U.S. patent application number 10/728246 was filed with the patent office on 2005-02-03 for detection of conformationally altered proteins and prions.
Invention is credited to Davidson, Eugene A., Grosset, Anne, Orser, Cindy.
Application Number | 20050026165 10/728246 |
Document ID | / |
Family ID | 34677130 |
Filed Date | 2005-02-03 |
United States Patent
Application |
20050026165 |
Kind Code |
A1 |
Orser, Cindy ; et
al. |
February 3, 2005 |
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; (McLean,
VA) ; Grosset, Anne; (La Croix-de-Rozon, CH) ;
Davidson, Eugene A.; (Washington, DC) |
Correspondence
Address: |
Henry D. Coleman
714 Colorado Avenue
Bridgeport
CT
06605-1601
US
|
Family ID: |
34677130 |
Appl. No.: |
10/728246 |
Filed: |
December 4, 2003 |
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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10161061 |
May 30, 2002 |
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60295456 |
May 31, 2001 |
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Current U.S.
Class: |
435/6.12 ;
435/7.1; 436/518 |
Current CPC
Class: |
C07K 14/4711 20130101;
G01N 33/542 20130101; G01N 2800/2828 20130101; G01N 33/582
20130101; G01N 33/6896 20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 436/518 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/543 |
Claims
What is claimed is:
1. A method for detecting .beta..beta.-sheet conformation of
insoluble 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 .beta..beta.-sheet
conformation insoluble proteins or prions in the sample and thereby
(i) undergo a conformational conversion to a predominately to
.beta..beta.-sheet conformation, and (ii) form detectable
aggregates with the .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.
2. A method of claim 1, wherein probe termini are bound to moieties
that are optically detectable when the probes form detectable
aggregates with the .beta..beta.-sheet conformation insoluble
proteins or prions in the sample.
3. A method of claim 2, wherein the moieties are fluorophores.
4. A method of claim 1, wherein probe termini are bound to
radionucleotide moieties that are detectable when the probes form
detectable aggregates with the .beta..beta.-sheet conformation
insoluble proteins or prions in the sample.
5. A method of claim 1, wherein the probes comprise at least two
amino acid sequences that are complimentary to amino acid sequences
of the .beta..beta.-sheet conformation insoluble proteins or
prions.
6. A method of claim 1, wherein one or more of the probes comprise
at least two amino acid sequences that are homologous to amino acid
sequences of the .beta..beta.-sheet conformation insoluble proteins
or prions.
7. A method claim 6, wherein one or more of the probes is a
palindromic probe.
8. A method of claim 1, wherein the .beta..beta.-sheet conformation
insoluble proteins or prions are selected from the group consisting
of low-density lipoprotein receptor, cystic fibrosis transmembrane
regulator, Huntingtin, Abeta peptide, prions, insulin-related
amyloid, hemoglobin, alpha synuclein, rhodopsin, crystallins, and
p53.
9. A method of claim 1, where one or more probes is a palindromic
33_mer comprising amino acid sequences that are homologous to amino
acids 122-104 and 109-122 of the PrP.sup.SC protein (SEQ ID NO: 1
or 29).33_mer palindrome
35 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
10. A method of claim 1, wherein one or more probes is a
palindromic 33_mer comprising amino acid sequences that are
equivalent to amino acids 122-104 and 109-122 of the PrP.sup.SC
protein (SEQ ID NO: 1 or 29).33 _mer palindrome
36 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
11. A method of claim 1, wherein one or more probes is 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 PrP.sup.SC protein (SEQ ID NO:1 or 29).33_mer palindrome
37 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
12. A method of claim 1, wherein one or more probes is a probe
comprising amino acid sequences that are homologous to amino acids
1-40 of the Abeta peptide Nref 00111747 (human)
38 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG SEQ ID NO: 4 GVV.
13. A method of claim 1, wherein one or more probes comprise amino
acid sequences that are equivalent to amino acids 1-40 of the Abeta
peptide (SEQ ID NO:4).
39 (SEQ ID NO:4). DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV GGVV.
14. A method of claim 1, wherein one or more probes comprise amino
acid sequences that are between about 70% to about 90% identical to
amino acids 1-40 of the A.beta peptide (SEQ ID NO:4)
40 (SEQ ID NO:4) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG GVV.
15. A method of claim 1, wherein one or more probes comprise an
amino acid sequence that has a helix-loop-helix conformation found
in polylysine and that is equivalent to
41 SEQ ID NO: 8. KKKKKKKKKKKKKKKKKKKKKKKKKKK.
16. A method of claim 1, wherein one or more probes comprise an
amino acid sequence that has a helix-loop-helix conformation found
in polylysine and that is homologous to
42 SEQ ID NO: 8. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK.
17. A method of claim 1, wherein one or more probes comprise an
amino acid sequence that has a helix-loop-helix conformation found
in polylysine and that is equivalent to
43 SEQ ID NO: 8 KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK.
18. A method of claim 1, wherein one or more probes comprise an
amino acid sequence that has a helix-loop-helix conformation found
in polylysine and that is between about 70% to about 90% identical
to SEQ ID NO: 8.
44 SEQ ID NO: 8. KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK.
19. A method of claim 1, wherein one or more probes comprise amino
acid sequences that are homologous to amino acids 104-122 of
wild-type (wt) TSE (SEQ ID NO:10)
45 (SEQ ID NO:10) KPKTNLKHVAGAAAAGAVV.
20. A method of claim 1, wherein one or more probes comprise amino
acid sequences that are equivalent to amino acids 104-122 of
wild-type (wt) TSE (SEQ ID NO:10).
46 (SEQ ID NO:10). KPKTNLKHVAGAAAAGAVV
21. A method of claim 1, wherein one or more probes comprise amino
acid sequences that are between about 70% to about 90% identical to
amino acids 104-122 of wild-type
47 (SEQ ID NO:10) KPKTNLKHVAGAAAAGAVV.
22. A method of claim 1, wherein one or more probes comprise an
amino acid sequence that: (a) is a selectively mutated TSE
sequence; and (b) is destabilized and noninfectious; and (c) has an
amino acid sequence that is homologous to SEQ ID NO: 10
48 SEQ ID NO: 10 KPKTNLKHVAGAAAAGAVV.
23. A method of claim 1, wherein one or more probes 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 SEQ ID NO: 10
49 SEQ ID NO: 10 KPKTNLKHVAGAAAAGAVV.
24. A method of claim 1, wherein one or more probes 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 SEQ ID NO: 10
50 SEQ ID NO: 10 KPKTNLKHVAGAAAAGAVV.
25. The method of claim 1, wherein the probes comprise an extrinsic
fluor.
