US008076089B2
(12) United States Patent
(10) Patent N0.:
(45) Date of Patent:
Tseng et a1.
(54)
BIOMARKERS FOR LIVER DISEASES AND
METHOD FOR USING THE SAME
6,329,198 B1
6,423,836 B1
2003/0153013 A1
(75) Inventors: Tzu Ling Tseng, Chiayi (TW); Ping Fu
Cheng, Changhua County (TW)
(73) Assignee: Industrial Technology Research
Institute, Hsinchu (TW)
(*)
Notice:
Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154(b) by 0 days.
US 8,076,089 B2
Dec. 13, 2011
12/2001 King et al.
7/2002 King et al.
8/2003 Huang
FOREIGN PATENT DOCUMENTS
JP
10319019
4/1998
OTHER PUBLICATIONS
Gonzalez et al. ‘Molecular cloning and sequencing of Zeta-crystallin/
quinone reductase cDNA from human liver.’ Biochem. Biophys. Res.
Commun. 191:902-907, 1993*
Tang et a1. ‘Identi?cation of C-Crystallin/NADPHzQuinone
Reductase as a Renal Glutaminase mRNA pH Response Element
(21) App1.No.: 12/763,453
binding Protein.’ J. Biol. Chem. 276(24):21375-211380, 2011.*
Schmits et al., Analysis of the antibody repertoire of astrocytoma
(22) Filed:
patients against antigens expressed by gliomas, Int. J. Cancer, vol. 98,
Apr. 20, 2010
pp. 73-77, 2002.
(65)
Prior Publication Data
US 2010/0261209 A1
Oct. 14,2010
Related US. Application Data
(60)
Continuation of application No. 12/073,603, ?led on
Mar. 7, 2008, noW abandoned, Which is a division of
Lerner et al., Tapping the immunological repertoire to produce anti
bodies of predetermined speci?city, Nature, vol. 299, pp. 592-596,
1982.
Le Naour et al., A distance repertoire of autoantibodies in hepatocel
lular carcinoma identi?ed by proteomic analysis, Molec & Cellular
Proteomics, vol. 1, pp. 197-203, 2002.
* cited by examiner
application No. 11/013,684, ?led on Dec. 17, 2004,
noW abandoned.
(30)
Primary Examiner * Nora Rooney
Foreign Application Priority Data
Dec. 19, 2003
(TW) ............................. .. 92136309A
(74) Attorney, Agent, or Firm * Bacon & Thomas, PLLC
(57)
ABSTRACT
Biomarkers for liver diseases and method for using the same
are provided. For detecting liver cirrhosis and liver cancer, the
(51)
Int. Cl.
G01N 33/53
(52)
US. Cl. ...................................................... .. 435/71
sequences With SEQ ID NO:1 to SEQ ID NO:24 or deriva
(58)
Field of Classi?cation Search ...................... .. None
tives or fragments or variants or the combination thereof or
See application ?le for complete search history.
the antibodies against the amino acid sequences. Then the
biomarkers are further developed into detection kits, such that
by detecting the existence of autoantibodies or autoantigens
in screened specimens, liver diseases are detected With higher
(56)
(2006.01)
References Cited
U.S. PATENT DOCUMENTS
4,487,830 A *
6,087,117 A
12/1984
biomarkers are selected from any one of the amino acid
accuracy and sensitivity.
Coates etal. .............. .. 435/723
7/2000 King etal.
8 Claims, 2 Drawing Sheets
US. Patent
umwor?mohum
Dec. 13, 2011
A.
E“mBx5E3oD%B
Sheet 2 of2
US 8,076,089 B2
N@E
US 8,076,089 B2
1
2
BIOMARKERS FOR LIVER DISEASES AND
METHOD FOR USING THE SAME
Cancer has been the leading cause of death in TaiWan since
1982, Whereas liver cancer is ranked among the top as the
CROSS REFERENCES TO THE RELATED
APPLICATIONS
?nd biomarkers With high accuracy and not susceptible to
cause of death in both men or Women. Thus it is important to
interference and use those biomarkers to develop detection
kits for liver cirrhosis and cancer to effectively screen patients
With liver diseases in the hope that early diagnosis and early
This is a continuation application of US. application Ser.
No. 12/073,603 ?led Mar. 7, 2008, noW abandoned, Which is
a division ofU.S. application Ser. No. 11/013,684 ?led Dec.
17, 2004, noW abandoned.
treatment can help loWer the mortality rate.
SUMMARY OF THE INVENTION
BACKGROUND OF THE INVENTION
in addressing the draWbacks of prior arts, the present inven
tion provides biomarkers for liver diseases, Which can be
developed into detection kits for diagnosis of liver cirrhosis
1. Field of the Invention
The present invention is related to biomarkers for liver
diseases and method for using the same, in Which a method
and liver cancer based on the knoWledge of the existence of
autoantibodies.
for screening autoantigens is employed to identify biomark
An objective of the present invention is to provide biom
ers that can be used in detecting liver diseases. The identi?ed
biomarkers are further developed into detection kits to detect
the presence of autoantibodies or autoantigens in specimens
for screening of liver diseases.
2. Description of Related Art
People With impaired immune functions are prone to
develop immune diseases. The etiology of many human dis
arkers for detecting liver cirrhosis and liver cancer, Which are
selected from any one of the amino acid sequences With SEQ
20
ID NO:1 to SEQ ID NO:24 or derivatives or fragments or
variants or the combination thereof or the antibodies against
the amino acid sequences.
According to the present invention, the aforesaid variants
are obtained by substituting, deleting, inserting and/ or adding
to the amino acid in the amino acid sequences of the biomar
eases may be traced to our immune system in any of the three
conditions described beloW. The ?rst is reduced immunity,
loWer activity of immune cells, or reduced quantity of
immune cells, such that the human body cannot ?ght off the
invading bacteria, virus or mold, and becomes susceptible to
contagious diseases, such as common cold, ?u, pneumonia,
25
ker With one or more amino acids; the amino acid sequence of
enteritis, or even hepatitis and AIDS. The second condition is
immunode?ciency or over-reaction of the immune system
30
the variant and that of the biomarker have sequence homology
greater than 80%.
Another objective of the present invention is to provide a
detection kit for liver diseases, comprising a set of biomarkers
selected from any one of the amino acid sequences With SEQ
ID NO:1 to SEQ ID NO:24 or derivatives or fragments or
variants or the combination thereof.
In one embodiment of the present invention, the aforesaid
Where the invading sub stances are not germs, but tiny pollens
or macromolecular proteins in the food ingested, against
Which the immune system releases a large amount of antibod
ies. Such attack and defense occur in our cells, causing a chain
of reactions Which is also called allergy. When real pathogens
35
detection kit may further include secondary antibodies that
can recogniZe the antibodies against any one of the amino
acid sequences With SEQ ID NO:1 to SEQ ID NO:24 or
such as bacteria, virus or mold attack the human body at this
derivatives or fragments or variants thereof.
time, the immune system is no longer able to put up resis
tance. The third condition of impaired immune system is the
immune cells attack normal cells in the human body, called
method for screening liver diseases, comprising the steps of:
providing a specimen; using biomarkers selected from any
autoimmune disorder as in the case of rheumatoid arthritis,
A further objective of the present invention is to provide a
40
one of the amino acid sequences With SEQ ID NO:1 to SEQ
lupus erythematous, and herpes. Such immune diseases arise
ID NO:24 or derivatives or fragments or variants or the com
from our oWn immune system having an identi?cation prob
bination thereof to capture the autoantibody in the specimen;
lem that autoantibodies are produced against human body’s
oWn cells, resulting in tissue damage and illnesses.
and detecting the autoantibody.
It is noW knoWn that autoantibodies are present not just in
autoimmune diseases. More and more studies indicate that in
the immune response to cancer, autoantigen (from the tumor)
and autoantibody (from the body) exist in some cases. Thus
the detection of tumor autoantigen that elicits body response
may be directed toWards and applied in the testing, diagnosis,
Yet another objective of the present invention is to provide
45
A further objective of the present invention is to provide a
method using the aforesaid detection kit to screen liver dis
50
specimen; and detecting the antibody-antigen complex.
US. Pat. No. 6,631,330, 5,137,807, 5,830,667, 6,264,949,
This invention is based on the use of autoantigen screening
55
lines (HepG2 C3A & SNU-387) in sequence through the
normal antibody column and patient antibody column to
60
US. Pat. No. 5,891,436 and Publication No. 20030138860
disclose the use of biomarkers to detect the presence of
autoantibodies in human serum as a diagnostic tool for pri
mary biliary cirrhosis or hepatocellular carcinoma. Those
patents con?rm the existence of autoantibodies in cancer
kers in cancer screening.
from normal persons, liver cirrhosis patients, and liver cancer
umns; passing the cell extracts from liver disease related cell
lular carcinoma as a diagnostic tool. But the biomarkers dis
closed in those patents lack accuracy or are susceptible to
patients and thereby establish the rational for using biomar
method, comprising the steps of: ?rstly purifying antibodies
patients respectively and immobilizing them in different col
arkers in the diagnosis ofhepatocellular carcinoma; US. Pat.
No. 6,410,724 uses DNA primer associated With hepatocel
interference to a certain extent.
eases, comprising the steps of: providing a specimen; using
the antibody against any one of the amino acid sequences With
SEQ ID NO:1 to SEQ ID NO:24 to capture the antigen in the
or prognosis of cancer, and furthermore, in the treatment of
disease.
and 5,985,542 disclose the use ofbiomarkers in the diagnosis
of cirrhosis, ?brosis or autoimmune hepatitis (All-I); US.
