phe
Val Gln Asp Trp Thr65 70 75 80Asp Leu Leu Met Val Asp Asp Thr Ala
Leu Asp Glu Gln Met Leu Glu 85 90 95Gly Pro Val Glu Glu Ile Val Phe
Arg Gly Ile Lys Asn Ser Cys Ala 100 105 110Phe Leu Arg Pro Pro His
Ala Asp Met Lys Lys Asp Lys Leu Arg Pro 115 120 125Ser Thr Arg Tyr
Ala Ala Lys Gly Gly Leu Ala Glu Trp Phe Gly Asp 130 135 140Pro Glu
Ser Gly Glu Ala Val Gln Val Pro Gln Ala Arg Leu Val Arg145 150 155
160Leu Leu Glu Gln Ala Gly Thr Lys Glu Ala Glu Ser Ala Gly Thr Gly
165 170 175Val Trp Leu Tyr Ala Ala Ser Pro Thr Lys Leu Val Phe Ala
Asp Asn 180 185 190Ser Asn Leu Ala Gly Ala Lys Pro Leu Val Glu Val
Ala Arg Thr Ala 195 200 205Gly Ala Ser Asn Ala Arg Val Gln Phe Leu
Arg Thr Ser Ala Pro Ala 210 215 220Thr Gln Gly Gly Glu Arg Ser Thr
Gly Gly Lys Tyr Ile Ile Met Pro225 230 235 240Lys Gly Gly Phe Leu
Ser Met Leu His Ala Pro Glu Lys Gly Gln Val 245 250 255Val Arg Ser
Gln Val Pro Thr Gly Gly Leu Glu Glu Asp Leu Gln Ala 260 265 270Thr
Leu Ile Ala Ser Ala Ala Val Glu Asp Gln Thr Ser Gln Ala Gly 275 280
285Gln Trp Asn Tyr Ile Ile Gln Gly Gln Arg Gly Gly His Arg Tyr Asn
290 295 300Tyr Leu Ile Ile Tyr Ser Asp Gly Gly Tyr Phe Gln Gly Tyr
Thr Ala305 310 315 320Pro Asp Val Pro Val Lys Asn Ser Gly Glu Ile
Arg Trp Ile Gln Lys 325 330 335Gln Gly Thr Gly Asp Asp Asp Met Gly
His Gln Ala Ala Met Ala Thr 340 345 350Ser Thr His Leu Thr Ala Ala
Asp Phe Pro Val Arg Glu Val Asn Ala 355 360 365Ile His Ser Ser Leu
Tyr Ser Leu Gly Leu Gly Asp Thr Gln Asp Pro 370 375 380Asp Arg Trp
Asn Lys Phe Phe Gln Lys Phe Leu Pro Thr Glu Gly Gly385 390 395
400Glu Pro Leu Val Ser Val Gln Thr Gln Lys Pro Tyr Asp Met Lys Thr
405 410 415Ile Phe Asp Ser Ala Thr Lys Leu Ala Ala Lys Arg Glu Glu
Thr Asn 420 425 430Ala Gln Lys Gly Lys Trp Val Phe Ile Lys Gly Asp
Lys Gly His Asp 435 440 445Leu Ile Phe Cys Asp Glu Ser Lys Leu Ala
Gly Gln Ala Phe Pro Asn 450 455 460Glu Asp Ala Val Leu Ser Val Ser
Met Thr Gly Ala Gly Asn Ser Val465 470 475 480Lys Tyr Leu Lys Ala
Leu Lys Arg Asn Ala Ala Asp Glu Lys Ala Thr 485 490 495Asp Glu Thr
Gly Ala Pro Leu Ala Pro Lys Pro Gly Leu Val Gln Leu 500 505 510Met
Ala Glu Pro Glu Thr Gly Glu Glu Ser Val His Val Arg Ala Arg 515 520
525Gly Ser Arg Glu Asn Asp Arg Ile Gly Lys Ser Val Gln Thr Ala Lys
530 535 540Leu Arg Glu Tyr Arg Asn Ser Asn Ser Gly Cys Trp Gln His
Ile Asn545 550 555 560Asn Gly Leu Asp Leu Ile Phe Cys Asp Gly Asn
Asn Phe Ser Glu Trp 565 570 575Ser Val Pro Val Glu Thr Ala Arg Val
Val Arg Arg Gly Lys Val Gln 580 585 590Phe Leu Arg Gln Val Val Val
Glu Asn Pro Val Val His Lys Phe Gly 595 600 605Ser Ala Val Gly Gly
Lys Lys Tyr Lys Leu Gly Ser Lys Phe Tyr Gly 610 615 620Leu Phe Thr
Ala Ser Glu Phe Gly Gln Val Glu Arg His Gln Val Ala625 630 635
640Arg Gly Asn Leu Tyr Asp Asp Leu Gln Val Thr Phe Ile Ala Ala Ala