26. The method of claim 25, wherein the extrinsic fluor is
pyrene.
27. A method of claim 1, further comprising reacting the sample and
probes prior to detecting with a probe that limits the formation of
detectable aggregates to detectable but non-infectious levels.
28. A method of claim 1, wherein levels of detectable aggregates
are compared to levels of .beta..beta.-sheet conformation insoluble
proteins or prions associated with amyloidogenic diseases.
29. A method of claim 1, wherein the .beta..beta.-sheet
conformation insoluble proteins or prions form amyloid plaques or
amyloid deposits associated with amyloidogenic diseases.
30. A method of claim 1, wherein the sample is disaggregated prior
to reaction with the probe.
31. A method of claim 1, wherein the sample is a tissue sample or
is a liquid biological material obtained from spinal fluid, saliva,
urine or other bodily fluids.
32. A method of claim 1, wherein excimers are formed by reacting
one or more .alpha.-helix or random coil conformational probes with
.beta..beta.-sheet conformation insoluble proteins or prions in the
sample.
33. A kit comprising one or more .alpha.-helix or random coil
conformational probes that interact with .beta.-sheet conformation
insoluble proteins or prions in a sample and thereby (a) undergo a
conformational conversion to a predominately to .beta..beta.-sheet
conformation, and (b) form detectable aggregates with the
.beta..beta.-sheet conformation insoluble proteins or prions in the
sample, wherein levels of detectable aggregates correlate to the
levels of .beta..beta.-sheet conformation insoluble proteins or
prions in the sample.
34. A kit of claim 33, wherein probe termini are bound to moieties
that are optically detectable when the probes form detectable
aggregates with .beta..beta.-sheet conformation insoluble proteins
or prions in a sample.
35. A kit of claim 34, wherein the moieties are fluorophores.
36. A kit of claim 33, wherein probe termini are bound to
radionuclide moieties that are detectable when the probes form
detectable aggregates with .beta..beta.-sheet conformation
insoluble proteins or prions in a sample.
37. A kit of claim 33, wherein the probes comprise at least two
amino acid sequences that are complementary to amino acid sequences
of .beta..beta.-sheet conformation insoluble proteins or
prions.
38. A kit of claim 33, wherein one or more of the probes comprise
at least two amino acid sequences that are homologous to amino acid
sequences of .beta..beta.-sheet conformation insoluble proteins or
prions.
39. A kit of claim 33, wherein one or more of the probes comprise
an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 15, 18, 20, 22, 23, 24, 25 or 27.
40. A kit of claim 33, wherein the .beta..beta.-sheet conformation
insoluble proteins or prions are selected from the group consisting
of low-density lipoprotein receptor, cystic fibrosis transmembrane
regulator, Huntingtin, Abeta peptide, prions, insulin-related
amyloid, hemoglobin, alpha synuclein, rhodopsin, crystallins, and
p53.
41. A kit of claim 33, where one or more probes is a palindromic
33_mer comprising amino acid sequences that are homologous to amino
acids 122-104 and 109-122 of the human or murine PrP.sup.SC protein
(SEQ ID NO: 1 or 29)
51 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
42. A kit of claim 33, wherein one or more probes is a palindromic
33_mer comprising amino acid sequences that are equivalent to amino
acids 122-104 and 109-122 of the PrP.sup.SC protein (SEQ ID NO: 1
or 29).
52 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
43. A kit of claim 33, wherein one or more probes is 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
PrP.sup.SC protein (SEQ ID NO: 1 or 29).
53 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
44. A kit of claim 33, wherein one or more probes is a probe
comprising amino acid sequences that are homologous to amino acids
1-40 of the Abeta peptide (SEQ ID NO: 4).
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV
45. A method of claim 1, wherein one or more probes comprise amino
acid sequences that are equivalent to amino acids 1-40 of the Abeta
peptide (SEQ ID NO:4).
54 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV.
46. A kit of claim 33, wherein one or more probes comprise amino
acid sequences that are between about 70% to about 90% identical to
amino acids 1-40 of the Abeta peptide (SEQ ID NO:4).
55 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV.
47. A kit of claim 33, wherein one or more probes comprise an amino
acid sequence that is equivalent or homologous to SEQ ID NO: 9 or
20.
48. A kit of claim 33, wherein one or more probes comprise an amino
acid sequence that has a helix-loop-helix conformation found in
polylysine and that is homologous to SEQ ID NO: 8.
49. A kit of claim 33, wherein one or more comprise an amino acid
sequence that has a helix-loop-helix conformation found in
polylysine and that is equivalent to SEQ ID NO: 8.
50. A kit of claim 33, wherein one or more probes comprise an amino
acid sequence that has a helix-loop-helix conformation found in
polylysine and that is between about 70% to about 90% identical to
SEQ ID NO: 9.
51. A kit of claim 33, wherein one or more probes comprise amino
acid sequences that are homologous to amino acid sequences 104-122
of wild-type (wt) TSE (SEQ ID NO: 10).
56 KPKTNVKHVAGAAAAGAVV.
52. A kit of claim 33, wherein one or more probes comprise amino
acid sequences that are equivalent to amino acid sequences 104-122
of wild-type (wt) TSE (SEQ ID NO: 10).
57 KPKTNVKHVAGAAAAGAVV.
53. A kit of claim 33, wherein one or more probes comprise amino
acid sequences that are between about 70% to about 90% identical to
amino acid sequences 104-122 of wild-type (wt) TSE (SEQ ID NO:
10).
58 KPKTNVKHVAGAAAAGAVV.
54. A kit of claim 33, wherein one or more probes 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 SEQ ID NO: 10.
55. A kit of claim 33, wherein one or more probes 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 SEQ ID NO: 10.
56. A kit of claim 33, wherein one or more probes 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 SEQ ID
NO: 10.
57. A kit of claim 33, wherein the probes comprise an extrinsic
fluor.
58. A kit of claim 57, wherein the extrinsic flour is pyrene.
59. A kit of claim 33, further comprising a pendant probe that
limits the formation of detectable aggregates to detectable but
non-infectious levels.
60. A method of diagnosing whether a subject suffers from, or is
predisposed to, a disease associated with conformationally altered
proteins or prion comprising: (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
.beta..beta.-sheet conformation insoluble proteins or prions in the
sample and thereby (i) undergo a conformational conversion to a
predominately to .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 .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.
61. A method of claim 60, wherein probe termini are bound to
moieties that are optically detectable when the probes form
detectable aggregates with the .beta..beta.-sheet conformation
insoluble proteins or prions in the sample.
62. A method of claim 61, wherein the moieties are
fluorophores.