Pat. Nos. 4,994,374 and 5,175,084 disclose the use of biom
a detection kit for liver diseases, comprising a set of antibod
ies against any one of the amino acid sequences With SEQ ID
N011 to SEQ ID NO:24.
65
obtain autoantigens associated With liver cirrhosis and liver
cancer; using those autoantigens as biomarker kits coupled
With enzyme-linked immunosorbent assay (ELISA), radio
immunoassay (RIA), or immuno?uorescence to detect the
presence of autoantibodies against said autoantigens in the
screened specimen, and based on Which, to determine
Whether the patient has liver cirrhosis or liver cancer. Since
those biomarkers are identi?ed based on existing autoanti
US 8,076,089 B2
3
4
bodies, they can be developed into diagnostic kits to deter
variants or the combination thereof. The aforesaid specimen
mine if the patient has such diseases based on the presence of
autoantibodies against the biomarkers. Such method is much
is Whole blood or serum, preferably serum.
To facilitate the detection, the aforesaid biomarker may
come in any form, including but not limited to, a detection kit
easier than direct screening of the antigen and offers greater
accuracy and sensitivity.
or pre-immobiliZed on a substrate, said substrate may be an
BRIEF DESCRIPTION OF THE DRAWINGS
immunoassay plate or a biochip, said substrate may be an
immunoassay plate or a biochip. The autoantibodies in the
specimen captured by the biomarkers can be recogniZed and
adsorbed by the secondary antibodies, Which are modi?ed
antibodies having special functional groups for color reac
FIG. 1 shoWs a ?oW chart of an autoantigen screening
method according to the present invention.
FIG. 2 shoWs a ?oW chart of using biomarkers for screen
tion, radio detection or ?uorescence detection.
ing autoantibody according to the present invention.
After autoantibody is adsorbed by the secondary antibody,
a special reagent is added to undergo color reaction and
DETAILED DESCRIPTION OF THE INVENTION
enZyme-linked immunosorbent assay (ELISA) is employed
to determine the presence of the secondary antibody, and
The present invention relates to the use of an autoantigen
from Which to learn the presence of the autoantibody as a
basis for determining if the patient has liver cancer or liver
screening method to identify biomarkers that may be used in
the detection of liver diseases, such as liver cirrhosis and liver
cancer. Said autoantigen screening method as shoWn in FIG.
1 comprises the folloWing steps: ?rstly obtaining serum
samples from normal persons and patients and passing the
cirrhosis. The presence of the secondary antibody and thereby
20
respective samples over a?inity columns that can capture
antibodies to purify the antibodies contained in the serum
marker (e.g. cy3 or Cy5) prior to reacting With the biomark
samples; next packing respectively the resulting puri?ed nor
mal antibodies and patient antibodies into columns to obtain
a column containing antibodies from normal persons (normal
ers. The ?uorescence-labeled autoantibodies screened by the
25
Whether a patient has liver cirrhosis or liver cancer. To achieve
30
To continue the procedure, passing the sample from the
extract of disease related cell lines or pathological tissues
over the normal antibody column Where non-speci?c anti
gens are captured and retained in the column through the
35
non-speci?c antigens are removed, the sample constitutes
only speci?c antigens. Next, passing the sample over the
40
EXAMPLE 1
45
Autoantibodies in the Serum Sample
spectrum technology; the aforesaid determination procedure
involves comparing the signals from mass spectrograph With
50
Autoantigens in liver disease related cell lines are puri?ed
and identi?ed according to the method described above.
Given that those autoantigens are identi?ed by the antibodies
in patient sera, the autoantigens or derivatives or fragments or
serum using a 0.45 pm ?lter membrane to prevent the block
55
arkers are selected from autoantigens screened by the autoan
tigen screening method or its derivatives or fragments or
age of column in subsequent steps; next rinsing a Protein G
a?inity column With the binding buffer ten times the column
volume at the rate of 1 ml/min, and then passing the ?ltered
serum sample over the Protein G af?nity column at the rate of
0.2 ml/min to retain the antibodies in the column through
a?inity; rinsing the Protein G a?inity column again using the
binding buffer 5-10 times the column volume at the rate of 1
ml/min to remove sub stances in the serum sample that do not
60
form af?nity bonding With the column. Eluting antibodies
from the column using an elution buffer (0.1 M Glycine-HCl,
pH 2.7) 2-5 times the column volume at the rate of 1 ml/min
and collecting the elated antibodies in a test tube Which is
cation of the detection method.
As shoWn in FIG. 2, the screening method utiliZing the
detection kits described above comprises the steps of: provid
ing a specimen; using biomarkers to capture the autoantibody
in the specimen; and detecting the autoantibody. Said biom
Firstly obtaining a serum of a patient With liver cirrhosis or
liver cancer, diluting the serum With a binding buffer (20 mM
PBS, pH 7.0) at the ratio of 1:10, and then ?ltering the diluted
ence of autoantibodies in screened specimen, it can be deter
mined Whether the patient has liver cirrhosis or other liver
diseases. In addition to biomarkers, the detection kits can
further include secondary antibodies that can recogniZe the
autoantibodies against the biomarkers to facilitate the appli
Screening of Autoantigens Using Autoantibodies in
Sera of Patients With Liver Diseases Puri?cation of
body column are subjected to determination by the mass
ers and developed into detection kits. By detecting the pres
tions made in the example should not be construed as a
limitation on the actual application of the present invention.
Finally, the autoantigens displaced from the patient anti
variants or combinations thereof can be utiliZed as biomark
The method of using the aforesaid detection kit for screen
ing liver cancer and liver cirrhosis comprises the steps of:
providing a serum specimen; using the aforesaid antibody to
The advantages of the present invention are further
depicted With the illustration of an example, but the descrip
related autoantigen. Since non-speci?c antigens have been
the database to obtain the information on the autoantigens.
kit containing antibodies that can recogniZe autoantigens
identi?ed by the autoantigen screening method for the screen
ing of liver diseases.
the antibody-antigen complex.
column packed With patient antibodies to screen disease
removed by normal serum antibodies, the autoantigens as
identi?ed by patient’s autoantibodies are more speci?c.
this purpose, the present invention also provides a detection
recogniZe and capture the antigen in the serum; and detecting
speci?c a?inity of normal antibodies; this step may be vieWed
as pre-treatment of the sample before the patient antibody
column is used to screen autoantigens in the sample. After
biomarkers can then be detected by a ?uorescence scanner
Without the use of the secondary antibody.
Besides detecting the presence of the autoantibody, detec
tion of the antigen may also be used as a basis for determining
antibody column) and a column containing antibodies from
patients (patient antibody column) in Which antibodies are
immobiliZed through the chemical bonding formed betWeen
the antibodies and chemical functional groups in the column;
obtaining a sample Which may be the extract of disease
related cell lines or pathological tissues; the aforesaid serum
sample may be that of a single patient or a mixture sample
containing the sera of a plurality of patients.
the presence of the autoantibody can also be determined by
radioimmunoassay (RIA) or immuno?uorescence.
If the screening method does not include the secondary
antibody, the specimen may be labeled With a ?uorescence
65
added beforehand With 60-200 ul Tris-HCL solution (1 M, pH
9.0). Finally displacing the sample in a coupling buffer (0.2M
NaHCO3, 0.5M NaCl, pH 8.3) to complete the puri?cation of
autoantibodies (IgG) in the serum sample.
US 8,076,089 B2
5
6
The method according to the present invention requires one
normal IgG and patient IgG column each. Thus sera from
normal persons and patients should be obtained and subject to
column is free of non- speci?c antigens. Injecting the resulting
cell extract into the patient antibody column. Eluting the
column With the binding buffer 5-10 times the column vol
ume at the rate of 1 ml/min. At this time, the autoantigens
the puri?cation steps described above.
Preparation of Columns Containing Autoantibodies
Pipette one drop of an acidi?cation solution (1 mM HCl,
ice bathed) into a NHS-activated column to prevent the for
mation of bubbles. After connecting the upper end of the
column With a syringe or pump, removing the adapter at the
bottom of the column. Rinsing out isopropanol in the column
using the acidi?cation solution tWo times the column volume.
present in the cell extract Will be captured by the autoanti
bodies from the patients and retained in the column. When the
cell extract passes over the normal antibody column, the
antigens captured by the normal antibodies are retained in the
column, Whereas the cell extract free of antigens can be iden
10
ti?ed and captured by the normal antibodies, only antigens
that can be identi?ed and captured by the patient antibodies
Will be retained by the column. The antigens retained in the
patient antibody column are eluted and collected using the
After repeating the Wash step three times, injecting the
sample containing autoantibodies into the column. Preparing
the aforesaid coupling buffer containing puri?ed autoanti
elution buffer 2-5 times the column volume at the rate of 1
the column volume and a concentration of 0.5-10 mg/ml.
ml/minl. Subjecting the ?ow-through to protein hydrolysis
using trypsin and the resulting peptides are assayed using the
After passing the aforesaid sample containing autoantibodies
mass spectrum technology. The resulting spectrographs are
over the column, sealing the column and let the reaction go on
for 15-30 minutes under 25° C. or 4 hours under 40 C. to
proteins.
bodies into a solution With a volume equivalent to one time
immobiliZe the antibodies in the column through chemical
bonding.
compared With the database to obtain the information on the
20
After the bonding betWeen the autoantibodies and the col
umn, eluting the column With a blocking buffer (0.5M etha
nolamine, 0.5M NaCl, pH 8.3) tWo times the column volume,
and repeating the steps three times. Then rinsing the column
With a Washing buffer (0.1M acetate, 0.5M NaCl, pH 4) tWo
cancer, the folloWing autoantigens are obtained:
1. Nucleoside diphosphate kinase (gi|1421609, SEQ ID
NO.1).
