Note: Descriptions are shown in the official language in which they were submitted.
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PROTElN WHICH INTERACTS WITH T~ HUNTINGTON'S DISEASE GENE
PROD~JCT, cDNA CODING THEREFOR, AND ANTIBODIES THERETO
13ACKGRO~IN~ OF T~F~NVENTION
This application relates to a protein design~ted as HIP1 which interacts with
the ~llntin~ton's Disease gene product, cDNA coding for HIPI, and methods and
compositions relating thereto.
"Interacting proteins" are proteins which associate in vivo to forrn specific
stable complexes. Non-covalent bonds, including hydrogen bonds, hydrophobic interactions
and other molecular associations form between the proteins when two protein surfaces are
matched or have affinity for each other. This affinity or match is required for the recognition
of the two proteins, and the formation of a stable interaction. Protein-protein interactions are
involved in the assembly of en~yme subunits; in antigen-antibody reactions; in forming the
supramolecular structures of ribosomes, fil~ment~, and viruses; in transport; and in the
interaction of receptors on a cell with growth factors and hormones.
Hllntington's disease is an adult onset disorder characterized by selective
neuronal loss in discrete regions of the brain and spinal chord that lead to progressive
movement disorder, personality change and intellectual decline. From onset, which generally
occurs around age 4O, the disease progresses with worsening symptoms, ending in death
approximately 18 years after onset.
The biochemical cause of Huntington's disease has thus far not been
determined. Various theories have been advanced, but each has failed to stand up to
~0 experimental evidence designed to test its validity. For example, it was sl1ggested that the
selective neuronal loss could be attributed to restricted expression of mRN!A or proteins in
cells undergoing degeneration. No obviously altered levels of n~A transcript or protein
expression has ever been observed in HD-affected tissues, however.
While the biochemical cause of Huntington's disease has remained elusive, a
mutation in a gene within chromosome 4pl6.3 subband has been identified and linked to the
disease. This gene, referred to as the Huntington's Disease or HD gene, contains three repeat
regions, a CAG repeat region and two CCG repeat regions. Testing of H-mtington's disease
patients has shown that the CAG region is highly polymorphic, and that the number of CAG
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repeat units in the CAG repeat region is a very reliable diagnostic indicator of having inherited
the gene for ~lmtington~s disease Thus, in control individuals and in individua}s suffering
from neuropsychiatric disorders other than 1 T~lntin~tQn~s disease, the number of CAG repeats
is between 9 and 35, while in individuals suffering from ~llntin,~on~s disease the number of
CAG repeats is expanded and is 36 or greater.
The protein product encoded by the HD gene has been localized to the
cytoplasm, including to the membranes of vesicles on the brain of both normal and HD-
affected individuals. To date, no differences have been observed at either the total RNA,
mRNA or protein levels between normal and HD-affected individuals. Thus, the function
of the HD protein and its role in the pathogenesis of Huntington's Disease remain to be
elucidated.
SU~MARY OF THE INV3~NTIQN
We have now identified a protein, (lesign~ted as HIP1, that interacts
differently with the gene product of a normal (16 CAG repeat) and an expanded (>44
CAG repeat) HD gene. The HIP1 protein originally isolated from the yeast two-hybrid
screen is encoded by a 1.2 kb cDNA, devoid of stop codons, that is expressed as a 400
amino acid polypeptide. By further screening of a human frontal cortex cDNA library,
and employing the protocol for 5' Rapid Amplification of cDNA ends (RACE), a total of
4795 nucleotides (with an open reading frame of 914 amino acids) of the 10 kb message
HIP1 have been isolated to date. Expression of the HIPl protein was found to be limited
to the brain, where the interaction of the HIP1 witll the HD protein appears to be n~cess~ry
for the association of the HD protein with the membrane or specific cytoskeletalcomponents to render it functional. Because HIP1 interacts with expanded HD protein less
well than with normal length HD, introduction of additional HIP1 or overexpression of
HIP-1 can lead to increased functionality of the defective or normal HD protein.Alternatively, modified forms of the HIP1 which bind more effectively to expanded HD
could be introduced to convert the expanded HD protein into a functional molecule.
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F DF.S~IPTION OF THE ~RAWING
Fig. 1 graphically depicts the amount o~ interaction between HIPI and
}luntingtin proteins with varying lengtlls of polygl~lt~mint~ repeat.
DETA~r Fl ) DF~CI~PTION OF THE 3NVF.NTION
The HIP I protein which interacts with the HD gene product was identified
using the yeast two-hybrid systern described in US Patent No. 5,283,173 which isincorporated herein by reference. Briefly, thi~ system utilizes two chimeric genes or plasmids
expressible in a yeast host. The yeast host is selected to contain a detect7~le marker gene
having a binding site for the DNA binding domain of a transcriptional activator. The first
chimeric gene or plasmid encodes a DNA-binding domain which recognizes the binding site of
the selectable marker gene and a test protein or protein fragment. The second chimeric gene
or plasmid encodes for a second test protein and a transcriptional activation domain. The two
chimeric genes or plasmids are introduced into the host cell and expressed, and the cells are
cultivated. Expression of the detectable marker gene only occurs when the gene product of
the first chimeric gene or plasmid binds to the DNA binding domain of the cletect~ble marker
gene, and a transcriptional activation domain is brought into sufficient proximity to the DNA-
binding domain, an occurrence which is filçilit~ed by protein-protein interactions between the
first and second test proteins. By selecting for cells expressing the detectable marker gene,
those cells which contain chimeric genes or plasmids for interacting proteins can be identified,
and the gene can be recovered and identified.
In testing for Hllntington Interacting Proteins, several dirrerellt plasmids were
prepared cont~ininf~; portions ofthe Hr) gene. The first four, identified as 16pGBT9,
44pGBT9, 80pGBT9 and 128pGBT9, were GAL4 DNA binding domain-HI~ in-frame
fusions cont~ining nucleotides 314 to 1955 (amino acids 1-540) ofthe published HD cDNA
sequences cloned into the vector pGBT9 (Clontech). These plasmids contain a CAG repeat
region of 16, 44, 80 and 128 glllt~mine-encoding repeats, respectively. A clone (DMK
BamHIpGBT9) was made by fusing acDNA encoding the first 544 amino acids of the
myotonic dystrophy gene ~a gift from R. Komeluk) in-frame with the GAL4-DNA BD of
pGBT9 and was used as a negative control.
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These plasmids have been used to identify and characterize HIP I, two
additional HD-interacting proteins, HIP2 and HIP3 proteins, and can be further used for the
identification of additional interacting proteins, and for tests to refine the region on the protein
in which the interaction occurs. Thus, a first aspect of the invention is these four plasmids,
S and the use of this plasmids in identifying HD-interacting proteins. Furthermore, it will be
appreciated that the GAL4 DNA-binding and activating domains are not the only domains
which can be used in the yeast two-hybrid assay. Thus, in a broader sense, the invention
encompa~ses any chimeric genes or plasmids cont~ining nucleotides 314 to 1955 of the HD
gene together with an activating or DNA-binding domain suitable for use in the yeast one,
10 two- or three-hybrid assay for proteins critical in either binding to the HD protein or
responsible for reg~ ted ~A~ ion of the HD gene.
After introducing the plasmids into Y190 yeast host cells, trazlsforming the host
cells with an adult human brain M~tchm~kerT'" (Clontech) cDNA library coupled with a GAL4
activating domain, and selecting for the expression of two ~letect~hle marker genes to identify
15 clones cont~ining genes for interacting proteins, the activating domain plasmids were
recove~ed and analyzed. As a result of this analysis, three ~ elll cDNA fragments were
identified as encoding for ~D-interacting proteins and desi~natecl as HIP1, HIP2 and HIP3.
The sequences of ~IP I and HIP3 are given in Seq. ID. Nos 1 and 3 . The polypeptides which
each encodes are given by Seq. ID Nos. 2 and 4. Further investigation of the HIP 1 cDNA
20 resulted in the characterization of an additional region of cDNA totaling 4795 bases and a
corresponding protein, the sequences of which are given by Seq ID Nos. 5 and 67 respectively.