63. A method of claim 60, wherein probe termini are bound to
radionuclide moieties that are detectable when the probes form
detectable aggregates with the .beta..beta.-sheet conformation
insoluble proteins or prions in the sample.
64. A method of claim 60, wherein the probes comprise at least two
amino acid sequences that are complimentary to amino acid sequences
of the .beta..beta.-sheet conformation insoluble proteins or
prions.
65. A method of claim 60, wherein one or more of the probes
comprise at least two amino acid sequences that are homologous to
amino acid sequences of the .beta..beta.-sheet conformation
insoluble proteins or prions.
66. A method claim 60, wherein one or more of the probes comprise
an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 15, 18, 20, 22, 23, 24, 25 or 27.
67. A method of claim 60, wherein the .beta..beta.-sheet
conformation insoluble proteins or prions are selected from the
group consisting of low-density lipoprotein receptor, cystic
fibrosis transmembrane regulator, Huntingtin, Abeta peptide,
prions, insulin-related amyloid, hemoglobin, alpha synuclein,
rhodopsin, crystallins, transthyretin, gelsolin, cystatins and
p53.
68. A method of claim 60, where one or more probes is a palindromic
33_mer comprising amino acid sequences that are homologous to amino
acids 122-104 and 109-122 of the PrP.sup.SC protein (SEQ ID NO: 1
or 29).
59 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
69. A method of claim 60, wherein one or more probes is a
palindromic 33_mer comprising amino acid sequences that are
equivalent to amino acids 122-104 and 109-122 of the PrP.sup.SC
protein (SEQ ID NO:1 or 29)
60 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
70. A method of claim 60, wherein one or more probes is 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 PrP.sup.SC protein (SEQ ID NO: 1 or 29).
61 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
71. A method of claim 60, wherein one or more probes is a probe
comprising amino acid sequences that are homologous to amino acids
1-40 of the Abeta peptide (SEQ ID NO:4)
62 (SEQ ID NO:4) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG GVV.
72. A method of claim 60, wherein one or more probes comprise amino
acid sequences that are equivalent to amino acids 1-40 of the Abeta
peptide (SEQ ID NO:4)
63 (SEQ ID NO:4) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG GVV
73. A method of claim 60, wherein one or more probes comprise amino
acid sequences that are between about 70% to about 90% identical to
amino acids 1-40 of the Abeta peptide
64 (SEQ ID NO:4). DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMV GGVV
74. A method of claim 60, wherein one or more probes comprise an
amino acid sequence that is an oligo or polylysine.
75. A method of claim 74, wherein said probe is homologous to SEQ
ID NO: 8.
76. A method of claim 60, wherein said probe is equivalent to SEQ
ID NO: 8.
77. A method of claim 60, wherein one or more probes comprise an
amino acid sequence that has a helix-loop-helix conformation found
in lysine and that is between about 70% to about 90% identical to
oligo- or polylysine.
78. A method of claim 61, wherein one or more probes comprise amino
acid sequences that are homologous or equivalent to amino acids
104-122 of wild-type (wt) TSE (SEQ ID NO:10).
79. A method of claim 60, wherein one or more probes 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 or equivalent to SEQ ID NO:
10.
80. A method of claim 61, wherein one or more probes 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 SEQ ID NO: 10.
81. A method of claim 60, wherein the probes comprise an extrinsic
fluor.
82. The method of claim 60, wherein the extrinsic flour is
pyrene.
83. A method of claim 60, further comprising reacting the sample
and probes prior to detecting with a pendant probe that limits the
formation of detectable aggregates to detectable but non-infectious
levels.
84. A method of claim 60, wherein levels of detectable aggregates
are compared to levels of .beta..beta.-sheet conformation insoluble
proteins or prions associated with amyloidogenic diseases.
85. A method of claim 60, wherein the .beta..beta.-sheet
conformation insoluble proteins or prions form amyloid plaques or
amyloid deposits associated with amyloidogenic diseases.
86. A method of claim 60, wherein the sample is disaggregated prior
to reaction with the probe.
87. A method of claim 60, wherein the sample is a tissue sample or
is a liquid biological material obtained from spinal fluid, saliva,
urine or other bodily fluids.
88. A method of claim 60, wherein exi_mers are formed by reacting
one or more .alpha.-helix or random coil conformational probes with
.beta..beta.-sheet conformation insoluble proteins or prions in the
sample.
89. A palindromic peptide probe comprising three peptide sections,
a first peptide section, a second peptide section and a third
peptide section, said first and said third sections comprising
peptide sequences each of which comprises at least 5 amino acids
identical to a peptide fragment from a target insoluble protein
which is responsible for .beta..beta.-sheet formation in said
target insoluble protein and wherein at least a portion of said
first peptide section is a palindrome of at least a portion of said
third peptide section, said first peptide section or said third
peptide section being identical to at least a five amino acid
peptide sequence in said peptide fragment from said target
insoluble protein, said second peptide sequence comprising between
1 and 10 amino acid units one of which is a proline residue.
90. The probe according to claim 89 wherein said first and said
third sections are endcapped with hydrophobic amino acids which can
be chemically modified or complexed to accommodate a chemical
moiety capable of being measured.
91. The probe according to claim 90 wherein said chemical moiety is
a chromophore and both said first and third peptide sections of
said probe comprise said chromophore.
92. The probe according to claim 90 wherein said chromophore is
selected from the group consisting of pyrene, tryoptophan,
fluresceing rhodamine.
93. The probe according to claim 92 which is in the form of an
excimer.
94. The probe according to claim 89 wherein said second proline
section comprises between 1 and 5 amino acid residues all of which
are proline residues.
95. The probe according to claim 89 wherein said target peptide is
selected from the group consisting of low-density lipoprotein
receptor, cystic fibrosis transmembrane regulator, Huntingtin,
Abeta peptide, prions, insulin-related amyloid, hemoglobin, alpha
synuclein, rhodopsin, crystallins, transthyretin, gelsolin,
cystatins and p53.
96. The probe according to claim 89 wherein said first peptide
section and said third peptide section consist of identical amino
acids.
97. The probe according to claim 89 wherein said first and said
second peptide sections each comprise about 10 to about 25 amino
acid residues.
98. The palindromic probe according to claim 89 selected from the
group consisting of SEQ ID NO: 1, 18, 23, 25, 27 and 29.