25
times the column volume and also repeating the steps three
times. Again eluting the column three times using the afore
said blocking buffer tWo times the column volume each time,
and then let the column react 15-30 minutes to block and
inactivate the functional groups in the column that are not
30
bound With autoantibodies. After completing the blocking
reaction, rinsing the column three times using the aforesaid
Washing buffer tWo times the column volume each time,
folloWed by eluting the column three times using the afore
said blocking buffer tWo times the column volume to make
35
blocked. Again rinsing three times the column using the
packed With the autoantibodies.
Identi?cation of Autoantigens from Extract of Liver Disease
40
Firstly rinsing 2.68 mg of HepG2 C3A cells With culture
mM Tris pH 7.5, 150 mM NaCl, 1.5 mM PMSF, phosphatase
inhibitors) tWice, then adding in 1 ml of Triton Extraction
solution (15 mM Tris pH 7.5, 120 mM NaCl, 25 mM KCl, 2
mM EGTA, 0.1 mM DTT, 0.5% Triton X-100, 10 ug/ml
leupeptin, 0.5 mM PMSF, and phosphatase inhibitors) and let
45
it stand for 30 minutes under 40 C. At this time, cells start to
50
isomerase-related
protein
5
12. Tumor necrosis factor type I receptor associated protein
TRAP-1 (gil 1082886, SEQ ID NO.12).
13. Tumor rejection antigen (gp96) 1; glucose regulated
protein (gil4507677, SEQ ID NO.13).
NO. 1 5).
16. Heat shock 60kDa protein 1 (gi|31542947, SEQ ID
NO. 1 6).
17. HMG-l (gil968888, SEQ ID NO.17).
18. KIAA0144 gene product (NICE-4 protein)
(gi|13111995, SEQ ID NO.18).
NO. 1 9).
55
quent steps. Prior to injecting the sample into the IgG column,
rinsing the normal and patient antibody columns With the
20. Glyceraldehyde 3-phosphate dehydrogenase, liver
(gil30157565, SEQ ID NO.20).
21. Cytokeratin (giI1419564, SEQ ID NO.21).
22. IGF-II mRNA-binding protein 1 (gi|4191608, SEQ ID
NO.22).
23. NADPH: quinone reductase (gi|13236495, SEQ ID
60
NO.23).
24. Crystal Structure of The Human Co-Chaperone P23
(hsp-90 co-chaperone) (gil9257073, SEQ ID NO.24).
antibody column With the binding buffer 5-10 times the col
step is to remove non-speci?c antigens in the HepG2 C3A
cells. As a result, the cell extract that has passed through the
disul?de
19. Valosin-containing protein (p97); transitional endo
plasmic reticulum ATPase (gil 6005942, SEQ ID
decompose and release proteins. Centrifuging (With a table
umn volume at the rate of 1 ml/min. At this time, antigens in
the cell extract that are identi?ed and captured by the normal
antibodies Will be retained in the column. The purpose of this
Protein
(gi|1710248, SEQ ID N06).
7. Unnamed protein product (gi|21750187, SEQ ID NO.7).
8. Tropomyosin alpha 3 (gil37403, SEQ ID NO.8).
9. Trypomyosin alpha 4 (gi|10435300, SEQ ID NO.9).
14. Heat shock protein 90-beta (gil72222, SEQ ID NO.14).
15. Heat shock protein 90-alpha (gil23678, SEQ ID
medium removed With an ice-bathed Iris saline solution (50
binding buffer ten times the column volume at the rate of 1
ml/min. Then passing the ?ltered cell extract over the normal
antibody column at the rate of 0.2 ml/min. Eluting the normal
N05).
10. Calreticulin precursor (gil4757900, SEQ ID NO.10).
1 1. Human pre-mRNA splicing factor SF2p32 (gil338043,
SEQ ID NO.11).
Related Cell Lines
top centrifuge) the solution at 14,000 rpm under 40 C. for 15
minutes to remove solid, insoluble cell structures. Collecting
the supematants to carry on immunoa?inity chromatography.
After diluting the cell extract collected With the binding
buffer at the ratio of 1:10, passing it through a 0.45p. ?lter
membrane to prevent the blockage of the column in subse
2. NM23 protein (gil35068, SEQ ID N02).
3. ATP synthase beta chain, mitochondrial [precursor]
(gil28940, SEQ ID N03).
4. 14-3-3 Zeta protein (tyrosine 3/tryptophan 5-monooxy
genase activation protein) (gil4507953, SEQ ID NO.4).
5. 14-3-3 epsilon protein (tyrosine 3/tryptophan 5-mo
nooxygenase activation protein) (gil4507953, SEQ ID
6.
sure all functional groups not bound With autoantibodies are
Washing buffer tWo times the column volume each time.
Finally eluting the column With a pH neutral buffer 2-5 times
the column volume to complete the preparation of the column
By screening liver disease related cell lines With autoanti
bodies in the serum of the patients With liver cirrhosis or liver
65
The autoantigens identi?ed With the antibodies from liver
disease related cell lines are shoWn in Table 1; the left side of
the Table 1 lists the GI number and name of the proteins and
the right side indicates the autoantigens that may be identi?ed
from cell lines using sera of patients With liver cirrhosis or
liver cancer. As shoWn, those autoantigens are not just present
US 8,076,089 B2
7
8
in one liver disease, they are repeatedly identi?ed in different
cell lines using autoantibodies in sera of different sources,
indicating their close correlation With liver diseases. Some
proteins listed in Table 1 have tWo GI numbers. That is
because the protein and its variant had similar results in the
Gelatin, 0.15M NaCl, 5 mM EDTA'2Na, 0.05% Tween-20,
50 mM Tris base, or b. 1% BSA-PBS, pH:7.4, or c. 5%
non-fat milk-PBS, pH:7.4) and let blocking reaction go on
for at least 2 hours under ambient temperature; after the
reaction is completed, Washing With a PBST buffer three
times and then depositing a 100 ul/Well serum solution to be
mass spectrometry.
TABLE 2
Autoantigens screened from liver disease related cell lines
Liver cirrhosis
Liver cancer
Liver cirrhosis
Liver cancer
serum vs.
serum vs.
serum vs.
serum vs.
HepG2 C3A
HepG2 C3A
SNU-387
SNU-387
.
.
.
.
.
.
.
.
GI number Name ofprotein
1421609
28940
4507953,
Nucleoside Diphosphate Kinase (=NM23 protein)
ATP synthase beta chain, mitochondrial [Precursor]
14-3-3 protein
5 803225
1710248
Protein disul?de isomerase-related protein 5
.
21750187 Gil21750187 Unnamed protein product (RANirecimot)
37403,
Tropomyosin
.
.
10435300
4757900
Calreticulin precursor
.
.
338043
Human pre-mRNA splicing factor SF2p32, complete
.
.
1082886
Tumor necrosis factor type 1 receptor associated
.
.
4507677
protein TRAP-1
Tumor protein antigen (gp96)1; glucose regulated
.
.
Heat shock protein 90
.
.
Heat shock 60 kDa protein 1 (chaperonin);
mitochondrial matrix protein P1
HMG-l (high-mobility group-1)
KIAAO 144 gene product (NICE-4 protein)
.
Valosin-containing protein (p97); transitional
.
sequence
protein
72222,
123 678
31542947
968888
13111995
6005942
.
.
endoplasmic reticulum ATPase
30157565
Glyceraldehyde 3-phosphate dehydrogenase, liver
.
14195 64
4191608
13236495
9257073
Cytokeratin
IGF-II mRNA-binding protein 1
NADPH-quinone reductase
Crystal Structure of The Human Co-Chaperone P23
.
.
.
.
(hsp-90 co-chaperone)
EXAMPLE 2
40
Determining the Availability of Autoanti gens
Identi?ed by the Autoantigen Screening Method
To demonstrate the availability of 24 autoantigens identi
autoantibodies in the serum Will react With immobiliZed
biomarkers. After reaction for at least 2 hours under ambient
45
?ed in Example 1, further assay of serum samples from nor
mal persons, liver cirrhosis patients and liver cancer patients
using immunoassay (ELISA, RIA or immuno?uorescence)
and the aforesaid 24 biomarkers is carried out. The assay
method includes the folloWing steps as shoWn in FIG. 2:
50
providing a specimen; using the biomarker selected from any
one of the amino acid sequences With SEQ ID N011 to SEQ
30 minutes. Afterwards, adding a 100 ul/Well 0.5M HZSO4
55
60
biomarker, preferably a buffer having pH 1~2 higher than pi.
Adding 100 ul/Well biomarker solution to ELISAplate and let
it stand overnight under 40 C. for immobilization.
To continue the procedure, removing an unattached biom
arker by Washing the plate With a PBST buffer tWice (PBST
buffer: PSB buffer+0.05% Tween-20), then adding a 200
ul/Well blocking buffer (choice of a. Gelatin-NET: 0.5%
and detecting absorbance at 450 nm.
To make sure the expression of the autoantibody can be
used for diagnosis of liver cirrhosis and/or liver cancer,
ELISA is employed to obtain the absorbance values of
biomarker With a coating buffer (choice of a. 50 mM
Na2HCO3, pH:9.6, or b. 20 mM Tris-HCl, pH:8.5, or c. 10
mM PBS, pH:7.4) to a concentration of 0.5~1 0 ug/ml, Where
the coating buffer is selected according to the PI value of the
tibody. After reaction for at least 1 hour under ambient tem
Then adding in a 100 ul/Well TMB to elicit color reaction for
bination thereof to capture the autoantibody in the specimen;
In the example of enZyme-linked immunosorbent assay
(ELISA), the folloWing steps are taken: ?rstly diluting the
temperature, Washing the plate four times With the PBST
buffer and then adding in a 1000 ul/Well secondary antibody
(diluted 5000 times With the blocking buffer). At this time, the
secondary antibody Would recogniZe and adsorb the autoan
perature, Washing the plate ?ve times With the PBST buffer.