The cDNA molecules, particularly those encoding portions of HIP1, can be
explored using oligonucleotide probes for example for amplification and sequencing. In
addition, oligonucleotide probes complementary to the cDNA can be used as diagnostic
25 probes to localize and quantify the presence of HIPI DNA. Probes ofthis type with a one or
two base mi~m~tch can also be used in site-directed mutagenesis to introduce variations into
the HIPl sequence which may increase. Thus, a further aspect of the present invention is an
oligonucleotide probe, preferably having a length of from 15-40 bases which specifically and
selectively hyl)ridizes with the cDNA given by Seq. ID No. 1 or 5 or a sequence complemen-
30 tary thereto. As used herein, the phrase "specifically and selectively hybridizes with" the
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cDNA refers to primers which will hybridize with the cDNA under stringent hybridization
conditions
DNA sequencing of the HIP 1 cDNA initially isolated from the yeast two-hybrid
screen revealed a 1.2 kb cDNA that shows no significant degree of nucleic acid identity with
5 any stretch o~DNA using the blastn program at ncbi (blast~ncbi nlm nih.gov). When the
entire HIPI cDNA sec~uence (SEQ ID NO 5) is tr~n~l~ted into a polypeptide, the entire HIP1
cDNA coding (nucleotides 328-3069) is observed to be devoid of stop codons, and to produce
a 914 amino acid polypeptide A polypeptide identity search revealed an identity match over
the entire length of the protein (46% conservation) with that of a hypothetical protein from C
elega~7s (ZK370 3 protein; C. elega~7s cosmid ZK370). This C. elega~ls protein shares iden-
tity with the mouse talin gene, wllicll encodes a 217 kDa protein implicated with m~int~in-
ing integrity of the cytoskeleton. It also shares identity with the SLA2/MOP2/ E~ND4 gene
from Saccharomyces cerevisiae, which is known to code for an essential cytosl~eletal
associated gene required for the accumulation and or maintenance of plasma membrane H+-
ATPase on the cell surface. When pairwise comparisons are performed between HIP1 and
the C. elegans ZK370.3 protein (Genpept accession number celzk370.3), it shows 26%
complete identity and an overall 46% level of conservation. Comparative analysis between
HIPI and SLA2/MOP2/ END4 (EMBL accession number Z22811) demonstrate similar
conservation (20% identity, 40% conservation).
HIP2 is a 2.0 kb cDNA that encodes all but the 5'-most 33 amino acids of
human E225k ubiquitin conjugating enzyme. The resulting peptide has 100% identity with
the previously characterized bovine E225k protein. The cDNA has 95% nucleotide identity
with the bovine cDNA. Ubiquitin-conjugating enzyme is an important component in
ubiquitin-medi~ted protein degradation pathways.
No difference in the strength of the interaction between HIP2 and HD
constructs cont~ining either 44 or 15 CAG repeats is detected using a qll~ntit~tive ~-
galactosidase assay. The expression pattern of HIP2 (E225k) in the various parts of the
brain and nervous system appears to follow the specific neuropathology observed in HD,
although there does not appear to be any difference in expression levels between HD-
affected and HD-non-affected individuals.
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The third cDNA encoding an HD-interacting protein is a 537 bp cDNA
coding for 187 amino acids. A search of known DNA ~t~ es did not identify the
sequence homology Witll any known genes. However, when a protein search was per-formed using the blatsp server, a strong identity between HIP3 and ankyrin-related proteins
was observed. The strongest identity was with the D2021.8 gene product of C. elegans, an
uncharacterized gene, but there is also a 41% identity with AKR1, a yeast ankyrin repeat-
containing protein. Furthermore, when analogous structures with charge conservation over
the same amino acid stretch are considered, there is 70% protein identity. HIP3 also shares
approximately 60% amino acid conservation with human brain specific ankyrins (ankyrin B
and ankyrin C). Thus, it is reasonable to conclude that HIP3, like known ankyrins, is a
cytoskeletal protein, and may be involved, like previously characterized ankyrins in
promoting interactions between the membrane skeleton and other membrane proteins.
Further exploration of these three HD interacting proteins revealed several
important facts about HIPl that implicate it in a significantly in the pathogenesis of
Huntillgton's Disease. First, as shown in Fig. 1, it was found that the strength of the
interactioll between HD protein and HIP1 is dependent on the number of CAG repeats.
Second, it was found that expression of the HIP1 protein is not ubiquitous, but is limited to
brain tissue. The highest amounts of expression are in the cortex, with lower levels being
seen in the cerebellum, caudate and putamen.
Both HIP1 and HIP3 appear to be proteins which are involved in the
nl~int~ining the structural integrity of the cytoskeleton and various components of the
cellular membrane, including microtubules and focal adl1esions. Based upon this, the HD
protein may be associated as part of the cytoskeletal matrix in cells where it is expressed,
and our work supports the conclusion that binding of HIP1 to the HD protein is necessary
for the functional incorporation of the HD protein into the cell membrane. In this
circumstance, the larger polyglutamine tract in huntingtin has a decreased ability for an
HIP1-HD interaction. This decreased affinity for each other disrupts the normally strong
HD-HlP1-cytoskeletal anchoring association. Further, the HIP1-HD interaction may be a
critical interaction at the membranes of synaptic ves;cles and a decrease in the affinity of
HIP1 for huntingtin may affect protein trafficking or membrane org~ni7~tion throughout
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the neuron. Finally, we have demonstrated that HIP1 and Hl~ are both found in the Triton
X-100 insoluble membrane co~ a, L.,lent of the cell, therefore, a decreased interaction
between HIP1 and huntingtill may allow an abnormally subtle amount of huntingtin to be
found in subcellular compartments in which it is normally found.
As a result of all three of these phenomenon, increased apoptosis can occur
in specific neurons witllin the striatum. Tllis increase in apoptosis arises from an increased
susceptibility of polyglut~mine-expanded l1untingtin to cleavage by apopain, and because
more of tlle expanded forms of the HD protein may be available for cleavage (andsubsequent apoptosis) due to the fact they are not as tightly associated at the HD-~IP1-
l O cytoskeletal complex.
This understanding of a biochemical basis for the pathogenesis of
Huntington's Disease opens the doorway to a therapeutic method to ameliorate thepathology in patients expressing huntingtin protein with expanded polyglut~mint- tracts. In
accordance with the method, the patient is treated to increase the amount of HIPl or an
l 5 equivalent polypeptide which interacts less well with expanded Huntingtin than with
Huntingtin having a CAG repeat region cont~ining 15 to 35 repeats and facilitates the
incorporation of Huntingtin into brain cell membranes.
Increasing expression of HIP1 or an equivalent polypeptide can be
accomplished using gene therapy approaches. In general, tllis will involve introduction of
DNA encoding HIPI in an expressable vector into the brain cells. Vectors which have
been shown to be suitable expression systems in m~mm~lian cells include the herpes
simplex viral based vectors: p~SVl (Gelleretal. Proc. Natl. Acad. Sci 87:8950-8954
(1990));recombinantretroviralvectors: MFG (Jaffeeetal. CancerRes.53:2221-2226
(1993)); Moloney-based retroviral vectors: LN, LNSX, LNCX, LXSN (Miller and
Rosman Bio~echniques 7:980-989 (1989)); vaccinia viral vector: MVA (Sutter and Moss
Proc. Natl. Acad. Sci. 89:10847-10851 (1992)); recombinant adenovirus vectors: pJM17
(Ali et al Gene Therapy 1:367-384 (1994)), (Berkner K. L. Biotechniques 6:616-624
~ 1988); second generation adenovirus vector: DE1/DE4 adenoviral vectors (Wang and
~iner Nature Medicine 2:714-716 (1996) 3; and Adeno-associated viral vectors:
AAV/Neo (Muro-Cacho etal. J. Immunotherapy 11:231-237 ~1992)).
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Delivery of retroviral vectors to brain and nervous system tissue has been
described in US Patents Nos. 4,866,042, 5,~82,670 and 5,529,774, which are incorporated
herein by references. These patents disclose tl1e use of cerebral grafts or implants as one
mech~llicm for introducing vectors bearing therapeutic gene sequences ;nto the brain, as
well as an approach in which the vectors are transmitted across the blood brain barrier.
In addition to increasing the amount of HIP1 present in brain cells of
affected individuals, HD lethal phenotype may be rescued by coexpression of a HIPl and
normal sized HD protein within the same cell, specifically within neurons. The over-
expression of the normal HD protein and the presence of excess HIP1 in the cell may be
able to override the damaging effects of a decreased interaction between HIP1 and an
expanded form of the HD protein. Therefore, a "normal state" of interaction of HD with
HIP1 will rescue the cell from premature apoptotic death. Thus, a therapeutically desirable
m~mm~lian expression vector may include both a region encoding HIP1 and a regionencoding normal (less than 35 repeats) HD protein.
To further illustrate tlle methods of making the materials which are the
subject of this invention, and the testing which has established their utility, the following
non-limiting experimental procedures are provided.