99. The method according to claim 60 wherein said disease is
Alzheimer's Disease, Prion diseases, Creutzfeld Jakob disease,
scrapie and bovine spongiform encephalopathy (PrP.sup.Sc); ALS (SOD
and neurofilament); Pick's disease; Parkinson's disease,
Frontotemporal dementia; Diabetes Type II (Amylin); Multiple
myeloma--plasma cell dyscrasias; Familial amyloidotic
polyneuropathy; Medullary carcinoma of thyroid; Chronic renal
failure, Congestive heart failure, Senile cardiac and systemic
amyloidosis (Transthyretin), Chronic inflammation, Atherosclerosis,
Familial amyloidosis, or Huntington's disease.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part application 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.
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
[0004] 1. Conformationally Altered Proteins and Prions and
Associated Diseases.
[0005] 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. Sci. 94:1-28; Haan et al. (1990)
Clin. Neurol. Neurosurg. 92(4):305-310).
[0006] 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).
[0007] 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
142 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.
[0008] 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.
[0009] 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).
[0010] 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).
[0011] 2. Prions.
[0012] 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 polymerizes (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.
[0013] 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,
PrPsc 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.
[0014] 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.
[0015] 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)).
[0016] 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)).
[0017] 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.
[0018] 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.
[0019] 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 PrPs 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.
[0020] 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
post-mortem tissue sampling, 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
[0021] 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.
[0022] 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:
[0023] 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.
[0024] 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.
[0025] In one embodiment, the invention provides a method for
detecting conformationally altered proteins or prions in a sample
comprising:
[0026] (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
[0027] (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.
[0028] 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.
[0029] 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.
[0030] A method of diagnosing whether a subject suffers from, or is
predisposed to, a disease associated with conformationally altered
proteins or prion comprises:
[0031] (a) obtaining a sample from the subject;
[0032] (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
[0033] (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.
[0034] These and other aspects of the invention are described
further in the following detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0035] 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.
[0036] FIG. 2 illustrates a diagnostic analysis of a sample
containing TSE protein comprised of beta-sheets 12.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] FIG. 7 illustrates energy changes associated with
conformational changes in proteinaceous material or prions.
[0042] FIG. 8 illustrates the alpha-helix and loop structure of
PrP.
[0043] FIG. 9 illustrates the predominantly beta-sheet secondary
structure of PrP.sup.Sc.
[0044] FIG. 10 illustrates a palindromic 33_mer probe used in the
methods of the instant invention.
[0045] FIG. 11 illustrates a circular dichroism graph of three
distinct common conformational forms that proteins and peptides can
assume (source: Woody RW (1996) In Circular Dichroism and the
Conformational Analysis of Biomolecules (Fasman, GD ed.) pp. 25-69,
Plenum press NY).
[0046] 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).
[0047] 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. 10J 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 dimer or excimer state of
the pyrene-labeled peptide has an emission max between 475 and 510
nm.
[0048] 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.
[0049] FIG. 15 illustrates the ratio of excimer formation ((ID) 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.
[0050] 1. pH 6-8, KCl (100-500 mM)
[0051] 2. pH 10-11, KCl (100-500 mM)
[0052] 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.
[0053] 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).
[0054] 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]).
[0055] 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
[0056] 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
[0057] As used herein, the following terms have the following
respective meanings. "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).
[0058] 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).
[0059] 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.
[0060] 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).
[0061] 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.
[0062] 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).
[0063] "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.
[0064] 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).
[0065] 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.
[0066] "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.
[0067] 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.
[0068] 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 PrPc (non-disease) or PrP.sup.Sc
(disease) form.
[0069] A "peptidomimetic" is a biomolecule that mimics the activity
of another biologically active peptide molecule.
[0070] "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, carboxylcarbon 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 carboxylcarbon 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."
[0071] 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."
[0072] "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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] "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.
[0079] "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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] "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).
[0084] 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.
[0085] 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.
[0086] "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:
[0087] a palindromic 33_mer comprising amino acid sequences that
are identical to amino acids 122-104 and 109-122 of the PrP.sup.SC
protein (SEQ ID NO: 13 and 14) (Swiss-Prot PO4156 (Pfam ID Prion
Pf00377 & 03991)
1 VVAGAAAAGAVHKLNTKPKLKHVAG SEQ ID NO: 29 AAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAG SEQ ID NO: 1 AAAAGAVV (human);
[0088] a palindromic 33_mer comprising amino acid sequences that
are equivalent to amino acids 122-104 and 109-122 of the PrP.sup.SC
protein SEQ ID NO: 13 and 14) (Swiss-Prot PO.sub.4156 (Pfam ID
Prion PfO0377 & 03991)
2 VVAGAAAAGAVHKLNTKPKLKHVAG SEQ ID NO: 29 AAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAG SEQ ID NO: 1 AAAAGAVV (human);
[0089] 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 PrP.sup.SC protein SEQ ID NO: 13 and 14)
(Swiss-Prot PO.sub.4156 (Pfam ID Prion Pf00377 & 03991)
3 VVAGAAAAGAVHKLNTKPKLKHVAG SEQ ID NO: 29 AAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAG AAAAGAVV (human)
[0090] a probe comprising amino acid sequences that are identical
to amino acids 1-40 of the Abeta peptide (Nref 00111747
(human))
4 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLM (SEQ ID NO: 4) VGGVV;
[0091] a probe comprising amino acid sequences that are equivalent
to amino acids 1-40 of the Abeta peptide (Nref 00111747
(human))
5 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLM (SEQ ID NO: 4) VGGVV;
[0092] 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))
6 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLM (SEQ ID NO: 4) VGGVV;
[0093] a probe comprising amino acid sequences that are identical
to amino acids 11-34 of the Abeta peptides (Nref 00111747
(human))
7 EVHHQKLVFFAEDVGSNKGAIIGL; (SEQ ID NO: 5)
[0094] 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
8 EVRHQKLVFFAEDVGSNKGAIIGL; (SEQ ID NO: 6)
[0095] a probe comprising amino acid sequences that are identical
to amino acids 25-35 of the Abeta peptides (Nref 00111747
(human))
9 GSNKGAIIGLM; (SEQ ID NO: 7)
[0096] 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
10 SEQ ID NO:8 KKKKKKKKKKKKKKKKKKKKKKKKKKK (27_mer);
[0097] a probe that has a conformation found in polyglutamine and
an amino acid sequence that is equivalent or homologous to SEQ ID
NO:9
11 SEQ ID NO:9 8QQQQQQQQQQQQQQQQQQQQQQQ;
[0098] a probe comprising amino acid sequences that are homologous
to amino acids 104-122 of wild-type (wt) TSE (Human NREF
00130350)
12 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO:10)
[0099] a probe comprising amino acid sequences that are equivalent
to amino acids 104-122 of wild-type (wt) TSE (Human NREF
00130350)
13 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO: 10)
[0100] 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)
14 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO: 10)
[0101] 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)
15 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO: 10)
[0102] 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)
16 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO: 10)
[0103] 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)
17 KPKTNLKHVAGAAAAGAVV; (SEQ ID NO: 10)
[0104] 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
18 MGILKLQVFLIVLSVALNHLKATPIESHQVEKRK (SEQ ID NO: 11) CNTA;
[0105] a probe comprising amino acid sequences that are identical
to at least 10 contiguous sidues within the sequence corresponding
to amino acids 1-38 of the human polypeptide precursor (amylin)
protein (Accession # NP.sub.--000406 (human) human diabetes
19 MGILKLQVFLIVLSVALNHLKATPIESHQVEKRK (SEQ ID NO: 11) CNTA;
[0106] 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
20 MAESHLLQWLLLLLPTLCGPGTAAW; (SEQ ID NO: 11)
[0107] 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 PO.sub.4156 (Pfam ID Prion
Pf00377 & 03991)
[0108] Human prion protein
21 Human prion protein Accession: PO4156 (SEQ ID NO: 13) 1 #2 Mouse
prion protein
[0109] Mouse prion protein
22 Accession: PO4925 (SEQ ID NO: 14) 2
[0110] 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)
(PO.sub.6396), Maury, et al. FEBS Lett., 260(1), pp. 85-87
(1990);
23 3 YERLKATQVSKGIRDNERSGRARVHVSEEG- TEPEAM; (SEQ ID NO: 16)
[0111] 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.771-8 (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;
24 4
[0112] Palindromic probe of cystatin C protein (from amino acids
39-47 of the above sequence with a four unit proline linker)
25 EEEVSADMPPPPMDASVEEE ((SEQ ID NO: 18)
[0113] 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: 19) ID NO: 19)(P42858) [gi:1170192]:
26 5
[0114] exemplary probe:
27 QQQQQQQQQQQQQQQQQ; (SEQ ID NO: 20)
[0115] 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).