ID NO:24 or derivatives or fragments or variants or the com
and detecting the autoantibody.
assayed (a serum solution is obtained by diluting the serum
sample 1000 times With the blocking buffer). At this time, the
65
autoantibodies in the sera of normal persons, liver cirrhosis
patients and liver cancer patients as identi?ed by respective
autoantigens. The data derived from ?ve proteins-GADPH,
NADPH, HMG-l, NM23 and Cytokeratin are subject to bio
statistical analysis and Wilcoxon-Mann-Whitney Test. The
folloWing results at a 95% con?dence level as shoWn in the
table beloW are obtained:
US 8,076,089 B2
9
Normal person vs.
10
GADPH
NADPH
HMG-1
NM23
Cytokeratin
p = 0.001
p = 0.001
p = 0.00006
p = 0.0001
p = 0.001
p = 0.017
p = 0.016
p = 0.015
p = 0.002
p = 0.016
p >0.05
p > 0.05
p >0.05
p > 0.05
Liver cirrhosis patient
Normal person vs.
Liver cancer patient
Liver cirrhosis patientvs. p > 0.05
Liver cancer patient
Normal person: N= 10; liver cirrhosis patient: N = 15; liver cancer patient: N = 21 (the assumption ofp < 0.05 is valid)
mal persons and liver cirrhosis patients differed by 24 folds,
Assuming there are differences between the expressions of
biomarker-detected autoantibodies in normal persons, liver
cirrhosis patients and liver cancer patients, the table above
.
.
.
while that in normal persons and liver cancer patients differed
by 8.545 folds. These results demonstrate that the expression
15
.
.
.
.
.
.
.
shows that such assumption was vahd 1n normal persons
levels of the ant1bod1es 1n hver cirrhosis and hver cancer
versus liver cirrhosis patients and normal persons versus liver
cancer patients, meaning the differences in the expression
patients as detected by the 24 autoantigens provided herein
were higherthan those in normal persons. Thus adetection kit
levels of biomarker-detected autoantibodies between normal
using those 24 autoantigens coupled with immunoassay may
persons and liver cirrhosis patients and between normal per- 20 be applied in the screening of liver cirrhosis and liver cancer
sons and liver cancer patients are statistically signi?cant.
based on the expression levels of autoantibodies in the
Statistics shows that the expression levels of GADPHscreened specimens.
detected autoantibodies in normal persons and liver cirrhosis
The preferred embodiment of the present invention as dis
patients differed by 8.375 folds, while that in normal persons
closed above is not meant to limit this invention. All modi?
and liver cancerpatients differed by 4.86 folds; the expression
cations and alterations made by those familiar with the skill
levels of HMG-14detected autoantibodies in normal per- 25 without departing from the spirits of the invention and
sons and liver cirrhosis patients differed by 74 folds; the
appended claims shall remain within the protected scope and
expression levels of NM23-detected autoantibodies in norclaims of the invention.
SEQUENCE LISTING
<l60> NUMBER OF SEQ ID NOS :
24
<211> LENGTH: 151
<212> TYPE: PRT
<213> ORGANISM: Human
<4oo> SEQUENCE:
1
Ala Asn Leu Glu Arg Thr Phe Ile Ala Ile Lys Pro Asp Gly Val Gln
1
5
1o
15
Arg Gly Leu Val Gly Glu Ile Ile Lys Arg Phe Glu Gln Lys Gly Phe
2o
25
3o
Arg Leu Val Ala Met Lys Phe Leu Arg Ala Ser Glu Glu His Leu Lys
35
4o
45
Gln His Tyr Ile Asp Leu Lys Asp Arg Pro Phe Phe Pro Gly Leu Val
5o
55
6o
Lys Tyr Met Asn Ser Gly Pro Val Val Ala Met Val Trp Glu Gly Leu
65
7o
75
so
Asn Val Val Lys Thr Gly Arg Val Met Leu Gly Glu Thr Asn Pro Ala
s5
9o
95
Asp Ser Lys Pro Gly Thr Ile Arg Gly Asp Phe Cys Ile Gln Val Gly
1oo
1o5
11o
Arg Asn Ile Ile His Gly Ser Asp Ser Val Lys Ser Ala Glu Lys Glu
115
12o
125
Ile Ser Leu Trp Phe Lys Pro Glu Glu Leu Val Asp Tyr Lys Ser Cys
13o
135
Ala His Asp Trp Val Tyr Glu
14 5
<2lO> SEQ ID NO 2
<211> LENGTH: 180
15o
14 0
US 8,076,089 B2
11
12
—cont inued
<2l2> TYPE: PRT
<2l3> ORGANISM: Human
<400> SEQUENCE: 2
Cys Cys Glu Pro Arg Gly
Ser
Arg
Ala
1
Arg
Phe
Gly Cys Trp Arg
10
Gln Pro Glu Phe
Lys
Pro
Lys
Gln Leu Glu
Leu
15
Gly
Thr Met Ala Asn
Cys
Arg Gly
Leu
25
Glu
Val
Arg
Gly
Thr Phe Ile Ala Ile
Lys
35
40
Glu Ile Ile
Lys Arg
50
Gly
Pro
Asp Gly
45
Phe Glu Gln
Lys Gly
55
Leu
Lys
Phe Met
Val
Phe
Arg
Lys
Glu His
Leu Val
60
Ala Ser Glu
Asp
Leu Leu
65
Val
Tyr
80
Asp
Leu
Lys Asp Arg
Pro Phe Phe Ala
Gly
Leu Val
Lys Tyr
Met
95
Lys
Ser
Gly
Thr
Gly Arg
Pro Val Val Ala Met Val
100
105
Val Met Leu
115
Pro
Gly
Thr Ile
Arg Gly Asp
130
Ile His
Gly
Ser
Asp
Tyr
Leu Asn Val Val
110
Glu Thr Asn Pro Ala
120
125
Phe
Cys
Ile Gln Val
Asp
Ser
Lys
Gly Arg
Asn Ile
Glu Ile
Gly
140
Ser Val Glu Ser Ala Glu
150
155
Phe His Pro Glu Glu Leu Val
Ile
Gly
Gly
Asp Tyr
165
Trp
Glu
135
145
Trp
Trp
Lys
Thr Ser
Cys
Ala
Leu
160
Asn
170
Glu
180
<210>
<211>
<2l2>
<2l3>
SEQ ID NO 3
LENGTH: 539
TYPE: PRT
ORGANISM: Human
<400> SEQUENCE: 3
Met Thr Ser Leu
1
Arg
Trp Gly Lys Gly
Thr
Gly Cys Lys
Val Ala Ala Ala Pro Ala Ser
Gly
Lys
Ser His Pro Val
50
Ala
Gly
Arg Asp Tyr
Ala Leu
Arg Arg
Arg
Leu Thr Pro
30
Ala Val
Arg Arg
45
60
Gly Arg
Ile Val Ala Val Ile
Gly
Ala Val
65
Val
80
Asp
Val Gln Phe
Glu
Gly
Leu Pro Pro Ile Leu Asn Ala Leu
90
Glu Thr
Arg
Leu Val Leu Glu Val Ala Gln His
105
Asp
85
Glu Val Gln
Leu
Phe
Ala Ala Gln Thr Ser Pro Ser Pro
55
Ala Ala Thr
Lys
15
Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu Leu
35
Arg
Leu Phe
10
Gly
Gly Arg
Glu Ser Thr Val
Arg
115