FXAMPLI~ 1
rnENTTFlcATIoN OF INTERACTrNG PROTElNS
~AT 4-Hl) cDNA con~tructs
An HD cDNA construct (44pGBT9), with 44 CAG repeats was generated
encompassing amino acids 1 - 540 of the published HD cDNA . This cDNA fragment was
2~ fused in frame to the GAL4 DNA-binding domain (BD) of the yeast two-hybrid vector
pGBT9 (Clontech). Other HD cDNA constructs, 16pGBT9, 80pGBT9 and 128pGBT9
were constructed, identical to 44pGBT9 but included only 16, 80 or 128 CAG repeats,
respectively.
Another clone (DMKDBamHlpGBT9) Cont~inin~ the first ~44 amino acids
30 of tlle myotonic dystrophy gene (a gift from R. Korneluk) was fused in-frame with the
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GAL4-DNA i~D of pGBT9 and was used as a negative control. Plasmids expressing the
GAL4-BDRAD7 (D. Gietz, unpublished) and SIR3 were used as a positive control for the
galactosidase filter assay.
The clones IT15-23Q, IT15-44Q and HAP1 were generous gifts from Dr. C.
5 Ross. These clones represent a previously isolated huntingtin interacting protein that has a
higher affinity for the expanded form of the E~D protein.
yPzlct strains~ transformations and ~-galactosidase ~ ys
The yeast strain Y 190 (MATa leu2-3,112, ura3 -52, trp 1 -901, his3-~200,
ade2-101, gal4~gal80A, UR~3::GAL-lacZ, LYS2::GAL-HIS3,cycr) was used for all
transformations and assays Yeast transformations were performed using a modified lithium
acetate transformation protocol and grown at 30 C using appropriate synthetic complete ~SC)
dropout media.
The ,B-galactosidase chromogenic filter assays were performed by transfer-
ring the yeast colonies onto Whatman filters. The yeast cells were lysed by submerging the
filters in liquid nitrogen for 15-20 seconds. Filters were allowed to dry at room tempera-
ture for at least five minutes and placed onto filter paper presoaked in Z-buffer (100 mM
sodium phosphate (pH7.0) 10 mM KCI, I mM MgSO4) supplemented with 50 mM
2-mercaptoethanol and 0.07 mg/ml 5-bromo-4-chloro-3-indolyl ~3-D-galactoside (X-gal).
Filters were placed at 37 C for up to 8 hours.
Yeast two-hybrid screenin~ for huntin~in interactin~ protein (HIP)
cDNAs from an human adult brain M~tcllm~kerrM cDNA library (Clontech)
was transformed into the yeast strain Y190 already harboring the 44pGBT9 construct The
transformants were plated onto one hundred 150 mm x 15 mm circular culture dishes
cont~inin~; SC media deficient in Trp, Leu and His. The herbicide 3-amino-triazole (3-AT)
(25mM) was utilized to limit the number of false His+ positives (31). The yeast transformants
~ were placed at 30 C for 5 days and ,B-galactosidase filter assays were performed on all
colonies found after this time, as described above, to identify ~-galactosidase+ clones.
Primary His+/~-galactosidase+ clones were then orderly patched onto a grid on SC
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-Trp/-Leu/-His (25 7mM 3AT) plates and assayed again for His+ growth and the ability to turn
blue with a filter assay. Secondary positives were identified for filrther analysis. Proteins
encoded by positive cDNAs were designated as HIPs ~untin~tin Interactive Proteins).
Approximately 4 0 x 107 Trp/Leu auxotrophic transformants were screened and of 14 clones
isolated 12 represented the same cDNA (HIP1), and the other 2 cDNAs, HIP2 and HIP3
were each represented only once.
The ~IP cDNA plasmids were isolated by growing the His+/~-
galactosidase~ colol1y in SC -Leu media overnight, Iysing the cells with acid-washed glass
beads and electroporating the bacterial strain, KC8 (leuB auxotrophic) with the yeast Iysate.
The KC8 ampicillin resistant colonies were replica plated onto M9 (-Leu~ plates. The
plasmid DNA from M9+ colonies was transformed into DH5-a for further manipulation.
CONFIRMATION OF INTERACTIONS
The HIPI-GAL4-AD cDNA activated both the lac-Z and His reporter genes in
the yeast strain Y190 only when co-transformed with the GAIA-BD-~D construct, but not
the negative controls (Figure 1) of the vector alone or a random fusion protein of the
myotonin killase gene. In order to assess the influence of the polyglutamine tract on the
interaction between HIP1 and HD, semi-quantitative ~-galactosidase assays were
performed. GAL4-BD-HD fusion proteins with 16, 44, 80 and 128 glllt~mine repeats were
assayed for their strength of interaction with the GAL,4-AD-HIP1 fusion protein.Liquid ~3-galactosidase assays were performed by inoc~ ting a single yeast
colony into appropriate synthetic complete (SC) dropout media and grown to OD6000.6-1.5. ~ive millilitres of overnight culture was pelleted and washed once with 1 ml of
Z-Buffer, then resuspended in 100 ml Z-Buffer supplemented with 38 mM 2-mercapto-
ethanol, and 0.05 % SDS. Acid washed glass beads (-100 ml) were added to each sample
and vortexed for four minutes, by repeatedly alternating a 30 seconds vortex, with 30
seconds on ice. Each sample was pelleted and 10 ml of Iysate was added to 500 ml of Iysis
buffer. The samples were incubated in a 30 C waterbath for 30 seconds and then 100 ml of
a 4 mg/ml o-nitrophenyl b-D galactopyranoside (ONPG) solution was added to each tube.
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The reactioll was allowed to continue for 20 minutes at 30 C and stopped by the addition of
500 ml of 1 M Na2CO3 and placing tlle samples on ice. Subsequently, OD420 was taken in
order tO calculate the ~-galactosidase activity with the equation 1000 x OD420/(t x ~I x
OD600) where t is the elapsed time (minutes) and V is the amount of Iysate used.The specificity of the HIPI-HD interaction can be observed using the
chromogenic filter assay. Only yeast cells harboring HIPI and HD activate both the HIS
and lacZ reporter genes in the Y190 yeast host. The cells that contain the HIP1 with HD
constructs with 80 or 128 CAG repeats turn blue approximately 45 minutes af~er the cells
with the smaller sized repeats (16 or 44).
No difference in the ,~-galactosidase activity was observed between the 16
and 44 repeats or between the 80 and 128 repeats. However, a significant difference
(p ~ 0.05) in activity is seen between the smaller repeats (16 and 44) and the larger repeats
(80 and 128). ~Figure 1)
EXAMPLE 3
DNA SEQUENCING. cDNA l.~OLATION AND 5' RACE
Oligonucleotide primers were synthesized on an ABI PCR-mate oligo-
synthesizer. DNA sequencing was performed using an ABI 373 fluorescent automatedDNA sequencer. The HIP cDNAs were confirmed to be in-frame with the GAL4-AD by
sequencing across the AD-HIP1 cloning junction using an AD oligonucleotide (5'GAA
GAT ACC CCA CCA AAC3').
Subsequently, primer walking was used to determine the rem~in~n~
sequences. A human frontal cortex >4.0 kb cDNA library (a gift from S. Montal) was
screened to isolate the full length HIPl gene. Fifty nanograms of a 558 base pair Eco RI
fragment from the original HIP1 cDNA was radioactively labeled with [o~37P]-dCTP using
nick-translation and the probe allowed to hybridized to filters containing > 105 pfu/ml of
the cDNA library overnight at 65 C in Churcll buffer (see Northern blot protocol). The
filters were washed at 65 C for 10 minutes with 1 X SSPE, 15 minutes at 65 C with 1 X
SSPE and 0.1 % SDS, then for thirty minutes and fifteen minutes with 1 X SSPE and 0.1%
SDS. The filters were exposed to X-ray film (Kodak, XAR5) overnight at -70 C. Primary
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positives were isolated and replated and subsequent secondary positives were hybridized
and washed as for the primary screen. The resulting positive phage were eonverted into
plasmid DNA by conventional methods (Stratagene) and the eDNA isolated and sequeneed.