28 6
[0116] 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:
29 LANFV; (SEQ ID NO: 22) VFNALPPPPLANFV (SEQ ID NO: 23)
(palindromic probe); FLVHSS; (SEQ ID NO: 24) SSHVLFPPPFLVHSS (SEQ
ID NO: 25) (palindromic probe);
[0117] 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)
30 7
[0118] a palindromic probe based upon the above referenced sequence
(amino acid residues 10-19):
31 ESVFVLGALPPPPLAGLVFVSE. (SEQ ID NO: 27)
[0119] Numerous other probes may be readily produced without undue
experimentation using standard laboratory techniques and peptide
and related chemical syntheses.
[0120] 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).
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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
.alpha.-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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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 .alpha.-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 .alpha.-sheet
formation) at 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.
[0131] 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 (where AAAV is a
hydrophobic amino acid sequence) then the palindrome would contain
a first peptide sequence which is VAAAKLRUVVTSRQ and a second
peptide sequence which is QRSTVVURLKAAAV (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 preferably most, if not all, of those amino acids,
being proline amino acids. The probe would therefore be:
32 VAAAKLRUVVTSRQPPPPQRSTVVURLKAAAV SEQ ID NO: 28 (hypothetical
palindromic probe)
[0132] 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.
[0133] 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.
[0134] The following rules may be applied to the instant invention
to produce preferred conformational peptide probes:
[0135] 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.
[0136] 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.
[0137] 3. One or more of the following additional criteria:
[0138] 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
[0139] b) Amino acid repeats of at least 20 and preferably 25 (such
as is present in huntingtin)
[0140] 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.
[0141] 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
[0142] Test criteria for peptide probe:
[0143] 1. The conformation of the palindrome peptide probe should
be that of an alpha helix or random coil but not a beta sheet.
[0144] 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.
[0145] 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. "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.
[0146] 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
run.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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).
[0152] 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.
[0153] 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).
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] Other structural techniques include equilibrium
ultracentrifugation or size-exclusion chromotography.
[0160] 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 as 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 can 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.
[0161] The invention is described further in the following
examples, which are illustrative and in no way limiting.
EXAMPLE 1
[0162] Materials and Methods
[0163] 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.
[0164] 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.
[0165] 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.
[0166] Illustrative Analysis
[0167] 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.
[0168] 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
[0169] 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.
[0170] 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.
[0171] As also illustrated in FIG. 3:
[0172] Sample 24, which was maintained at pH 7, 25.degree. C.,
exhibited a minimum at approximately 205 nanometers (nm),
indicating a random coil structure;
[0173] 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);
[0174] 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;
[0175] 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
[0176] 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.
[0177] 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:
[0178] Sample 32 (pH 11, 25.degree. C.) evidenced a plateau at
approximately 0.12, indicating a pre-dominantly .alpha.-helical
structure;
[0179] Sample 34 (maintained at pH 7, 50.degree. C.) evidenced a
plateau at approximately 0.22, which indicated a predominantly
random coil structure;
[0180] 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;
[0181] 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.
[0182] 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
[0183] 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)
33 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
[0184] 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.
[0185] 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
[0186] 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)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAG- AVV (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.
[0187] In the experiment that lead to the results illustrated in
FIG. 16, a specific .lambda. (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--Figure
11).
[0188] 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.
[0189] Referring to FIG. 16, for solvent conditions above 50% (far
left dashed line), the 33_mer target peptide (SEQ ID NO: 1 and
29)
34 VVAGAAAAGAVHKLNTKPKLKHVAGAAAAGAVV (murine)
VVAGAAAAGAMHKMNTKPKMKHMAGAAAAGAVV (human)
[0190] 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 P sheet structure.
[0191] 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).
[0192] 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
[0193] The experiment that led to the results illustrated in FIG.
17 was conducted as follows.
[0194] 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.
[0195] 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.
[0196] 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
[0197] The experiment that led to the results illustrated in FIG.
18 was conducted as follows.
[0198] 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.