Thr Ile Ala Met
Asp Gly
120
Leu Val
130
Arg Gly
Pro Val
145
Gly
Pro Ile
Asp
Gln
Lys
Val Leu
Asp
Ser
Gly
135
Pro Glu Thr Leu
150
Glu
Arg Gly
Thr Glu
Gly
Lys
Ile
Gly
Glu
125
Ala Pro Ile
140
Gly Arg
Pro Ile
Lys
Ile Met Asn Val Ile
Thr
155
160
Lys
Gln Phe Ala Pro Ile
US 8,076,089 B2
14
13
—cont inued
165
170
Ala Glu Ala Pro Glu Phe Met Glu Met Ser Val Glu
180
Leu Val Thr
195
Gly
Gly Gly Lys
Ile
185
Ile
Gly
210
Lys
Val Val
Leu Phe
215
Asp
200
Leu Leu Ala Pro
205
Gly Gly
Ala
Gly
Val
Gly
Val
Gly
Glu
Arg
245
Tyr His
Gly Lys
Glu Met Ile Glu Ser
Arg
Lys
Ala
Arg
Gly
Tyr Gly
Val Ala Leu Thr
290
Asp
Gly
Gln
305
Phe Thr Gln Ala
Gly
325
Ser Ala Val
Arg
Gly Tyr
Tyr
Val Ile Asn Leu
Gln Pro Thr Leu Ala Thr
Lys Lys Gly
360
Val Pro Ala
Asp Asp
Leu Thr
Tyr
Pro Ala Val
Asp
Gly
Lys
Asp Tyr Lys
Ile Leu Gln
Arg
Ala
Met
Arg Lys
Arg
Asn Ile Phe
Arg
Gly Arg
Ile Pro
335
Met
Gly
Thr Met
Asp
Pro Ala Pro Ala Thr
380
Asp
Arg
Ala Ile Ala
Ser Glu His
425
400
Asp
Ser Thr Ser
415
Tyr Asp
Asp
Ser Leu
Gly
Asp Lys
460
His Met
Ile Ile
Leu Thr Val
Phe Leu Ser Gln Pro Phe Gln Val
Gly Lys
485
Thr Ile
Lys Gly
Pro Glu
Phe Gln Gln Ile Leu Ala
Tyr
515
Ala
530
<210>
<211>
<2l2>
<2l3>
Asp Lys
Leu Val Pro Leu
Met Val
520
Gly
Lys
Glu
495
Gly
505
Ala Phe
480
490
500
Arg
445
455
Arg
Arg
430
Glu Leu Ser Glu Glu
Ile Gln
Val Ala
475
Ala Glu Val Phe Thr
Lys
Asp
440
450
Ser
465
Phe
Tyr
Ser Ile Thr Ser Val Gln
365
Pro Leu
410
Pro Asn Ile Val
420
Gly
Ala
395
435
Ala Ile Leu
Ala Thr
320
Ala Thr Thr Val Leu Ser
405
Val
Leu
345
390
Gly
Asp
Ser Glu Val Ser Ala Leu Leu
330
Asp
Asp
Asp
Gly
Leu Thr Val Ala Glu
300
315
385
Ile Met
Asn
Lys Asp
Gly
Val Leu Leu Phe Ile
Ile Thr Thr Thr
Ile
Gly
240
Gln Met Asn Gln Pro Pro
310
Thr Phe Ala His Leu
Gly
Thr Val
270
375
Glu Leu
Lys
255
Asp
355
Ala Ile
Glu
285
340
Gln Glu
Arg
280
295
Gln Glu
Thr
265
Val Ala Leu Val
Ala
Gly Gly Tyr
250
260
Ser
Tyr
220
Leu Ile Met Glu Leu Ile Asn Asn Val Ala Lys Ala His
225
230
235
Ser Val Phe Ala
Glu Ile
19O
Glu
Tyr Asp His
Leu
510
Pro Ile Glu Glu Ala Val Ala
525
Leu Ala Glu Glu His Ser Ser
535
SEQ ID NO 4
LENGTH: 245
TYPE: PRT
ORGANISM: Human
<400> SEQUENCE: 4
Met Asp Lys Asn Glu Leu Val Gln Lys Ala Lys Leu Ala Glu Gln Ala
US 8,076,089 B2
15
16
—cont inued
10
Glu
Arg Tyr Asp Asp
Met Ala Ala
Cys
Met
15
Lys
25
Gly
Ala Glu Leu Ser Asn Glu Glu
35
Lys
Asn Val Val
Ile Glu Gln
Arg
40
Lys
Gly
Ala
Thr Glu
Arg Arg
Gly
Asn Leu Leu Ser Val Ala
45
Ser Ser
Ala Glu
Ser Val Thr Glu Gln
30
Trp Arg
Lys Lys
Val Val Ser Ser
Gln Gln Met Ala
65
Glu
Tyr Arg
Glu
Lys
Ile Glu Thr Glu Leu
Lys
Lys
105
Val Phe
Tyr
Leu
115
Leu Ala Glu Val Ala Ala Gly
130
135
Tyr
Lys
Met
Asp Asp Lys Lys Gly
Leu
Gly
Lys
Ala
Glu Ala Ile Ala Glu Leu
Asp
Asp
Tyr Lys Asp
Gly Gly
Cys
185
210
Glu
Glu Met
160
200
Trp
Lys Lys
Asp
Ser Leu Ala
190
Thr Leu Ser Glu
205
Arg Asp
Asn
Glu Ala Glu Ala
Gly
Ser Thr Leu Ile Met Gln Leu Leu
215
220
Thr Ser
Gln
Leu Ala Leu Asn Phe Ser Val
170
175
Glu Ile Leu Asn Ser Pro Glu
195
Leu Thr Leu
225
Asp
155
180
Glu Ser
Ile Val
140
Glu Ala Phe Glu Ile Ser
Arg
Asp
125
165
Thr Ala Phe
Asn
95
Lys Gly Asp Tyr Tyr Arg Tyr
150
Gln Pro Thr His Pro Ile
Tyr Tyr
Cys
110
120
Ser Gln Gln Ala
145
Ile
Phe Leu Ile Pro Asn Ala Ser Gln Ala
100
Glu Ser
Arg Asp
90
Val Leu Ser Leu Leu Glu
Lys
Arg
80
85
Phe
Tyr
Thr Gln
Gly Asp
230
235
240
Glu Asn
245
<210>
<211>
<212>
<213>
SEQ ID NO 5
LENGTH: 255
TYPE: PRT
ORGANISM: Human
<400> SEQUENCE:
Met Asp Asp Arg Glu Asp
Leu Val
Tyr
Gln Ala
Lys
1
Ala Glu
Arg Tyr Asp
Glu Met Val Glu Ser Met
20
Met
Asp
Val Glu Leu Thr Val Glu Glu
Asn Val Ile
Gly
Ala
Arg Arg
Gly
Arg
Asn Leu Leu Ser Val Ala
45
Ala Ser
Trp Arg
Ile Ile Ser
55
Ser Ile Glu Gln
65
Arg
Glu
Lys
Glu Glu Asn
Lys Gly Gly
Cys Cys Asp
Gly
115
Arg Tyr
Glu
Asp Lys
Leu
Lys
80
Tyr Arg
Gln Met Val Glu Thr Glu Leu
85
Thr
Val Ala
30
40
50
Met Ile
Lys Lys
25
35
Tyr Lys
Leu Ala Glu Gln
15
Ile Leu
Glu Ser
Lys
90
Asp
Lys
Val Leu
Val Phe
Asp Lys His
Leu Ile
95
105
Leu Ile Pro Ala Ala
110
Tyr Tyr Lys
Met
120
Leu Ala Glu Phe Ala Thr
Lys Gly Asp Tyr
125
Gly
Asn
Asp Arg Lys
Glu Ala
US 8,076,089 B2
17
18
—cont inued
130
135
Ala Glu Asn Ser Leu Val Ala
145
140
Tyr Lys
Ala Ala Ser
150
Thr Glu Leu Pro Pro Thr His Pro Ile
Arg
165
170
Phe Ser Val Phe
Tyr Tyr
Leu Ala
195
Lys
Ala Ala Phe
Arg Asp
225
Gly
Lys
Asp Arg
190
Asp Asp
Ala Ile Ala Glu Leu
215
Trp
Leu Ala Leu Asn
175
185
Tyr Lys Asp
Asn Leu Thr Leu
230
Glu Gln Asn
Leu
200
Leu Ser Glu Glu Ser
210
Ile Ala Met
160
Glu Ile Leu Asn Ser Pro
180
Arg
Asp
155
Cys
Asp
Thr
205
Ser Thr Leu Ile Met Gln Leu Leu
220
Thr Ser
Glu Ala Leu Gln
Ala
Asp
245
Asp
Met Gln
235
Gly Asp Gly
Asp
Glu Asn Gln
Val Glu
Glu
240
250
255
SEQ ID NO 6
LENGTH: 421
TYPE: PRT
ORGANISM: Human
SEQUENCE: 6
Leu Tyr Ser Ser Ser
1
Asn
Asp Asp
Val Ile Glu Leu Thr Pro Ser Asn Phe
5
Arg
10
Glu Val Ile Gln Ser
Asp
20
Ala Pro
Trp Cys Gly His Cys
35
Ala Ala Thr Ala Leu
Asp Lys His His
Lys Asp
Ser Leu
Ser Leu
25
Arg
15
Trp
Leu Val Glu Phe
30
40
Leu Thr Pro Glu
45
Val Val
Lys
Gly Gly
Gln
Val
Tyr Gly
Gly
Trp Lys Lys
Ala Val
Val Gln
Gly
Asp
80
Thr Ile
Lys
Ile Phe
Gly
Ser Asn
Lys
Asn
Arg
Pro Glu
Asp Tyr
85
Gly Gly Arg
Thr
Gly
Gln Leu Val
Glu Ala Ile Val
Gln
Lys Asp Arg
Gly Arg
130
Glu Leu Thr
Asp Asp
145
Val
Trp
Leu Glu Pro Glu
180
Trp
Lys Gly Arg
Lys
Lys Gly
Gly Gly Arg
Asp
Ser Ser Ser
Gly Gly Tyr
Tyr
Ala Pro
Lys Lys Asp
Asn Val Leu
155
Asp
Ser Glu
Asp
160
Trp Cys Gly His Cys Lys