In order to obtain the most 5' sequenee of the HIP1 gene, a Rapid
Amplffllcation of cDNA Ends (RACE) protocol was performed aeeording to the
manufacturers reeommendations (BRL). First strand eDNA was synthesized using theoligo HIP1-242R (5' GCT TGA CAG TGT AGT CAT AAA GGT GGC TGC AGT CC
3'). After dCTP tailing the cDNA with terminal deoxy transferase, two rounds of 3~
eyeles (94 C I minute; 53 C 1 minute; 72 C 2 minutes) of PCR Usillg HIP1-R2 (5' GGA
CAT GTC CAG GGA GTT GAA TAC 3') and an anehor primer (5' (CUA)4 GGC CAC
GCG TCG ACT AGT ACG GGI IGG GII GG& IIG3') (BRL) were performed. The
subsequent 650 base pair PCR produet was cloned using the TA eloning system
(Invitrogen) and sequenced using T3 and T7 primers. Sequences ID Nos. 1 and 5 show the
sequence of t'he HIP1 cDNAs obtained.
EXAMPLE 4
DNA AND A~INO ACID ANALYSF,.~
Overlapping DNA sequence was assembled using the program MacVector
and sent via email or Netscape to the BLAST server at NIH (http://www.ncbi.nlm.nih.gov)
to search for sequence similarities with known DNA (blastn) or protein (tblastn) sequences.
Amino acid alignments were performed with the program Clustalw.
F,XAMPLE 5
FISH DETECTION SYSTEM AND IMAGE ANALYSIS
The HIP1 cDNA isolated from the two-hybrid screen was mapped by
fluorescent in situ hybridization (FISH) to normal human lymphocyte chromosomes
counterstained with propidium iodide and DAPI. Biotinylated probe was detected with
avidin-fluorescein isothiocyanate (FITC). Images of metaphase preparations were captured
by a thermoelectrically cooled charge coupled camera (Photometrics). Separate images of
DAPI banded chromosomes and FITC targeted chromosomes were obtained. Hybridization
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signals were acquired and merged using image analysis software and pseudo colored blue
(DAPI) and ~ellow (FITC) as described and overlaid electronically. This study showed
tl1at HIP 1 maps tO a single genomic locus at 7q 1 1.~ .
FX~MPLF 6
NORTHERN BLOT ANALYSIS
RNA was isolated using tlle single step method of homogenization in
guanidinium isothiocyante and fractionated on a 1.0% agarose gel cont~ining 0.6 M
formaldehyde. The RNA was transferred to a hybol1d N -membrane (Amersham) and
crosslinked with ultraviolet radiation.
Hybridization of the Northern blot with b-actin as an internal control probe
provided confirmation that the RNA was intact and had transferred. The 1.2 kb HIP1
cDNA was labeled using nick translation and incorporation of o~32P-dCTP. Hybridization
of the original 1.2 kb 1~IP1 cDNA was carried out in Church buffer (0.5 M sodiumphosphate buffer, pH 7.2, 2.7% sodium dodecyl sulphate, 1 mM EDTA) at 55 C over-night. Following hybridization, Northern blots were washed once for 10 minutes in 2.0 X
SSPE, 0.1 % SDS at room temperature and twice for 10 minutes in 0.15 X SSPE, 0.1 %
SDS. Autoradiography was carried our from one to three days using Hyperfilm
(Amersham) ~ilm at -70 C.
Analysis of the levels of RNA ievels of HIP1 by Northern blot data revealed
tllat the 10 kilo base HIP1 message is present in all tissue assessed. However, the levels of
l~NA are not uniform, with brain having highest levels of expression and peripheral tissues
having less message. No apparent differences in RNA expression was noted betweencontrol samples and HD affected individuals.
EXA~LE 7
TISSUE LOCALIZATION OF ~TTPI
'rissue localization of HIP1 was studied using a variety of techniques as
described below. Subcellular distribution of HIP-1 protein in adult human and mouse
brain Biochemical fractionation studies revealed the HIP1 protein was found to be a
,
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membrane-associated protein. No immunoreactivity was seen by Western blotting incytosolic fractions, using the anti-HIP1-pepl polyclonal antibody. HIP1 immunoreactivity
was observed in all membrane fractions including nuclei (P1~, mitochondria and synapto-
somes (P2), microsomes and plasma membranes (P3). The P3 fraction contained the most
5 HIPl compared to other membrane fractions. HIP1 could be removed from membranes by
high salt (O.SM NaCI) buffers indicating it is not an integral membrane protein, however,
since low salt (0.1- 0.25M NaCI) was only able to partially remove HIP1 from membranes,
its membrane association is relatively strong. The extraction of P3 membranes with the
non-ionic detergent, Triton X-100 revealed HIP1 to be a Triton X-100 insoluble protein.
10 This characteristic is shared by many cytoskeletal and cytoskeletal-associated membrane
protehls including actin, which was used as a control in this study. The biochemical
characteristics of HIP1 described were found to be identical in mouse and human brain and
was the same for both forms of the protein (both bands of the HIP1 doublet). HIP1
co-localized with l1l~ntingtin in the P2 and P3 membrane fractions, including the high-salt
15 membrane extractions, as well as in the Triton X-100 insoluble residue. The subce}lular
distribution of HIP1 was unaffected by the expression of polygl~lt~mine-expandedhuntingtin in transgenic mice and H~ patient brain samples.
The localization of HIP1 protein was further investigated by immunohisto-
chemistry in normal adult mouse brain tissue. Immunoreactivity was seen in a patchy,
20 reticular pattern in the cytoplasm, appeared excluded from tlle nucleus and stained most
intensely in a discontinuous pattern at tlle membrane. These results are consistent with the
association of HIP1 with the cytoskeletal matrix and further indicate an enrichment of HIP1
at plasma membranes. Immulloreactivity occurred in all regions of the b.~ain, including
cortex, striatum, cerebellum and brainstem, but appeared most strongly in neurons and
25 especially in cortical neurons. As described previously, huntingtin immunoreactivity was
seen exclusively and uniformly in the cytosol.
The in situ hybridization studies showed HIPI mRNA to be ubiquitously and
generally expressed throughout the brain. This data is consistent with the immunohisto-
chemical results and was identical to the distribution pattern of huntingtin mRNA in
30 trallsgenic mouse brains expressing full-length human huntingtin.
CA 02238075 1998-05-19
WO 97/18825 PCT/US96/18370
- 15 -
Protein Preparation And Western Blottin~ For Fxpression Studies
Frozen human tissues were homogellized using a Polytron in a buffer
cont~inin~ 0.25M sucrose, 20mM Tris-HCl (pH 7.5), 10mM E~GTA, 2mM EDTA
supplemented with lOug/ml of leupeptin, soybean trypsin inhibitor and lmM PMSF, then
centrifuged at 4,000rpm for 10' at 4 C to remove cellular debris. 100-lSOug/lane of protein
was separated on 8% SDS-PAGE mini-gels and then transferred to PVDF membranes.
Huntingtin and HIP1 were electroblotted overnight in Towbin's transfer buffer (25 mM
Tris-HCI, 0.192M glycine, pH8.3, 10~ methanol) at 30V onto PVDF membranes
(Immobilon-P, Millipore) as described (Towbin et al, Proc. Nat'l Acad. Sci. fUSA) 76:
4350-4354 (1979)). Membranes were blocked for 1 hour at room temperature in 5% skim
milk/ TBS (1nmM Tris-HCl, 0.15M NaCl, pH7.5). Antibodies against hulltingtin (pAb
BKP1, 1:500), actin (mAb A-4700, Sigma, 1:500) or HIP1 ~pAb HIP-pepl, 1:200) were
added to blocking solution for 1 hour at room temperature. After 3 x 10 minll~es washes
in TBS-T (0.05 % Tween-20/TBS), secondary Ab (horseradish peroxidase conjugated IgG,
Biorad) was applied in blocking solution for 1 hour at room temperature. Membranes were
washed and then incubated in chemilllmill~scent E~CL solution and visualized using
Hyperfilm-ECL film (Amersham).
~eneration of Antibodies
The generation of huntingtin specific antibodies GHM1 and BKP1 is des-
cribed elsewhere (Kalchman, et al., J. Biol. Chem. 271: 19385-19394 (1996)). The HIPl
peptide (VLEKDDLMDMDASQQN, a.a. 76-91 of Seq. ID No. 2) was synthesized with
Cys on the N-terminus for the coupling, and coupled to Keyhole }impet hemocyanin(KLH) (Pierce) with succinimidyl 4-(N-maleimidomethyl) cyclohexame- 1 -carboxylate
(Pierce). Female New 7~ nd White rabbits were injected with HIP1 peptide-KLH andFreund's adjuvant. Antibodies against the HIP1 peptide were purified from rabbit sera
using affinity column Wit]l low pH elution. Affinity column was made by incubation of
HIP1 peptide with activated thio-Sepharose (Pharmacia).