[0199] 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
29 1 33 PRT Homo sapiens 1 Val Val Ala Gly Ala Ala Ala Ala Gly Ala
Met His Lys Met Asn 1 5 10 15 Thr Lys Pro Lys Met Lys His Met Ala
Gly Ala Ala Ala Ala Gly 20 25 30 Ala Val Val 2 19 PRT Artificial
Sequence Synthetic Peptide 2 Lys Pro Lys Thr Asn Leu Lys His Val
Ala Gly Ala Ala Ala Ala 1 5 10 15 Gly Ala Val Val 3 14 PRT
Artificial Sequence Synthetic Peptide 3 Leu Lys His Val Ala Gly Ala
Ala Ala Ala Gly Ala Val Val 1 5 10 4 40 PRT Artificial Sequence
Synthetic Peptide 4 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val
His His Gln 1 5 10 15 Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala 20 25 30 Ile Ile Gly Leu Met Val Gly Gly Val Val 35
40 5 24 PRT Artificial Sequence Synthetic Peptide 5 Glu Val His His
Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly 1 5 10 15 Ser Asn Lys
Gly Ala Ile Ile Gly Leu 20 6 24 PRT Artificial Sequence Synthetic
Peptide 6 Glu Val Arg His Gln Lys Leu Val Phe Phe Ala Glu Asp Val
Gly 1 5 10 15 Ser Asn Lys Gly Ala Ile Ile Gly Leu 20 7 11 PRT
Artificial Sequence Synthetic Peptide 7 Gly Ser Asn Lys Gly Ala Ile
Ile Gly Leu Met 1 5 10 8 28 PRT Artificial Sequence Synthetic
Peptide 8 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys 1 5 10 15 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
20 25 9 23 PRT Artificial Sequence Synthetic Peptide 9 Gln Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 1 5 10 15 Gln Gln
Gln Gln Gln Gln Gln Gln 20 10 19 PRT Artificial Sequence Synthetic
Peptide 10 Lys Pro Lys Thr Asn Leu Lys His Val Ala Gly Ala Ala Ala
Ala 1 5 10 15 Gly Ala Val Val 11 38 PRT Artificial Sequence
Synthetic Peptide 11 Met Gly Ile Leu Lys Leu Gln Val Phe Leu Ile
Val Leu Ser Val 1 5 10 15 Ala Leu Asn His Leu Lys Ala Thr Pro Ile
Glu Ser His Gln Val 20 25 30 Glu Lys Arg Lys Cys Asn Thr Ala 35 12
25 PRT Artificial Sequence Synthetic Peptide 12 Met Ala Glu Ser His
Leu Leu Gln Trp Leu Leu Leu Leu Leu Pro 1 5 10 15 Thr Leu Cys Gly
Pro Gly Thr Ala Ala Trp 20 25 13 253 PRT Artificial Sequence
Synthetic Peptide 13 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu
Phe Val Ala Thr 1 5 10 15 Trp Ser Asp Leu Gly Leu Cys Lys Lys Arg
Pro Lys Pro Gly Gly 20 25 30 Trp Asn Thr Gly Gly Ser Arg Tyr Pro
Gly Gln Gly Ser Pro Gly 35 40 45 Gly Asn Arg Tyr Pro Pro Gly Gly
Gly Gly Gly Trp Gly Gln Pro 50 55 60 His Gly Gly Gly Trp Gly Gln
Pro His Gly Gly Gly Trp Gly Gln 65 70 75 Pro His Gly Gly Gly Trp
Gly Gln Pro His Gly Gly Gly Trp Gly 80 85 90 Gly Gly Gly Gly Thr
His Ser Gln Trp Asn Lys Pro Ser Lys Pro 95 100 105 Lys Thr Asn Met
Lys His Met Ala Gly Ala Ala Ala Ala Gly Ala 110 115 120 Val Val Gly
Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser 125 130 135 Arg Pro
Ile Ile His Phe Gly Ser Asp Tyr Glu Asp Arg Tyr Tyr 140 145 150 Arg
Glu Asn Met His Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro 155 160 165
Met Asp Glu Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val 170 175
180 Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr Thr Lys Gly 185
190 195 Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg Val Val
200 205 210 Glu Gln Met Cys Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala
Tyr 215 220 225 Tyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser Pro
Pro Val 230 235 240 Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val
Gly 245 250 14 254 PRT murine 14 Met Ala Asn Leu Gly Tyr Trp Leu
Leu Ala Leu Phe Val Thr Met 1 5 10 15 Trp Thr Asp Val Gly Leu Cys
Lys Lys Arg Pro Lys Pro Gly Gly 20 25 30 Trp Asn Thr Gly Gly Ser
Arg Tyr Pro Gly Gln Gly Ser Pro Gly 35 40 45 Gly Asn Arg Tyr Pro
Pro Gln Gly Gly Thr Trp Gly Gln Pro His 50 55 60 Gly Gly Gly Trp
Gly Gln Pro His Gly Gly Ser Trp Gly Gln Pro 65 70 75 His Gly Gly
Ser Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln 80 85 90 Gly Gly
Gly Thr His Asn Gln Trp Asn Lys Pro Ser Lys Pro Lys 95 100 105 Thr
Asn Leu Lys His Val Ala Gly Ala Ala Ala Ala Gly Ala Val 110 115 120
Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg 125 130
135 Pro Met Ile His Phe Gly Asn Asp Trp Glu Asp Arg Tyr Tyr Arg 140
145 150 Glu Asn Met Tyr Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val
155 160 165 Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val
Asn 170 175 180 Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr Thr Lys
Gly Glu 185 190 195 Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg
Val Val Glu 200 205 210 Gln Met Cys Val Thr Gln Tyr Gln Lys Glu Ser