Ala Ala Ala Ala Ser Glu Val
Asn
Lys
Leu Ala Ala Val
200
Ile
Arg Gly
Ala Leu
Glu Gln Thr
19O
Asp
Ala Thr Val Asn Gln Val
205
Phe Pro Thr Ile
Lys
Ile Phe
Thr
Arg
220
Asp Tyr Asp Gly Gly Arg
230
245
Val Ile
175
Glu Ser Pro Val
Arg
Ser
140
215
Ile Val Ser
Ser
125
Asp Lys
Arg Tyr Gly
225
Asp
Ser
135
110
185
195
Leu Ala Ser
210
Ala Ala Leu Ser Ala Leu
Leu
120
Ser Phe
150
Met Val Glu Phe
165
Val
Asp
105
115
Gly Lys
Gln
95
100
Ser
Ala
Phe Pro
65
Arg
Tyr
235
Asp
Leu Phe Ser
250
Pro Glu Leu Leu Glu Ile Ile Asn Glu
Asp
Asp
Ile Ala
Ser
240
Asn Ala Pro Pro
255
Lys Arg
Thr
Cys
US 8,076,089 B2
19
20
—cont inued
260
265
Glu Glu His Gln Leu
Cys
Val Val Ala Val Leu Pro His Ile Leu
275
Thr
Gly
280
Ala Ala
Gly Arg
290
Ala
270
Asn Ser
295
Asp Lys Tyr Lys Lys Lys
Met
Tyr
Leu Glu Val Leu Leu
300
Trp Gly Trp
305
310
Gly
Ala Gln Ser Glu Leu Glu Thr Ala Leu
Leu
Trp
Gly
320
Ile
Gly Gly
330
Arg Lys
Leu
Lys Gly
Ile Asn Glu Phe Leu
Gly Arg Gly
Asp Asp
Ile
Asp
Gly
Lys
Phe Ala Leu
Arg
Glu
365
Gly Gly
Ala Phe
Trp Asp Gly Arg Asp Gly
Glu Leu
395
Leu Ser
405
Gly Lys Asp
Met
Ser Thr Ala Pro Val Gly
375
380
Pro Thr Ile Val Glu Arg Glu Pro
385
390
Pro Val Glu
Gly
Phe
335
Pro Ala Met Ala Ala Ile Asn Ala
340
345
Ser Phe Ser Glu Gln
360
Leu
Thr Glu Ala
Tyr
Leu Ser Phe
370
Lys
315
325
355
Asp
285
Asp
400
Val Glu Leu
Asp Asp
410
Leu
415
Glu Leu
420
<211> LENGTH: 687
<2l2> TYPE: PRT
<213> ORGANISM: Human
<400> SEQUENCE:
Met Val Lys Leu Ala
Lys
Ala
Gly Lys
1
Met Ala Pro Pro Pro
20
Met Ser Glu
35
Asp
Lys
Gly Asp
Asp
Ser Glu
Glu Val Glu Glu
Glu Glu
Asp Asp
Ser Ser
Gly
Lys Lys
Asp
Glu Glu
Glu Glu Val Val Ile
45
Ala Ala Ala Thr Ser Ala
55
Val Val Ser Pro Thr
Lys Lys
30
40
50
Pro
15
25
Lys Lys Gly Lys Lys
Pro
Asn Gln
10
Lys Lys
Val
Lys
Ala
60
Val Ala Val Ala Thr Pro Ala
65
Val Thr Thr Pro
Gly Lys Lys Gly
Ala Thr Pro
85
Ala Thr Pro
Gly Lys Lys Gly
Gly Lys
Lys Lys
115
Asp
Asp
Glu
Asp
Asp
Glu Glu
Ser
Glu
Glu Glu Glu
Glu
Asp Asp
Asp
Glu
Asp
Glu
Lys
Glu Glu
155
Glu
Asp Asp
Lys Gly Lys Lys
Asp Asp
Glu
160
Asp Asp
Glu
Asp Asp Asp
Ser Glu Glu Glu Ala Met Glu Thr Thr Pro
185
190
Ala Ala
195
Val Ala Glu
Lys
Val Val Pro Val
200
Asp
Glu
Glu
175
180
Ala
Asp
Ala Ala Ala Ala Ala Pro Ala
165
Asp Asp
Asp Asp
140
150
Asp
Lys
125
Asp Asp
Glu Ile Glu Pro Ala Ala Met
Ser Glu
Asp
135
145
Ala
11O
120
Ser Glu Glu
130
Lys Gly
105
Asn Ala
Ala Leu Val
95
Ala Ala Ile Pro Ala
100
Asn
Gly Lys
90
Asp
Glu Glu Glu
Lys
Ala
Lys
Glu
Asp Asp
Asn
205
Asp Asp
Glu
Asp
US 8,076,089 B2
21
22
—cont inued
210
215
Asp Asp Asp
Glu
Asp Asp
225
Glu
220
Asp Asp Asp Asp
230
Glu Glu Glu Glu Glu Glu Glu Glu Pro Val
245
Arg Lys Lys
Glu Met Ala
Lys
Gln
260
Gln
Lys
Val Glu
Glu
Asp Asp
Glu Glu
235
Lys
Lys
240
Glu Ala Pro
Gly Lys
250
255
Ala Ala Pro Glu Ala
Lys Lys
265
Gly
Thr Glu Pro Thr Thr Ala Phe Asn Leu Phe Val
280
285
Gly
Asn Leu Asn Phe Asn Lys Ser Ala Pro Glu Leu
290
295
300
Lys
Thr
Gly
Ser
305
Asp
Asp
Val
Arg
Gly
Met Thr
Val Phe Ala
Lys
Asn
Asp
Leu Ala Val Val
310
Arg Lys
Phe
Ile
Ala
Lys
Arg
Lys
Gly Tyr
Val
Ala Leu Glu Leu Thr
Gly
340
345
Leu Glu
355
Lys
Pro
Glu Leu
Thr Leu Leu Ala
Lys
335
Lys
Val Phe
Gly
Asn Glu
Lys Gly Lys Asp
Ser
Lys Lys
Glu
Arg Asp
Lys
365
Asn Leu Pro
Asp
Ala Ala Glu Ile
Ala Glu
Ser
405
Lys Gly
Lys
Thr Phe Glu Glu
Ile Ala
Asp Tyr Arg Gly Gly Lys
450
Tyr Tyr
Lys
Thr
Asn Ser Thr
455
Tyr
Ile Glu Phe
Lys
Lys
Gln
Gly
Gly
Glu
Lys Gly
Trp
Ser
Gly
Lys Gly Tyr
Lys
Cys
Asn
Lys
Lys Arg
Gly
Glu Ile
Ser Pro Asn Ala
540
Arg
Thr Leu Phe Val
Lys Gly
Leu Ser Glu
Asp
Thr
Lys
555
Glu Ser Phe
Glu Thr
Phe Asn Ser Glu Glu
Ser Val
Gly
Asp
Ala
Ser Ser
585
Lys
Lys Gly
Asp Gly
Gly Gly
Arg
Ala
Arg
Gly
Phe Val
Ala Ala
Lys
Glu Ala Met Glu
605
Lys
Pro
Phe
Gly Gly Arg Gly Gly Gly Arg Gly Gly
Phe
630
635
640
Asn
615
Lys
Phe
590
600
610
Glu
Asp Gly
560
570
595
625
Gly Gly Arg
Arg Gly
580
Lys Gly
Asp
Pro
550
Asp Arg
Glu Ile
Asn
525
565
Asp Gly
Thr
495
Ala Phe Ile Glu Phe Ala Ser Phe Glu
Leu Glu Leu
Thr Glu Glu Thr Leu
Asp
Lys
Val Pro Gln Asn
535
Ile Val Thr
Glu Ser
505
530
Ser Gln Pro Ser
545
Asp
Gln Asn Gln
490
Glu Ala Leu Asn Ser
Arg
Thr Glu Ile
460
520
Ala Ile
Thr Glu
415
Ser Ala Thr Glu Glu Thr Leu Gln
475
480
Ala Thr Phe Ile
500
Lys
Leu Val Ser
400
445
485
Ser
Tyr
Arg
395
440
Leu Val Leu Ser Asn Leu Ser
465
470
Gly Lys
Asp
425
Ser Ile Ser Leu
435
Glu Val Phe Glu
Val Thr Gln
410
420
Gly Arg
Tyr Lys
380
390
Asp
Asp
Leu
Glu Val Phe Glu
Lys Asp Gly Lys
Ala
Phe Glu Ser Ala Glu
330
375
385
Ala
Asp
360
370
Ile
320
325
Leu Glu
Ile
Val Thr Leu
Asp Trp
Ala
620
US 8,076,089 B2
23
—cont inued
Gly Gly Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly Phe Gly Gly Arg
645
650
655
Gly Arg Gly Gly Phe Gly Gly Arg Gly Gly Phe Arg Gly Gly Arg Gly
660
665
670
Gly Gly Gly Asp His Lys Pro Gln Gly Lys Lys Thr Lys Phe Glu
675
<2lO>
<211>
<2l2>
<213>
680
685
SEQ ID NO 8
LENGTH: 641
TYPE: PRT
ORGANISM: Human
<400> SEQUENCE: 8
Met Ala Gly Ile Thr Thr Ile Glu Ala Val Lys Arg Lys Ile Gln Val
1
5
1O
15
Leu Gln Gln Gln Ala Asp Asp Ala Glu Glu Arg Ala Glu Arg Leu Gln
2O
25
3O
Arg Glu Val Glu Gly Glu Arg Arg Ala Arg Glu Gln Ala Glu Ala Glu
35
4O
45
Val Ala Ser Leu Asn Arg Arg Ile Gln Leu Val Glu Glu Glu Leu Asp
5O
55
6O
Arg Ala Gln Glu Arg Leu Ala Thr Ala Leu Gln Lys Leu Glu Glu Ala
65
7O
75
8O
Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met Lys Val Ile Glu Asn
Arg Ala Leu Lys Asp Glu Glu Lys Met Glu Leu Gln Glu Ile Gln Leu
100
105
110