Western blotting of various peripheral and brain tissues were consistent with
the RNA data. The HIP1 protein levels observed was not ubiquitous. The protein
CA 0223807~ 1998-0~-l9
WO 97/18825 PCT/US96/18370
- 16-
expression is limited to brain tissue, witl1 highest arnounts seen in the cortex and lower
levels seen in the cerebellum and caudate and putamen.
More regio-specific analysis of HIP1 expression in the brain revealed no
differential expression pattern in affected individuals when compared to normal controls,
S with highest levels of expression seen in both controls and HD patients in the cortical
regions.
FXAMPI F 8
CO-IMMUNOPRECIP~T~TION OF HIPl WITH HUNTIN~TIN
Confirmation o~ the HD-HIP1 interaction was performed using coimmuno-
prepitation as follows. Control human brain (frontal cortex) Iysate was prepared in the
same manner as for subcellular localization study. Prior to immunoprecipitation, tissue
Iysate was centrifuged at 5000 rpm for 2 minutes at 4 C, then the supernatant was
pre-cleared by the incubated with excess amount of Protein A-Sepharose for 30 minutes at
15 4 C, and centrifuged at the same condition. Fifty microlitres oF supernatant (500 mg
protein) was incubated with or witl1out antil~odies (10 ug of anti-huntingtin G~M 1
(Kalchman, et al. 1996) or anti-synaptobrevin antibody) in the total 500 ul of incubation
buffer (20mM Tris-Cl (pH7.5), 40mM NaCI, lmM MgC12) for 1 hour at 4 C. Twenty
microlitres of Protein A-Sepharose ~1:1 suspension, for GHM1 and no antibody control) or
20 Protein G-Sepharose (for anti-synaptobrevin antibody; Pharmacia) was added and
incubated for 1 hour at 4 C. The beads were washed with washing buffer (incubation
buffer cont~inin~ 0.5 % Triton X-100) three times. The samples on the beads wereseparated using SDS-PAGE (7.5% acrylamide) and transferred to PVDF membrane
(Immobilon-P, Millipore). The membrane was cut at about 150 kDa after transfer for
25 Western blotting (as described above). The upper piece was probed with anti-huntingtin
BKPl (1/1000) and lower piece with anti-HlPl antibody (1/300).
The results showed that when an anti-HIP1 polyclonal antibody was
immunoreacted against a blot containing the GHM1 immunoprecipitates from the brain
lysate a doublet was observed at approximately 100 kDa was. When GHM1 was immuno-
30 reacted against the same immunoprecipitate the 350 kDa HD protein was also seen The
CA 0223807~ 1998-0~-19
WO 97/1882~; PCT/US96/18370
specificity of the HD-HIP1 interaction is seen as no immunoreactive bands seen are as a
result of the proteins adsorbing to the Protein-A-Sepharose (Lysate + No Antibody) or
when a randcm, non related antibody (Lysate + anti-Synaptobrevin) is used as theimmunoprecipitating antibody.
EXAMPT.F. 9
Subcellular fractionation of brain tissue
Cortical tissue (20-100 mg/ml) was homogenized, on ice, in a 2 ml
pyrex-teflon IKA-RW15 homogenizer (Tekmar Company) in a buffer containing 0.303Msucrose, 20mM Tris-HCl pH 6.9, lmM MgCl2, 0.5mM EDTA, lmM PMSF, lmM
leupeptin, soybean trypsin inhibitor and lmM benzamidine (Wood et al., Hun1a~tMolec.
Ge~7ef. 5: 481-487 (1996)).
Crude membrane vesicles were isolated by two cycles of a three-step differ-
ential centrifugation protocol in a Beckman TLA 120.2 rotor at 4 C based on the methods
15 of Wood et al (1996). Tlle first step precipitated cellular debris and nuclei from tissue
homogenates for 5 minutes at 1300 x g (P1). The 1300 x g supernatant was subse(luently
centrifuged for 20 minutes at 14 000 x g to isolate synaptosomes and mitocl1ondria (P2).
Finally, microsomal and plasma membrane vesicles were collected by a 35 minute
centrifugation at 142 000 x g (P3). The rem~ining supernatant was defined as the cytosolic
20 fraction.
Hi~h salt extraction of membranes
Aliquots of P3 membranes were twice suspended at 2mg/ ml in 0.5M NaCI,
10mM Tris-HCI, 2mM MgCI2, pH7.2, con~ining protease inhibitors (see above). The
25 same buffer witl1out NaCl was used as a control. The membrane suspensions were
incubated on ice for 30 minutes and then centrifuged at 142 000 x g for 30 minutes.
~ Fxt~action of cytoskelet~l and cytoskelet~ soci~t~-d proteins.
To extract cytoskeletal proteins, crude membrane vesicles from the P3
30 fraction membrane were suspended in a volume of Triton X-100 extraction buffer to give a
CA 0223807~ 1998-0~-19
WO 97/18825 PCT/US96/18370
protein: detergent ratio of 5:1. The composition of the Triton X-100 extraction buffer was
based on the methods of Arai et al., J. Neuroscience 38: 348-357 (1994) and contained
2% Triton X-100, lOmM Tris-HCI, 2mM MgCl2, lmM leupeptin, soybean trypsin
inhibitor, PMSF and benzamidine. Membrane pellets were suspended by hand with a
round-bottom teflon pestle, and placed on ice for 40 minutes. Insoluble cytoskeletal
matrices were precipitated for 35 minutes at 142 000 x g in a Beckman TLA 120.2 rotor.
The supernatant was defined as non-cytoskeletal-associated membrane or membrane--associated protein and was removed. The rem~ining pellet was extracted with Triton
X-100 a second time using the same conditions. We defined the final pellet as cytoskeletal
and cytoskeletal-associated protein.
Solubili7~ti~n of protein alld analysis by SDS-PAGE~ and Western Blotting
Membrane and cytoskeletal protein was solubilized in a minimum volume of
1% SDS, 3M urea, 0. lmM dithiothreitol in TBS buffer and sonicated. Protein concen-
tration was determined using the BioRad DC Protein assay and samples were diluted at
least 1 X with 5 X sample buffer (250mM Tris-HCI pH 6.8, 10% SDS, 25% glycerol,
0.02~ bromophenol blue and 7% 2-mercaptoethanol) and were loaded on 7.5%
SDS-PAG~ gels (Bio-Rad Mini-PROTEIN II Cell system) without boiling. Western
blotting was performed as described above.
Imnlullollistochemistry
Brain tissue was obtained from a normal C57BL/6 adult (6 months old) male
mouse sacrificed with chloroform then perfusion-fixed with 4% v/v paraformaldehyde/0.01
M phosphate buffer (4% PFA). The brain tissues were removed, immersion fixed in 4~
PFA for 1 day, washed in 0.01M phosphate buffered saline, pH 7.2 (PBS) for 2 days, and
then equilibrated in 25% w/v sucrose PBS for 1 week. The sam~les were then snap-frozen
in Tissue Tek molds by isopentane cooled in liquid nitrogen. After warming to -20 C,
frozen blocks derived from frontal cortex, c~ te/putamen, cerebellum and brainstem
were cut into 14 mm sections for immunohistochemistry. Following washing in PBS, the
tissue sections were blocked using 2.5~ v/v normal goat serum for 1 hour at room
CA 0223807~ 1998-0~-19
WO 97/18825 PCT/US96/18370
- 19-
temperature. Primary antibodies diluted witll PBS were applied to sections overnigllt at 4
C. Optimal dilutions for the polyclonal antibodies BKP1 and HIP1 were 1:50. Using
washes of 3 x 5 mhlutes in PBS at room temperature, sections were sequentially incubated
with biotinylated secondary antibody and then an avidin-biotin complex reagent (Vecta
Stain ABC Kit, Vector) for 60 minutes each at room temperature. Color was developed
using 3-3'-diaminobenzidine tetrahydrocholoride and ammonium nickel sulfate.
For controls, sections were treated as described above except that HIP1
antibody aliquots were preabsorbed with an excess of HIPl peptide as well as a peptide
unrelated to HIP1 prior to incubation with the tissue sections.