645 650 655Gly Ser Glu Asp Gln Ser Cys Glu Asn Gly Leu Trp Tyr His
Leu Asp 660 665 670Tyr Gln Leu Asn Gly Asn Arg Leu Gln Val Thr Lys
Leu Ile Val Tyr 675 680 685Ala Asp Gly Thr Phe Phe Asp Gly Tyr Leu
Asn Thr Pro Val Pro Val 690 695 700 Leu Asp Phe Lys Glu Val Arg Trp
Ile Gln Leu Gly Pro Glu Lys Gly705 710 715 720Ala Lys Leu Phe Glu
Pro His Glu Val Val Met Ser Asn Pro Arg Ala 725 730 735Glu Pro Val
Arg Gly Gln Pro Ala Gly Ala Gln Ser Ala Gly Arg Ser 740 745 750Ala
Thr Ala Ala Arg Val Pro Leu Ser Leu Ala Cys Leu Ala Leu Ser 755 760
765Leu Ala Cys Leu Leu Ala Pro Ala Pro Arg His Pro Ala Met 770 775
7804836PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Met Ala Glu Pro Glu Thr Gly Glu Glu Ser Val His
Val Arg Ala Arg1 5 10 15Gly Ser Arg Glu Asn Asp Arg Ile Gly Lys Ser
Val Gln Thr Ala Lys 20 25 30Leu Arg Glu Tyr 3549146PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
49Ala Asp Gln Cys Thr Ser Lys Ser Leu Thr Met Thr Gly Gln Trp Pro1
5 10 15Val Ala Tyr Ile Gln Phe Ser Cys Phe Ala Lys Arg Lys Leu His
Pro 20 25 30Gln Asp His Phe Pro Cys Asn Asp Leu Asn Pro Gln Thr Lys
Thr Cys 35 40 45Thr Thr Arg Gly Leu Glu Val Asp Leu Phe Tyr Asn Val
Gly Ala Val 50 55 60Ile Gln Lys Arg Ala Arg Val Val Gln Leu Ala Arg
Ser His Tyr Met65 70 75 80Asp Asn Ser Ala Lys Asn Tyr Glu Gly Val
Ala Phe Asp Leu Ala Arg 85 90 95Arg Val Gly Glu Glu Glu Val Ser Ala
Asp Met Pro Gly Gly Val Leu 100 105 110Arg Pro Pro Lys Gly Pro Ser
Ser Gly Ala Ala Pro Ser Val Ala Leu 115 120 125Ala Val Ala Leu Ile
Ala Leu Leu Leu Leu Pro Ala Arg Leu Pro Gly 130 135 140Ala
Met1455020PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Glu Glu Glu Val Ser Ala Asp Met Pro Pro Pro Pro
Met Asp Ala Ser1 5 10 15Val Glu Glu Glu 2051315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
51Gly Leu Ile Leu Leu Thr Ser His Glu Asp Glu Val Pro Val Leu Leu1
5 10 15Gly Leu Leu Val Asn Leu Leu Trp Ser Tyr Phe Tyr Gln Thr Arg
Arg 20 25 30Ser His Gln Cys Ile Ser Val Ala Ser Gly Ala Ala Thr Arg
Arg Ile 35 40 45Thr Pro Ser Ser Ser Lys Leu Asn Ala Ile Phe Ala Lys
Leu Leu Val 50 55 60Lys Ile Glu Asn Asp Asn Ala Phe Asn Gly Phe Ser
Ala Met Ile Lys65 70 75 80Pro Val Ala Ala Ala
Leu Thr Glu Gln Val Ser Glu Glu Pro Arg Lys 85 90 95Ser Thr Arg Thr
Leu Cys Pro Leu Leu Asn Val Leu Tyr Pro Arg Cys 100 105 110Lys Gln
Pro Arg Val Leu His Ala Leu Glu Ala Phe Arg Trp Leu Ala 115 120
125Ala Arg Leu Ser Arg Pro Ala Gly Asn Lys Lys Ile Glu Lys Tyr Leu
130 135 140Glu Leu Gln Leu Arg Pro Leu Asn Ser Asp Met Leu Ala Lys
Ile Val145 150 155 160Lys Asn Leu Cys Glu Asp Ala Val Met Arg Val
Asp Ser Glu Ala Asp 165 170 175Asp Ser Cys Leu Leu Phe Leu Glu Met
Ala Ile Gly Leu Leu Lys Gln 180 185 190Phe Glu Pro Ser Asn Arg Val
Ser Gln Ala Val Ile Asn Glu Cys Ile 195 200 205Thr Leu Cys His Asn
Val Arg Asp Lys Lys Thr Ala Ser Leu Glu Lys 210 215 220Lys Pro Arg