Gln Ala Tyr Tyr 215 220 225 Asp Gly Arg Arg Ser Ser Ser Thr Val Leu
Phe Ser Ser Pro Pro 230 235 240 Val Ile Leu Leu Ile Ser Phe Leu Ile
Phe Leu Ile Val Gly 245 250 15 782 PRT Artificial Sequence
Synthetic Peptide 15 Met Ala Pro His Arg Pro Ala Pro Ala Leu Leu
Cys Ala Leu Ser 1 5 10 15 Leu Ala Leu Cys Ala Leu Ser Leu Pro Val
Arg Ala Ala Thr Ala 20 25 30 Ser Arg Gly Ala Ser Gln Ala Gly Ala
Pro Gln Gly Arg Val Pro 35 40 45 Glu Ala Arg Pro Asn Ser Met Val
Val Glu His Pro Glu Phe Leu 50 55 60 Lys Ala Gly Lys Glu Pro Gly
Leu Gln Ile Trp Arg Val Glu Lys 65 70 75 Phe Asp Leu Val Pro Val
Pro Thr Asn Leu Tyr Gly Asp Phe Phe 80 85 90 Thr Gly Asp Ala Tyr
Val Ile Leu Lys Thr Val Gln Leu Arg Asn 95 100 105 Gly Asn Leu Gln
Tyr Asp Leu His Tyr Trp Leu Gly Asn Glu Cys 110 115 120 Ser Gln Asp
Glu Ser Gly Ala Ala Ala Ile Phe Thr Val Gln Leu 125 130 135 Asp Asp
Tyr Leu Asn Gly Arg Ala Val Gln His Arg Glu Val Gln 140 145 150 Gly
Phe Glu Ser Ala Thr Phe Leu Gly Tyr Phe Lys Ser Gly Leu 155 160 165
Lys Tyr Lys Lys Gly Gly Val Ala Ser Gly Phe Lys His Val Val 170 175
180 Pro Asn Glu Val Val Val Gln Arg Leu Phe Gln Val Lys Gly Arg 185
190 195 Arg Val Val Arg Ala Thr Glu Val Pro Val Ser Trp Glu Ser Phe
200 205 210 Asn Asn Gly Asp Cys Phe Ile Leu Asp Leu Gly Asn Asn Ile
His 215 220 225 Gln Trp Cys Gly Ser Asn Ser Asn Arg Tyr Glu Arg Leu
Lys Ala 230 235 240 Thr Gln Val Ser Lys Gly Ile Arg Asp Asn Glu Arg
Ser Gly Arg 245 250 255 Ala Arg Val His Val Ser Glu Glu Gly Thr Glu
Pro Glu Ala Met 260 265 270 Leu Gln Val Leu Gly Pro Lys Pro Ala Leu
Pro Ala Gly Thr Glu 275 280 285 Asp Thr Ala Lys Glu Asp Ala Ala Asn
Arg Lys Leu Ala Lys Leu 290 295 300 Tyr Lys Val Ser Asn Gly Ala Gly
Thr Met Ser Val Ser Leu Val 305 310 315 Ala Asp Glu Asn Pro Phe Ala
Gln Gly Ala Leu Lys Ser Glu Asp 320 325 330 Cys Phe Ile Leu Asp His
Gly Lys Asp Gly Lys Ile Phe Val Trp 335 340 345 Lys Gly Lys Gln Ala
Asn Thr Glu Glu Arg Lys Ala Ala Leu Lys 350 355 360 Thr Ala Ser Asp
Phe Ile Thr Lys Met Asp Tyr Pro Lys Gln Thr 365 370 375 Gln Val Ser
Val Leu Pro Glu Gly Gly Glu Thr Pro Leu Phe Lys 380 385 390 Gln Phe
Phe Lys Asn Trp Arg Asn Pro Asn Gln Thr Asn Gly Leu 395 400 405 Gly
Leu Ser Tyr Leu Ser Ser His Ile Ala Asn Val Glu Arg Val 410 415 420
Pro Phe Asp Ala Ala Thr Leu His Thr Ser Thr Ala Met Ala Ala 425 430
435 Gln His Gly Met Asp Asp Asp Gly Thr Gly Gln Lys Gln Ile Trp 440
445 450 Arg Ile Glu Gly Ser Asn Lys Val Pro Val Asp Pro Ala Thr Tyr
455 460 465 Gly Gln Phe Tyr Gly Gly Asp Ser Tyr Ile Ile Leu Tyr Asn
Tyr 470 475 480 Arg His Gly Gly Arg Gln Gly Gln Ile Ile Tyr Asn Trp
Gln Gly 485 490 495 Arg Gln Ser Thr Gln Asp Glu Val Ala Ala Ser Ala
Ile Leu Thr 500 505 510 Ala Gln Leu Asp Glu Glu Leu Gln Gln Thr Pro
Val Gln Ser Arg 515 520 525 Val Val Gln Gly Lys Glu Pro Ala His Leu
Met Ser Leu Phe Gly 530 535 540 Gly Lys Pro Met Ile Ile Tyr Lys Gly
Gly Thr Ser Arg Glu Gly 545 550 555 Gly Gln Thr Ala Pro Ala Ser Thr
Arg Leu Phe Gln Val Arg Ala 560 565 570 Asn Ser Ala Gly Ala Thr Arg
Ala Val Glu Val Leu Pro Lys Ala 575 580 585 Gly Ala Leu Asn Ser Asn
Asp Ala Phe Val Leu Lys Thr Pro Ser 590 595 600 Ala Ala Tyr Leu Trp
Val Gly Thr Gly Ala Ser Glu Ala Glu Lys 605 610 615 Thr Gly Ala Gln
Glu Leu Leu Arg Val Leu Arg Ala Gln Pro Val 620 625 630 Gln Val Ala
Glu Gly Ser Glu Pro Asp Gly Phe Trp Glu Ala Leu 635 640 645 Gly Gly
Lys Ala Ala Tyr Arg Thr Ser Pro Arg Leu Lys Asp Lys 650 655 660 Lys
Met Asp Ala His Pro Pro Arg Leu Phe Ala Cys Ser Asn Lys 665 670 675
Ile Gly Arg Phe Val Ile Glu Glu Val Pro Gly Glu Leu Met Gln 680 685
690 Glu Asp Leu Ala Thr Asp Asp Val Met Leu Leu Asp Thr Trp Asp 695
700 705 Gln Val Phe Val Trp Val Gly Lys Asp Ser Gln Glu Glu Glu Lys
710 715 720 Thr Glu Ala Leu Thr Ser Ala Lys Arg Tyr Ile Glu Thr Asp
Pro 725 730 735 Ala Asn Arg Asp Arg Arg Thr Pro Ile Thr Val Val Lys
Gln Gly 740 745 750 Phe Glu Pro Pro Ser Phe Val Gly Trp Phe Leu Gly
Trp Asp Asp 755 760 765 Asp Tyr Trp Ser Val Asp Pro Leu Asp Arg Ala
Met Ala Glu Leu 770 775 780 Ala Ala 16 36 PRT Artificial Sequence
Synthetic Peptide 16 Tyr Glu Arg Leu Lys Ala Thr Gln Val Ser Lys
Gly Ile Arg Asp 1 5 10 15 Asn Glu Arg Ser Gly Arg Ala Arg Val His
Val Ser Glu Glu Gly 20 25 30 Thr Glu Pro Glu Ala Met 35 17 146 PRT
Artificial Sequence Synthetic Peptide 17 Met Ala Gly Pro Leu Arg
Ala Pro Leu Leu Leu Leu Ala Ile Leu 1 5 10 15 Ala Val Ala Leu Ala
Val Ser Pro Ala Ala Gly Ser Ser Pro Gly 20 25 30 Lys Pro Pro Arg
Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu 35 40 45 Glu Glu Gly