In situ hybridization
In situ hybridization was performed as previously described with some
modification ~Suzuki et al, BBRC219: 708-713 (1996)). The RNA probes were prepared
using the plasmid gtl49 (Lin, B., et al., Human Molec. Genet. 2: 1541-1545 (1994)) or a
558 subclone of HIPl. The anti-sense and sense single-stranded RNA probes were
synthesized using T3 and T7 RNA polymerases and the In Vitro Transcription Kit
(Clontech) witll the addition of [o~35S]-CTP (Amersham) to the reaction mixture. Sense
RNA probes were used as negative controls. For HIP1 studies normal C57BL/6 mice were
used. Hulltingtin probes were tested on two different transgenic mouse strains expressing
full-length huntingtin, cDNA HD10366(44CAG) C57BL/6 mice and YAC
HD10366(18CAG) FVB/N mice. Frozen brain sections (lOum thick) were placed onto
silane-coated slides under RNase-free conditions. The hybridization solution contained
4~)% w/v formamide, 0.02M Tris-HCI (pH 8.0), 0.005M EDTA, 0.3 M NaCI, 0.01M
sodium phosphate (pH 7.0), lx Denhardt's solution, 10% w/v dextran sulfate (pH 7.0),
0.2% w/v sarcosyl, yeast tRNA (50Qmg/ml) and salmon sperm DNA (200mg/ml). The
radiolabelled RNA probe was added to the hybridization solution to give 1 x 106 cpm/200
ul/ section. Sections were covered with hybridization solution and incubated on
~ formamide paper at 65 C for 18 hours. After hybridization, the slides were washed for 30
minutes se~uentially witll 2x SSC~, lx SSC and high stringency wash solution (50%
formamide, 2x SSC and 0.1M dithiothreitol) at 65 C, followed by treatment with Rnase A
CA 02238075 1998-05-19
WO 97/18825 PCT/US96/18370
- 20 -
(lmg/ml) at 37 C for 30 minutes, then washed again and air-dried. The siides were first
exposed on autoradiographic film (b-max, Amersham, UK) for 4~ hours and developed for
4 minutes in Kodak D-19 followed by a 5 minute fixation in Fuji-fix. For longer
exposures, the slides were dipped in autoradiographic emulsion (50% w/v in distilled
5 water, NR-2, Konica, Japan3, air-dried and exposed for 20 days at 4 C then developed as
described. Sections were counterstained with methyl green or Giemsa solutions.
CA 02238075 1998-05-l9
WO 97/18825 PCT/US96/18370
- 2t -
SEQUENCF T ~ TlNG
(I) GENERAL INFORMATION:
(i) APPLICANT: Kalchman, Michael
Goldberg, Paul
Hayden. Michael R.
(ii) TrTLE OF INVENTION: Protein Which Interacts with the H-lntin~ton's Disease Gene
Product, cDNA Coding Therefor, and Antibodies Thereto
(iii) NUMBER OF SEQUENCES: 8
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Oppedahl & Larson
(B) STREET: 1992 Commerce Street Suite 309
(C) CITY: Yorktown
(D) STATE: NY
(E) COUNTRY: USA
(F) ZIP: 10598
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.50 inch, 1.44 Kb storage
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: MS DOS 5.0
(D) SOFTWARE: WordPerfect
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Larson, Marina T.
(B) REGIST~ATION NUMBER: 32038
(C) REFERENCE/DOCKET NUMBER: UBC.P-013
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (914) 245-3252
(B) TELEFAX: (914) 962-4330
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 1164
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: cDNA
(iii) ~YPOTHETICAL: no
(iv) ANTI-SENSE: no
(vi~ ORIGINAL SOURCE:
(A) ORGANISM: human
(ix) FEATURE: cDNA for HllntinFtin-interacting protein
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CA 02238075 lsss-05-ls
W097/18825 PC~S96/18370
ACAGCTGACA CCCTGCAAGG CCACCGGGAC CGCTTCATGG AGCAGTTTAC 50
AAAGTTGAAA GATCTGTTCT ACCGCTCCAG CAACCTGCAG TACTTCAAGC 100
GGGTCATTCA GATCCCCCAG CTGCCTGAGA ACCCACCCAA CTTCCTGCGA 150
GCCTCAGCCC TGTCAGAACA TATCAGCCCT GTGGTGGTGA TCCCTGCAGA 200
GGCCTCATCC CCCGAC~GCG AGCCAGTCCT AGAGAAGGAT GACCTCATGG 250
ACATGGATGC CTCTCAGCAG AATTTATTTG ACA~CAAGTT TGATGACNTC 300
TTTGGCAGTT CATCCAGCAG TGATCCCTTC AATTTCAACA GTCAAAATGG 35O
TGTGAACAAG GATGAGAAGG ACCACTTAAT TGAGCGACTA TACAGAGAGA 400
TCAGTGGATT GAAGGCACAG CTAGAAAACA TGAAGACTGA GAGCCAGCGG 450
GTTGTGCTGC AGCTGAAGGG CCACGTCAGC GAGCTGGAAG CAGATCTGGC 500
CGAGCAGCAG CACCTGCGGC AGCAGGCGGC CGACGACTGT GAATTCCTGC 550
GGGCAGAACT GGACGAGCTC AGGNGGCAGC GGGAGGACAC CGAGAAGGCT 600
CAGCGGAGCC TGTCTGAGAT AGAAAGGAAA GCTCAAGCCA ATGAACAGCG 650
ATATAGCAAG CTAAAGGAGA AGTACAG~A GCTGGTTCAG AACCACGCTG 700
ACCTGCTGCG GAAGAATGCA GAGGTGACCA AACAGGTGTC CATGGCCAGA 750
CAAGCCCAGG TAGATTTGGA ACGAGAGAAA AAAGAGCTGG AGGATTCGTT 800
GGAGCGCATC AGTGACCAGG GCCAGCGGAA GACTCAAGAA CAGCTGGAAG 850
TTCTAGAGAG CTTGAAGCAG GAACTTGGCA CAAGCCAACG GGAGCTTCAG 9OO
GTTCTGCAAG GCAGCCTGGA AACTTCTGCC CAGT QGAAG CAAACTGGGC 950
AGCCGAGTTC GCCGAGCTAG AGAAGGAGCG GGACAGCCTG GTGAGTGGCG 1000
CAGCTCATAG GGAGGAGGAA TTATCTGCTC TTCGGAAAGA ACTGCAGGAC 1050
ACTCAGCTCA AACTGGCCAG CACAGAGGAA TCTATGTGCC AGCTTGCCAA llOO
AGACCAACGA A~AATGCTTC TGGTGGGGTC CAGGAAGGCT GCGGAGCAGG 1150
TGATACAAGA CGCG 1164
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) l_ENGTH: 386
(B) T~PE: protein
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: protein .