Glu Glu Ala Lys His Ile Ala Glu Glu Ala Asp Arg Lys Tyr Glu Glu
115
120
125
Val Ala Arg Lys Leu Val Ile Ile Glu Gly Asp Leu Glu Arg Thr Glu
130
135
140
Glu Arg Ala Glu Leu Ala Glu Ser Arg Cys Arg Glu Met Asp Glu Gln
145
150
155
160
Ile Arg Leu Met Asp Gln Asn Leu Lys Cys Leu Ser Ala Ala Glu Glu
165
170
175
Lys Tyr Ser Gln Lys Glu Asp Lys Tyr Glu Glu Glu Ile Lys Ile Leu
180
185
190
Thr Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala Glu Phe Ala Glu Arg
195
200
205
Ser Val Ala Lys Leu Glu Lys Thr Ile Asp Asp Leu Glu Asp Thr Asn
210
215
220
Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe Gly
225
230
235
240
Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu Ser
245
250
255
Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe Gly
260
265
270
Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met Ser
275
280
285
Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu Gly
290
295
300
Lys Gly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr Phe
305
310
315
320
Ser Asp Ala Cys Val His His Ile Lys Arg Arg Asp Ile Val Leu Lys
325
330
335
US 8,076,089 B2
25
26
—cont inued
Trp
Glu Leu
Gly
Glu
Gly
Ala Phe
340
Leu
Gly Lys
Val Phe Leu Ala Glu
Cys
Lys
Ala
345
Asn Leu Leu Pro Glu Gln Asp
355
360
Lys
Met Leu Val Ala Val
365
Lys
Arg
Gln
Glu Ala Ser Glu Ser Ala
370
Asp
375
Phe Gln
Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val
385
Val
390
Cys
Thr Glu
Gly Arg
Arg
Arg
Phe Phe
395
Leu Leu Ala
435
Leu Asn
420
Arg
Tyr
Met
415
Arg
Asp
Ala
Lys
Gly
Leu
Gly
Met Val
Tyr
Leu
Pro Leu Leu Met Val Phe Glu
Gly Gly
Glu
Phe Leu
Arg
Ser His
Pro
430
Val Ala Pro
Gly
440
Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala
Ala
Gly
425
Asp
450
455
Gly
Leu His Phe Val His
Gly
Gln
Pro Leu
445
Gly
460
Arg Asp
Leu Ala Thr
Arg
Asn
Cys
Leu
480
Lys
Gly Asp
Gly
Met Ser
495
Arg
465
Val
Asp
Ile
Pro Ile
Tyr
Gly
Ser Thr
500
Arg Trp
515
Thr Glu Ser
Leu Val Val
485
Asp
Thr
Tyr Gly Lys
Ile
Val
Trp
Gln Pro
545
Ile Thr Gln
Thr Met Leu
510
Tyr Arg Lys
Gly
Trp Tyr
Gln Leu Ser Asn Thr Glu Ala Ile
Val Val Leu
Trp
Glu Ile Phe
540
555
Gly Arg
Glu Leu Glu
Arg
560
Pro
Arg
Ala
565
Pro Glu Val
Tyr
Arg His
Ala Ile Met
Arg Gly Cys Trp
Leu
Gln
Arg
585
Pro
Arg
Met
Cys
Thr Pro
Gly Cys Lys
600
Leu Leu Ser Thr
Trp
Glu Pro Ser
590
Pro
Trp
Pro
605
Met Ser
Trp
Ala
610
Gly
Cys
575
580
Asn Ala Thr Ala Ser
595
Phe Thr
525
Ser Phe
535
550
Asp Cys
Gly Gly Arg
505
Met Pro Pro Glu Ser Ile Leu
520
Val
Phe
490
Asp Tyr Tyr Arg
530
Gly
400
405
Gly Asp
Glu Ala
380
Arg Gly
Pro Ala Gln
620
Gly
Val Val Ser
625
Arg
Asn Thr
630
Gly
Ala
Cys
Pro Gln His Pro
640
Lys
Leu
635
Pro
284
<2l2> TYPE: PRT
<2l3> ORGANISM: Human
<400> SEQUENCE:
Met Glu Ala Ile
l
Asn Ala Ile
Lys Lys Lys
Asp Arg
Met Gln Met Leu
10
Ala Glu Gln Ala Glu Ala
20
Glu
Lys
Leu
50
Lys Asp
Lys Gly
Asp Lys Lys
Ala Ala
30
Gln Val Glu Glu Glu Leu Thr His Leu Gln
35
40
Thr Glu
Asp
Glu Leu
Glu
15
25
Asp Lys Cys Lys
Asp Lys
Lys
45
Asp Lys Tyr
Asp
Leu
Ala Ser
Asp
Ser Glu
60
Ala Gln Glu
Lys
Leu Glu Leu Thr Glu
Lys Lys
US 8,076,089 B2
27
28
—cont inued
65
Ala Glu
Gly Asp
Glu Glu Leu
Val Ala Ala Leu Asn
Arg Arg
85
90
Asp Arg
Ala Gln Glu
100
Leu Glu Glu Ala Glu
115
Val Ile Glu Asn
Arg
Lys
Ala Ala
Arg
105
Leu Ala Thr Ala Leu Gln
110
Asp
Glu Ser Glu
Ala Met
Lys
Glu Ala
Lys Asp
Glu Glu
Met
Lys
Lys
Met Glu Ile Gln
140
Lys His
Ile Ala Glu Glu Ala
150
Glu Glu Val Ala
Arg Gly
Lys
125
135
145
Lys Tyr
95
120
130
Glu Met Gln Leu
Ile Gln Leu Val Glu
Asp Arg
155
Arg Lys
160
Leu Val Ile Leu Glu
Gly
Glu Leu
165
Glu
Arg
Ala Glu Glu
Arg
Ala Glu Val Ser Glu Leu
180
Leu Glu Glu Glu Leu
195
Ala Ala Ser Glu
Lys
Lys Tyr
210
Ile
Lys
Leu Leu Ser
225
190
Asn Val Thr Asn Asn Leu Lys Ser Leu Glu
200
205
Ser Glu
215
Asp Lys
Leu
Lys
Glu
Arg
Thr Leu
Lys
Asp
Thr Val Ala
Leu Ala Gln Ala
260
Lys
Glu Ala Glu Thr
Lys
Lys
Leu Glu
250
Arg
Ala Glu
Lys
240
Thr Ile
Asp Asp
Leu
255
Glu Glu Asn Val
Gly
Leu His Gln
270
Gly
Leu Ala Val
15
265
Gln Thr Leu Asn Glu Leu Asn
275
<210>
<211>
<212>
<213>
Glu Glu Glu
235
245
Glu Glu
Asp Lys Tyr
220
230
Phe Ala Glu
Lys Cys Gly Asp
185
Cys
Ile
280
SEQ ID NO 10
LENGTH: 417
TYPE: PRT
ORGANISM: Human
<400> SEQUENCE: 10
Met Leu Leu Ser Val Pro Leu Leu Leu Gly Leu Leu
1
10
Ala Glu Pro Ala Val
Tyr
Phe
Lys
Glu Gln Phe Leu
Asp Gly Asp Gly
25
Trp
Thr Ser
Arg Trp
Ile Glu Ser
35
Phe Val Leu Ser Ser
Gly
Lys His Lys
Ser
40
Gly Lys
50
55
Leu Gln Thr Ser Gln
Asp
Phe
Asp
Phe
Gly Lys
45
Tyr Gly Asp
Glu Glu
Lys Asp Lys
60
Ala
Arg
Phe
Tyr
Ala Leu Ser Ala Ser
65
80
Phe Glu Pro Phe Ser Asn
Lys Gly
Gln Thr Leu Val Val Gln Phe Thr
90
Val
Lys His
Glu Gln Asn Ile
95
Asp Cys Gly Gly Gly Tyr
105
Phe Pro Asn Ser Leu
Asp
Gln Thr
115
Asn Ile Met Phe
130
Asp
Pro
Asp
Ile
Leu
Ser Glu
Tyr
Lys Lys
Val
11O
Met His
Gly Asp
120
Gly
Lys
Val
125
Cys Gly
Pro
135
His Val Ile Phe Asn
Tyr Lys Gly Lys
145
150
Ile
Arg Cys Lys Asp Asp
Gly
Thr
140
Asn Val Leu Ile Asn
155
Glu Phe Thr His Leu
Tyr
Lys Asp
160
Thr Leu Ile Val
US 8,076,089 B2
29
30
—cont inued
165
Arg
Ser
Pro
Gly
Lys Asp
Asp
Asn Thr
180
Ser Leu Glu
195
Pro
Asp
175
Tyr
Asp Asp Trp Asp
Asp Asp
Lys
Pro Thr
Asp
Ser
Asp Gly
Asp
Glu
Ile
Lys
Lys Lys
Pro
Arg
Gln Ile
Tyr
Asn Phe
310
Gly
Thr Ile Phe
Asp
Tyr Asp
Ala Glu Glu Phe
Gly
340
Ala Glu
Lys
Asp Trp Asp Lys
Pro Glu
250
Asp
Asn Pro
Gln Met
Asp Asp
Val Leu
Glu
Asp
Ser Pro
300
Asp
Pro Ser
Gly
Leu
Asp
Leu
Trp
Gln
320
Gln
Trp Gly
Ala
Leu
Lys
Glu Glu Glu Glu Ala Glu