Val Arg Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr 50 55 60 Asn Lys
Ala Ser Asn Asp Met Tyr His Ser Arg Ala Leu Gln Val 65 70 75 Val
Arg Ala Arg Lys Gln Ile Val Ala Gly Val Asn Tyr Phe Leu 80 85 90
Asp Val Glu Leu Gly Arg Thr Thr Cys Thr Lys Thr Gln Pro Asn 95 100
105 Leu Asp Asn Cys Pro Phe His Asp Gln Pro His Leu Lys Arg Lys 110
115 120 Ala Phe Cys Ser Phe Gln Ile Tyr Ala Val Pro Trp Gln Gly Thr
125 130 135 Met Thr Leu Ser Lys Ser Thr Cys Gln Asp Ala 140 145 18
20 PRT Artificial Sequence Synthetic Peptide 18 Glu Glu Glu Val Ser
Ala Asp Met Pro Pro Pro Pro Met Asp Ala 1 5 10 15 Ser Val Glu Glu
Glu 20 19 315 PRT Artificial Sequence Synthetic Peptide 19 Met Ala
Thr Leu Glu Lys Leu Met Lys Ala Phe Glu Ser Leu Lys 1 5 10 15 Ser
Phe Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 20 25 30
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Pro Pro Pro Pro 35 40
45 Pro Pro Pro Pro Pro Pro Gln Leu Pro Gln Pro Pro Pro Gln Ala 50
55 60 Gln Pro Leu Leu Pro Gln Pro Gln Pro Pro Pro Pro Pro Pro Pro
65 70 75 Pro Pro Pro Gly Pro Ala Val Ala Glu Glu Pro Leu His Arg
Pro 80 85 90 Lys Lys Glu Leu Ser Ala Thr Lys Lys Asp Arg Val Asn
His Cys 95 100 105 Leu Thr Ile Cys Glu Asn Ile Val Ala Gln Ser Val
Arg Asn Ser 110 115 120 Pro Glu Phe Gln Lys Leu Leu Gly Ile Ala Met
Glu Leu Phe Leu 125 130 135 Leu Cys Ser Asp Asp Ala Glu Ser Asp Val
Arg Met Val Ala Asp 140 145 150 Glu Cys Leu Asn Lys Val Ile Lys Ala
Leu Met Asp Ser Asn Leu 155 160 165 Pro Arg Leu Gln Leu Glu Leu Tyr
Lys Glu Ile Lys Lys Asn Gly 170 175 180 Ala Pro Arg Ser Leu Arg Ala
Ala Leu Trp Arg Phe Ala Glu Leu 185 190 195 Ala His Leu Val Arg Pro
Gln Lys Cys Arg Pro Tyr Leu Val Asn 200 205 210 Leu Leu Pro Cys Leu
Thr Arg Thr Ser Lys Arg Pro Glu Glu Ser 215 220 225 Val Gln Glu Thr
Leu Ala Ala Ala Val Pro Lys Ile Met Ala Ser 230 235 240 Phe Gly Asn
Phe Ala Asn Asp Asn Glu Ile Lys Val Leu Leu Lys 245 250 255 Ala Phe
Ile Ala Asn Leu Lys Ser Ser Ser Pro Thr Ile Arg Arg 260 265 270 Thr
Ala Ala Gly Ser Ala Val Ser Ile Cys Gln His Ser Arg Arg 275 280 285
Thr Gln Tyr Phe Tyr Ser Trp Leu Leu Asn Val Leu Leu Gly Leu 290 295
300 Leu Val Pro Val Glu Asp Glu His Ser Thr Leu Leu Ile Leu Gly 305
310 315 20 17 PRT Artificial Sequence Synthetic Peptide 20 Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 1 5 10 15 Gln
Gln 21 89 PRT Artificial Sequence Synthetic Peptide 21 Met Gly Ile
Leu Lys Leu Gln Val Phe Leu Ile Val Leu Ser Val 1 5 10 15 Ala Leu
Asn His Leu Lys Ala Thr Pro Ile Glu Ser His Gln Val 20 25 30 Glu
Lys Arg Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu 35 40 45
Ala Asn Phe Leu Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu 50 55
60 Ser Ser Thr Asn Val Gly Ser Asn Thr Tyr Gly Lys Arg Asn Ala
65
70 75 Val Glu Val Leu Lys Arg Glu Pro Leu Asn Tyr Leu Pro Leu 80 85
22 5 PRT Artificial Sequence Synthetic Peptide 22 Leu Ala Asn Phe
Val 1 5 23 14 PRT Artificial Sequence Synthetic Peptide 23 Val Phe
Asn Ala Leu Pro Pro Pro Pro Leu Ala Asn Phe Val 1 5 10 24 6 PRT
Artificial Sequence Synthetic Peptide 24 Phe Leu Val His Ser Ser 1
5 25 15 PRT Artificial Sequence Synthetic Peptide 25 Ser Ser His
Val Leu Phe Pro Pro Pro Phe Leu Val His Ser Ser 1 5 10 15 26 147
PRT Artificial Sequence Synthetic Peptide 26 Met Ala Ser His Arg
Leu Leu Leu Leu Cys Leu Ala Gly Leu Val 1 5 10 15 Phe Val Ser Glu
Ala Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys 20 25 30 Pro Leu Met
Val Lys Val Leu Asp Ala Val Arg Gly Ser Pro Ala 35 40 45 Ile Asn
Val Ala Val His Val Phe Arg Lys Ala Ala Asp Asp Thr 50 55 60 Trp
Glu Pro Phe Ala Ser Gly Lys Thr Ser Glu Ser Gly Glu Leu 65 70 75
His Gly Leu Thr Thr Glu Glu Glu Phe Val Glu Gly Ile Tyr Lys 80 85
90 Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys Ala Leu Gly Ile Ser 95
100 105 Pro Phe His Glu His Ala Glu Val Val Phe Thr Ala Asn Asp Ser
110 115 120 Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser Pro Tyr
Ser 125 130 135 Tyr Ser Thr Thr Ala Val Val Thr Asn Pro Lys Glu 140
145 27 22 PRT Artificial Sequence Synthetic Peptide 27 Glu Ser Val
Phe Val Leu Gly Ala Leu Pro Pro Pro Pro Leu Ala 1 5 10 15 Gly Leu
Val Phe Val Ser Glu 20 28 32 PRT Artificial Sequence unsure (8)
(25) Synthetic Peptide 28 Val Ala Ala Ala Lys Leu Arg Xaa Val Val
Thr Ser Arg Gln Pro 1 5 10 15 Pro Pro Pro Gln Arg Ser Thr Val Val
Xaa Arg Leu Lys Ala Ala 20 25 30 Ala Val 29 33 PRT murine 29 Val
Val Ala Gly Ala Ala Ala Ala Gly Ala Val His Lys Leu Asn 1 5 10 15
Thr Lys Pro Lys Leu Lys His Val Ala Gly Ala Ala Ala Ala Gly 20 25
30 Ala Val Val
* * * * *
References