(iii) HYPOTHETICAL: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: human
(ix) FEATURE: l~lntin~;tin-interacting protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Thr Ala Asp Thr Leu Gln Gly His Arg Asp Arg Phe Met Glu Gln
1 5 10 15
~he Thr Lys Leu Lys A~p Leu Phe Tyr Arg Ser Ser Asn Leu Gln
~yr Phe Lys Arg Val Ile Gln Ile Pro Gln Leu Pro Glu Asn Pro
~ro Asn Phe Leu Arg Ala Ser Ala Leu Ser Glu His Ile Ser Pro
Val Val Val Ile Pro Ala Glu Ala Ser Ser Pro Asp Ser Glu Pro
CA 0223807~ 1998-0~-19
WO 97/18825 PCT/US96/1837Q
- 23 -
Val Leu Glu Lys Asp Asp Leu Met Asp Met Asp Ala Ser Gln Gln
Asn Leu Phe Asp Asn Lys Phe Asp Asp Phe Gly Ser Ser Ser Ser
100 105
Ser Asp Pro Phe Asn Phe Asn Ser Gln Asn Gly Val Asn Lys Asp
110 115 120
Glu Lys A~p His Leu Ile Glu Arg Leu Tyr Arg Glu Ile Ser Gly
125 130 135
Leu Lys Ala Gln Leu Glu Asn Met Lys Thr Glu Ser Gln Arg Val
140 145 150
Val Leu Gln Leu Lys Gly His Val Ser Glu Leu Glu Ala Asp Leu
155 160 165
Ala Glu Gln Gln His Leu Arg Gln Gln Ala Ala Asp Asp Cys Glu
170 175 180
Phe Leu Arg Ala Glu Leu Asp Glu Leu Arg Gln Arg Glu Asp Thr
185 190 195
Glu Lys Ala Gln Arg Ser Leu Ser Glu Ile Glu Arg Lys Ala Gln
200 205 210
Ala Asn Glu Gln Arg Tyr Ser Lys Leu Lys Glu Lys Tyr Ser Glu
215 220 225
Leu Val Gln Asn His Ala Asp Leu Leu Arg Lys Asn Ala Glu Val
230 235 240
Thr Lys Gln Val Ser Met Ala Arg Gln Ala Gln Val Asp Leu Glu
245 250 255
Arg Glu Lys Lys Glu Leu Glu Asp Ser Leu Glu Arg Ile Ser Asp
260 265 270
Gln Gly Gln Arg Lys Thr Gln Glu Gln Leu Glu Val Leu Glu Ser
275 280 285
Leu Lys Gln Glu Leu Gly Thr Ser Gln Arg Glu Leu Gln Val Leu
290 295 300
Gln Gly Ser Leu Glu Thr Ser Ala Gln Ser Glu Ala Asn Trp Ala
305 310 315
Ala Glu Phe Ala Glu Leu Glu Lys Glu Arg Asp Ser Leu Val Ser
320 325 330
Gly Ala Ala His Arg Glu Glu Glu Leu Ser Ala Leu Arg Lys Glu
335 340 345
CA 02238075 l998-05-l9
W097/18825 PC~S96/18370
- 24 -
Leu Gln A~p Thr Gln Leu Lys Leu Ala Ser Thr Glu Glu Ser Met
350 355 36Q
Cys Gln Leu Ala Lys A~p Gln Arg Lys Met Leu Leu Val Gly Ser
365 370 375
Arg Lys Ala Ala Glu Gln Val Ile Gln Asp Ala
380 385 386
(2) rNFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(vi~ ORIGINAL SOURCE:
(A) ORGANISM: human
(ix) FEATURE: cDNA for H~ntingtin-interacting protein
(xi)SEQUENCE DESCRIPTION: SEQ ID NO:3:
ACCGATACCG AAGCGGGCTG TGTGCCCCTT CTCCACCCAG AGGAAATCAA 50
ACCCCAAAGC CATTATAACC ATGGATAT~-G TGAACCTCTT GGACGGAAAA lOO
CTCATATTGA TGATTACAGC ACATGGGACA TAGTCAAGGC TACACAATAT 150
GGAATATATG AACGCTGTCG AGAATTGGTG GAAGCAGGTT ATGATGTACG 2Q0
GCAACCGGAC AAAGAAAATG TTACCCTCCT CCATTGGGCT GCCATCAATA 250
ACAGA~TAGA TTTAGTCAA~ TACTATATTT CGA~AGGTGC TATTGTGGAT 300
CAACTTGGAG GGGACCTGAA TTC'AACTCCA TTGCACTGGG ACACAAGACA 350
AGGCCATCTA TCCATGGTTG TGCAACTAAT GAAATATGGT GCAGATCCTT 400
CATTAATTGA TGGAGAAGGA TGTAGCTGTA TTCATCTGGC TGCTCAGTTC 450
GGACATACCT CAAll~Ll~C TTATCTCATA GCAAAAGGAC AGGATGTG 498
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A)LENGTH: 154
(B) TYPE: protein
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: protein
(iii) HYPOTHETICAL: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: human
(ix) FEATURE: E~llntin~tin-interacting protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CA 02238075 l998-05-l9
WO 97/18825 PCT/US96/18370
- 25 -
Thr Asp Thr Glu Ala Gly Cys Val Pro Leu Leu His Pro Glu Glu
Ile Lys Pro Gln Ser His Tyr Asn His Gly Tyr Gly Glu Pro Leu
Gly Arg Lys Thr His Ile Asp Asp Tyr Ser Thr Trp Asp Ile Val
Lys Ala Thr Gln Tyr Gly Ile Tyr Glu Arg Cys Arg Glu Leu Val
Glu Ala Gly Tyr Asp Val Arg Gln Pro Asp Lys Glu Asn Val Thr
Leu Leu His Trp Ala Ala Ile Asn Asn Arg Ile Asp Leu Val Lys
Tyr Tyr Ile Ser Lys Gly Ala Ile Val Asp Gln Leu Gly Gly Asp
100 105
Leu Asn Ser Thr Pro Leu His Trp Asp Thr Arg Gln Gly His Leu
110 115 120
Ser Me~ Val Val Gln Leu Met Lys Tyr Gly Ala Asp Pro Ser Leu
125 130 135
Ile ASp Gly Glu Gly Cys Ser Cys Ile His Leu Ala Ala Gln Phe
140 145 150
~ly His Thr Ser
154
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4846
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(vi) ORIGINAL SOVRCE:
(A) ORGANISM: human
(ix) FEATURE: cDNA for Hllntingtin-interacting protein
(xi)SEQUENCE DESCRIPTION: SEQ ID NO:S:
CAGTGTACGG TTGATCATAT AACGCCGCGG GCGGGGATTG GTTTATATAT 50
CGCAAATTGA TNTAGGGGGG GGGGGATGGN CAGAGATTTC GCTTCATTAG 100
GCCATTATAA GCAGGAAGGG TTTCAAGGAA AAAAACCCAG A~AGTGCATA 150
TTGCACCCAC CATGAGAAAG GGGCAACAGA C~ N TGTTNTCAAC 200
CA 0223807~ l998-0~-l9
WO97/18825 PCT~S96/18370
- 26 -
CGCCTGCTTC ~ lAGCA ACGCAGTGTT TTGGTGGAAG TTGTGCCATG 250
TGTTCCACAA ANTCTTCCGA GATGGACACC CGAACGTCCT GAAGGACTTT 300
GTGAGATACA GAAATGAATT GAGTGACATG AGCAGGATGT GGGGCCACCT 350
GAGCGAGGGG TATGGCCAGC TGTGCAGCAT CTACCTGAAA CTGCTAAGAA 400
CCAAGATGGA GTACCACACC AA~AATCCCA GGTTCCCAGG CAACCTGCAG 450
ATGAGTGACC GCCAGCTGGA CGAGGCTGGA GAAAGTGACG TGAACAACTT 500
TTTCCAGTTA ACAGTGGAGA T~lLl~ACTA CCTGGAGTGT GAACTCAACC 550
TCTTCCAAAC AGTATTCAAC TCCCTGGACA TGTCCCGCTC TGTGTCCGTG 600
ACGGCAGCAG GGCAGTGCCG CCTCGCCCCG CTGATCCAGG TCATCTTGGA 650
CTGCAGCCAC CTTTATGACT ACACTGTCAA GCTTCTCTTC AAACTCCACT 700
CCTGCCTCCC AGCTGACACC CTGCAAGGCC ACCGGGACCG CTTCATGGAG 750
CAGTTTACAA AGTTGAAAGA TCTGTTCTAC CGCTCCAGCA ACCTGCAGTA 800
CTTCAAGCGG CTCATTCAGA TCCCCCAGCT GCCTGAGAAC CCACCCAACT 850