Asp
Asp
Val Thr
350
Arg
Glu Glu
365
380
Asp Lys Asp
Glu
Asp
Glu Glu
Asp
Glu Glu
395
Glu Glu Glu
Thr
Lys
Asn Glu Thr
345
390
Glu Glu
Asp Tyr Lys Gly
Asn Phe Leu Ile Thr Asn Asp Glu
330
335
Asp Lys Lys Arg Lys
385
Lys
Tyr Lys
Tyr
375
Glu
255
360
Glu Glu Glu Glu
Glu Glu
315
Lys Asp Lys
355
Lys
Asp Trp Asp
Pro Glu
240
285
Asn Pro Glu
325
Ala
Pro Glu
280
Gly
Lys
Ala
265
295
Ser
Arg
Glu Pro Pro Val Ile Gln Asn Pro Glu
Ala
Asp
Lys
Glu
220
Trp
305
Val
Asp Trp Asp
Asp
Ile His Pro Glu Ile
Ala
Ile
Pro
245
290
Tyr
Lys Lys
235
275
Trp
Phe Leu Pro Pro
205
Pro Glu
260
Trp Lys
Val Glu
Asn Ser
190
230
Glu
Asp
215
Ile Pro
Gly
Ile
200
Ala Ser
225
Met
Lys
185
210
Ile
Glu Val
Asp
Val Pro
405
Gly
Asp
400
Gln Ala
Lys Asp
410
Glu
415
Leu
<210>
<211>
<212>
<213>
SEQ ID NO 11
LENGTH: 278
TYPE: PRT
ORGANISM: Human
<400> SEQUENCE: 11
Val Leu
Gly
Ser Ser Val Ala
10
Ala Ala Ala Pro Ala Ser Pro Phe
Arg
Gln Leu Leu Gln Pro Ala Pro
Leu
1
Arg Cys Val
Pro
Arg
20
Arg
Leu
Cys
25
Gly
Leu
15
Arg
30
Thr
Arg
Pro Phe
Gly
Leu Leu Ser Val
Arg
Ala
Gly
Pro
Gly
Leu Leu
55
Arg
Pro
Arg Gly
Cys
Ala
Cys Gly
Ser
35
Glu
Arg Arg
50
Cys Gly Cys Gly
Ser Leu His Thr
Pro
60
Asp Gly Asp Lys
Ala Phe Val
65
80
Phe Leu Ser
Asp
Glu Ile
Lys
Glu Glu
85
Thr Leu Pro
Lys
Lys
115
Met Ser
Leu Val
Arg Lys
Ile Gln
Lys His Lys
90
95
Gly Gly Trp
Glu Leu Glu Leu Asn
105
110
100
Glu Ala
Asp
Arg Lys
Val Ala
120
Gly
Glu
Lys
Gly
Thr
Ile Thr Val Thr
125
US 8,076,089 B2
31
32
—cont inued
Phe Asn Ile Asn Asn Ser Ile Pro Pro Thr Phe
130
135
Pro Ser Gln
145
Gly
Gln
Lys
Val Glu Glu Gln Glu Pro Glu Leu Thr Ser
Thr Pro Asn Phe Val Val Glu Val Ile
Lys
165
170
Asp Cys His Tyr
Glu Ala Glu Ser
Asp
Asp
Gly
Asn
Asp Asp Gly Lys Lys
175
Ile Phe Ser Ile
Arg
Glu Ser Glu
Asp Trp
Thr Asn
Tyr Asp His
Val
Asp
Asn Thr Phe Ala
245
Tyr
260
Lys
Tyr
Thr Leu Asn Thr
Asp
Leu Met
235
Asp
Phe Leu Ala
240
Glu Leu Val Glu Leu Ser
250
Thr Ala Leu Glu His Gln Glu
Ser Phe Val
2'75
Glu Val Ser Phe Gln
220
230
Asp Arg Gly
Gln Glu
205
Trp Lys Asp
Ala Leu
Gly
190
215
Ser Leu
Glu Val
200
225
<210>
<211>
<212>
<213>
160
Asp
195
Ser Thr
210
155
Pro Glu
185
180
Asp
Glu Glu Glu
140
150
Ala Leu Val Leu
Asp Gly
255
Ile Thr Phe Leu Glu
Asp
265
270
Leu
Lys
Ser Gln
SEQ ID NO 12
LENGTH: 661
TYPE: PRT
ORGANISM: Human
<400> SEQUENCE: 12
Arg Ala Leu Arg Arg
Ala Pro Ala Leu Ala Ala Val Pro
Gly Gly Lys
1
Pro Ile Leu
Cys
Pro
Arg Arg
Thr Thr Ala Gln Leu
20
Asn Pro Ala
35
Ala Glu
Trp
Asp Lys
Gly
25
Ser Leu Gln Ala
40
Gly Arg
Pro
30
Arg Arg
Leu Phe Ser Thr Gln Thr
45
Glu Glu Pro Leu His Ser Ile Ile Ser Ser Thr Glu
50
55
Ser Val Gln
Gly
Ser Thr Ser
Lys His
Glu Phe Gln Ala Glu Thr
Lys
80
Lys
Leu Leu
Asp
Ser Leu
90
Tyr
Glu Leu Ile Ser Asn Ala Ser
Asp
Ile Val Ala
Arg
85
Phe Ile
Arg
Ser Glu
Lys
Glu Val
95
Ala Leu Glu
Lys
Leu
105
Arg His Lys
Leu Val Ser
Asp Gly
115
Leu Gln Thr Asn Ala Glu
130
135
Gly
Ile
Gly
Met Thr
145
Ala
125
Lys Gly
Thr Ile Thr Ile Gln
Arg
Ser
Gly
Glu Glu Leu Val Ser Asn Leu
Ser
165
Lys
Lys
Ile Ile
Ala Phe Leu
210
Ser Leu
Asp
Gly
Thr Ile
155
160
Ala Leu Gln Asn
Ala
Gly
Phe
Gly
Val
Gly
185
Asp Arg
195
Gly
Thr
170
180
Ala Phe Met Val Ala
Asp
140
150
Glu Ala Ser Ser
Pro
Gln Ala Leu Pro Glu Met Glu Ile
120
Val Glu Val
Gln
215
Trp
Tyr
Ser
19O
Tyr
Ser
200
Gly Tyr
Phe
Arg
Ser Ala Ala
205
Leu Ser
Asp Gly
220
Ser
Gly
Val Phe
US 8,076,089 B2
33
34
—cont inued
Glu Ile Ala Glu Ala Ser
225
Gly
Val
Arg
Leu
Lys
Ser
Val Val Thr
Asp Cys Lys
Gly Arg Arg
275
Lys Asp
Val
Gly
Lys Tyr
Asp Lys
Pro Leu Asn Ile
Arg
His Glu Glu Phe
Asp
Val Ser
Pro
310
Arg Tyr
Ser Ile Phe
Leu
Arg
Glu Leu
Thr Leu His
Tyr
Gly
345
Val Leu Ile Gln Thr
Lys
355
360
Phe Ile
Val Pro
Asp
Met Met
285
Tyr Arg Tyr
Tyr Lys
Thr
Arg Gly
Asp
Met
Lys
Val Val
Ala Thr
Asp
Ile Leu Pro
Arg
Arg
Asp
Ser Glu
Asp
Glu Leu Leu Gln Glu Ser Ala Leu Ile
390
Arg
Ala Glu
Leu Ile
Glu
Lys Tyr
Ala
Lys
Lys
Arg Lys
Lys
Phe Phe Ile
Asp
Phe Phe Glu
Ile Val Thr Ala Thr Glu Gln Glu Val
Leu Leu
Tyr Tyr
Lys Lys Lys
Leu
485
Arg Tyr
Cys
Tyr
Gly
Gln
Thr
Arg
Ala Ser
Ala Pro Asn
Arg
Met
475
Arg His
Lys Lys Lys Asp
Arg
Asp Arg
550
Leu Ala Glu His Ser
495
Thr Glu Val Leu Phe
Asp
Lys
Cys
Leu Ser
560
Arg
Asn Val Leu
575
Val Thr Leu
Arg
Leu
Asp
Gly
Gly
Thr His Pro
590
Ala Ala
Arg His
Phe Leu
Arg
605
Thr Gln Glu Glu
Arg
615
Pro Thr Leu Glu Ile Asn Pro
625
630
Asp His Tyr
Met
600
Lys
Asp
540
585
595
Glu Phe
Trp
Ala Met Val Thr Val Leu Glu Met
Gln Leu Ala
Arg
Ile Val Val
565
580
Cys
510
Ser Pro Ala Ala Glu
555
Glu Thr Glu Glu Leu Met Ala
Val Thr Asn Val
Gly
525
Lys
Cys
Ala
480
Glu Leu Thr Leu Leu His Leu
Phe Glu
Leu Phe
460
Glu Glu
610
Lys
Glu Ser Ser Ala Leu Pro Ser
505
545
Arg
Asp
490
Glu Ala Met
Asp
Leu
Glu
Leu Ile Ser Val Glu Thr
535
Met
Lys Trp
445
530
Arg
Ser
Lys
440
520
Ser
Tyr
430
455
Tyr Tyr
Lys
Ala
Pro Ser
335
Gln Ser
Asp Tyr Gly
Gly
Phe Glu Gln Phe
Glu
Asp
400
425
500
Lys
Val Ala
Ile Pro Leu Asn
420
Leu Thr Ser Leu Ser Glu
465
Pro
Pro
415
450
Asn Ile
Asp
395
435
Ile Ala
Leu
365
405
Met
Tyr
380
Val Leu Gln
Lys Asp
Arg
320
Ser Ser Val Ala Leu
350
370
Leu Ser
385
Trp
330
Arg
Val
315
340
Arg Lys
Arg
300
325
Met Phe
240
255
295
Ala His
Ile Ile Ile
Ser Asn Phe Val Ser Phe Pro Leu
265
270
Trp
290
305
Lys
Glu Phe Ser Ser Glu Ala
Met Asn Thr Leu Gln Ala Ile
280
Glu
Thr
250
260
Asn
Gly
235
245
Asp
Thr
230
Ala Gln Leu Leu Gln
620
Arg His
Ala Gln Ala Ser Leu Ala
Ala Leu Ile
Lys Lys
635
Trp
Leu Ser
Cys Trp Trp
Leu Asn
640
Ile
Arg