His Leu Pro Glu Glu Ala Val Ala Pro Gly Pro Pro Pro225 230 235
240Pro Pro Pro Pro Pro Pro Pro Gln Pro Gln Pro Leu Leu Pro Gln Ala
245 250 255Gln Pro Pro Pro Gln Pro Leu Gln Pro Pro Pro Pro Pro Pro
Pro Pro 260 265 270Pro Pro Pro Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln 275 280 285Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Phe Ser Lys Leu Ser Glu 290 295 300Phe Ala Lys Met Leu Lys Glu Leu
Thr Ala Met305 310 3155217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 52Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln1 5 10 15Gln5389PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 53Leu
Pro Leu Tyr Asn Leu Pro Glu Arg Lys Leu Val Glu Val Ala Asn1 5 10
15Arg Lys Gly Tyr Thr Asn Ser Gly Val Asn Thr Ser Ser Leu Ile Ala
20 25 30Gly Phe Asn Asn Ser Ser His Val Leu Phe Asn Ala Leu Arg Gln
Thr 35 40 45Ala Cys Thr Ala Thr Asn Cys Lys Arg Lys Glu Val Gln His
Ser Glu 50 55 60Ile Pro Thr Ala Lys Leu His Asn Leu Ala Val Ser Leu
Val Ile Leu65 70 75 80Phe Val Gln Leu Lys Leu Ile Gly Met
85545PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Val Phe Asn Ala Leu1 55514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Val
Phe Asn Ala Leu Pro Pro Pro Pro Leu Ala Asn Phe Val1 5
10566PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Ser Ser His Val Leu Phe1 55715PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Ser
Ser His Val Leu Phe Pro Pro Pro Phe Leu Val His Ser Ser1 5 10
1558147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 58Glu Lys Pro Asn Thr Val Val Ala Thr Thr Ser
Tyr Ser Tyr Pro Ser1 5 10 15Leu Leu Ala Ala Ile Thr Tyr Arg Arg Pro
Gly Ser Asp Asn Ala Thr 20 25 30Phe Val Val Glu Ala His Glu His Phe
Pro Ser Ile Gly Leu Ala Lys 35 40 45Trp Tyr Ser Lys Thr Asp Ile Glu
Val Lys Tyr Ile Gly Glu Val Phe 50 55 60Glu Glu Glu Thr Thr Leu Gly
His Leu Glu Gly Ser Glu Ser Thr Lys65 70 75 80Gly Ser Ala Phe Pro
Glu Trp Thr Asp Asp Ala Ala Lys Arg Phe Val 85 90 95His Val Ala Val
Asn Ile Ala Pro Ser Gly Arg Val Ala Asp Leu Val 100 105 110Lys Val
Met Leu Pro Cys Lys Ser Glu Gly Thr Gly Thr Pro Gly Ala 115 120
125Glu Ser Val Phe Val Leu Gly Ala Leu Cys Leu Leu Leu Leu Arg His
130 135 140Ser Ala Met1455922PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 59Glu Ser Val Phe Val Leu Gly
Ala Leu Pro Pro Pro Pro Leu Ala Gly1 5 10 15Leu Val Phe Val Ser Glu
206032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Val Ala Ala Ala Lys Leu Arg Xaa Val Val Thr Ser
Arg Gln Pro Pro1 5 10 15Pro Pro Gln Arg Ser Thr Val Val Xaa Arg Leu
Lys Ala Ala Ala Val 20 25 306133PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 61Val Val Ala Gly Ala Ala
Ala Ala Gly Ala Val His Lys Leu Lys Pro1 5 10 15Lys Thr Asn Leu Lys
His Val Ala Gly Ala Ala Ala Ala Gly Ala Val 20 25 30Val
* * * * *
References