TCCTGCGAGC CTCAGCCCTG TCAGAACATA TCAGCCCTGT GGTGGTGATC 900
CCTGCAGAGG CCTCATCCCC CGACAGCGAG CCAGTCCTAG AGAAG~TGA 950
CCTCATGGAC ATGGATGCCT CTCAGCAG~A TTTATTTGAC AACAAGTTTG 1000
ATGACATCTT TGGCAGTTCA TTCAGCAGTG ATCCCTTCAA TTTCAACAGT 1050
CAAAATGGTG TGAACAAGGA TGAGAAGGAC CACTTAATTG AGCGACTATA 1100
CAGAGAGATC AGTGGATTGA AGGCACAGCT AGAAAACATG AAGACTGAGA 1150
GCCAGCGGGT TGTGCTGCAG CTGAAGGGCC ACGTCAGCGA GCTGGAAGCA 1200
GATCTGGCCG AGCAGCAGCA CCTGCGGCAG CAGGCGGCCG ACGACTGTGA 1250
ATTCCTGCGG GCAGAACTGG ACGAGCTCAG GAGGCAGCGG GAGGACACCG 1300
AGAAGGCTCA GCGGAGCCTG TCTGAGATAG AAAGGAAAGC TCAAGCCAAT 1350
GAACAGCGAT ATAGCAAGCT AAAGGAGAAG TACAGCGAGC TGGTTCAGAA 1400
CCACGCTGAC CTGCTGCGGA AGAATGCAGA GGTGACCAAA CAGGTGTCCA 1450
TGGCCAGACA AGCCCAGGTA GATTTGGAAC GAGAGAAAAA AGAGCTGGAG 1500
GATTCGTTGG AGCGCATCAG TGACCAGGGC CAGCGGAAGA CTCAAGAACA 1550
GCTGGAAGTT CTAGAGAGCT TGAAGCAGGA ACTTGGCACA AGCCAACGGG 1600
AGCTTCAGGT TCTGCAAGGC AGCCTGGAAA CTTCTGCCCA GTCAGAAGCA 1650
AACTGGGCAG CCGAGTTCGC CGAGCTAGAG AAGGAGCGGG ACAGCCTGGT 1700
GAGTGGCGCA GCTCATAGGG AGGAGGAATT ATCTGCTCTT CGGAAAGAAC 1750
TGCAGGACAC TCAGCTCAAA CTGGCCAGCA CAGAGGAATC TATGTGCCAG 1800
CTTGCCAAAG ACCAACGAAA AATGCTTCTG GTGGGGTCCA GGAAGGCTGC 1850
GGAGCAGGTG ATACAAGACG CCCTGAACCA GCTTGAAGAA CCTCCTCTCA 19oO
TCAGCTGCGC TGGGTCTGCA GATCACCTCC TCTCCACGGT CACATCCATT 1950
TCCAGCTGCA TCGAGCAACT GGAGA~AAGC TGGAGCCAGT ATCTGGCCTG 2000
CCCAGAAGAC ATCAGTGGAC TTCTCCATTC CATAACCCTG CTGGCCCACT 2050
TGACCAGCGA CGCCATTGCT CATGGTGCCA CCACCTGCCT CAGAGCCCCA 2100
CCTGAGCCTG CCGACTCACT GACCGAGGCC TGTAAGCAGT ATGGCAGGGA 2150
AACCCTCGCC TACCTGGCCT CCCTGGAGGA AGAGGGAAGC CTTGAGAATG 2200
CCGACAGCAC AGCCATGAGG AACTGCCTGA GCAAGATCAA GGCCATCGGC 2250
GAGGAGCTCC TGCCCAGGGG ACTGGACATC AAGCAGGAGG AGCTGGGGGA 2300
CCTGGTGGAC AAGGAGATGG CGGCCACTTC AGCTGCTATT GAAACTTGCA 2350
CGGCCAGAAT AGAGGAGATG CTCAGCAAAT CCCGAGCAGG AGACACAGGA 2400
GTCAAATTG~ AGGTGAATGA AAGGATCCTT CGTTGCTGTA CCAGCCl'CAT 2450
GCAAGCTATT CAGGTGCTCA TCGTGGCCTC TAAGGACCTC CAGAGAGAGA 2500
TTGTGGAGAG CGGCAGGGGT ACAGCATCCC CTA~AGAGTT TTATGCCAAG 2550
AACTCTCGAT GGACAGAAGG ACTTATCTCA GCCTCCAAGG CTGTGGGCTG 2600
GGGAGCCACT GTCATGGTGG ATGCAGCTGA TCTGGTGGTA CAAGGCAGAG 2650
GGAAATTTGA GGAGCTAATG GTGTGTTCTC ATGAAATTGC TGCTAGCACA 2700
GCCCAGCTTG TGGCTGCATC CAAGGTGAAA GCT~-~T~GG ACAGCCCCAA 2750
CCTAGCCCAG CTGCAGCAGG CCTCTCGGGG AGTGAACCAG GCCACTGCCG 2800
G~ll~lGGC CTCAACCATT TCCGGCAAAT CACAGATCGA AGAGACAGAC 2850
AACATGGACT TCTCAAGCAT GACGCTGACA CAGATCAAAC GCCAAGAGAT 2900
CA 02238075 1998-05-19
WO97/18825 PCT~S96/18370
- 27 -
GGATTCTCAG GTTAGGGTGC TAGAGCTAGA AAATGAATTG CAGAAGGAGC 2950
GTCAAAAACT GGGAGAGCTT CGGAAAAAGC ACTACGAGCT TGCTGGTGTT 3000
GCTGAGGGCT GGGAAGAAGG AACAGAGGCA TCTCCACCTA CACTGCAAGA 3050
AGTGGTAACC GAAAAAGAAT AGAGCCAAAC CAACACCCCA TATGTCAGTG 3100
TAAATCCTTG TTACCTATCT CGT~l~'l'~'l"l ATTTCCCCAG CCACAGGCCA 3150
AATCCTTGGA GTCCCAGGGG CAGCCACACC ACTGCCATTA CCCAGTGCCG 3200
AGGACATGCA TGACACTTCC CAAAGATCCC TCCATAGCGA CACCCTTTCT 3250
GTTTGGACCC ATGGTCATCT CTGTTCTTTT CCCGCCTCCC TAGTTAGCAT 3300
CCAGGCTGGC CAGTGCTGCC CATGAGCAAG CCTAGGTACG AAGAGGGGTG 3350
GTGGGGGGCA GGGCCACTCA ACAGAGAGGA CCAACATCCA GTCCTGCTGA 3400
CTATTTGACC CCCACAACAA TGGGTATCCT TAATAGAGGA GCTGCTTGTT 3450
~lll~llGAC AGCTTGGAAA GGGAAGATCT TATGCCTTTT CTTTTCTGTT 3500
TTCTTCTCAG TCTTTTCAGT TTCATCATTT GCACAAACTT GTGAGCATCA 3550
GAGGGCTGAT GGATTCCAAA CCAGGACACT ACCCTGAGAT CTGCACAGTC 3600
AGAAGGACGG CAGGAGTGTC CTGG~l~'l'~A ATGCCAAAGC CATTCTCCCC 3650
CTCTTTGGGC AGTGCCATGG ATTTCCACTG CTTCTTATGG TGGTTGGTTG 3700
G~l"l"L'l"l"l'~G TTTT~'l-llll -l-llL-l-llAAG TTTCACTCAC ATAGCCAACT 3750
CTCCCAAAGG GCACACCCCT GGGGCTGAGT CTCCAGGGCC CCCCAACTGT 3800
GGTAGCTCCA GCGATGGTGC TGCCCAGGCC TCTCGGTGCT CCATCTCCGC 3850
CTCCACACTG ACCAAGTGCT GGCCCACCCA GTCCATGCTC CAGGGTCAGG 3900
CGGAGCTGCT GAGTGACAGC TTTCCTCAAA AAGCAGAAGG AGAGTGAGTG 4000
CCTTTCCCTC CTAAAGCTGA ATCCCGGCGG AAAGCCTCTG TCCGCCTTTA 4050
CAAGGGAGA~ GACAACAGAA AGAGGGACAA GAGGGTTCAC ACAGCCCAGT 4100
TCCCGTGACG AGGCTCAAAA ACTTGATCAC ATGCTTGAAAT GGAGCTGGTG 4150
AGATCAACAA CACTACTTCC CTGCCGGAAT GAACTGTCCG TGAATGGTCT 4200
CTGTCAAGCG GGCCGTCTCC CTTGGCCCAG AGACGGAGTG TGGGAGTGAT 4250
TCCCAACTCC TTTCTGCAGA CGTCTGCCTT GGQTCCTCT TGAATAGGAA 4300
GATCGTTCCA CTTTCTACGC AATTGACAAA CCCGGAAGAT CAGATGCAAT 4350
TGCTCCCATC AGGGAAGAAC CCTATACTTG GTTTGCTACC CTTAGTA'l'll' 4400
ATTACTAACC TCCCTTAAGC AGQA QGCC TA QAAGAGA TGCTTGGAGC 4450
AATCAGAACT TCAGGTGTGA CTCTAGCAAA GCTCATCTTT CTGCCCGGCT 4500
ACATCAGCCT TCAAGAATCA GAAGAAAGCC AAGGTGCTGG A~'l'~'l'-lACTG 4550
ACTTGGATCC CAAAGQAGG AGAT QTTTG GAGCTCTTGG GTCAGAGAAA 4600
ATGAGAAAGG ACAGAGCCAG CGGCTCCAAC TCCTTTCAGC CACATGCCCC 4650
AGGCTCTCGC TGCCCTGTGG AC~GGATGAG GACAGAGGGC ACATGAACAG 4700
CTTGCCAGGG ATGGGCAGCC CAA QGCACT TTTCCTCTTC TAGATGGACC 4750
CCAG QTTTA AGTGACCTTC TGATCTTG~G AAAACAGCGT CTTCCTTCTT 4800
TATCT~T~GC AACTCATTGG TGGTAGCCAT CAAGCACTTC GGAATT 4846
(2) INFORMATION FOR SEQ ID NO:6
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 924
(B) TYPE: protein
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: protein
(iii) HYPOTHETICAL: no
(vi) ORIGrNAL SOURCE:
(A) ORGANISM: human
(ix) FEATURE: ~llntinntin-interacting protein
(xi) SEQUE~CE DESCRIPTION: SEQ ID NO:6:
CA 0223807~ Isss-o~-l9
WO97/18825 PCT~S96/18370
- 28 -
Met Ser Arg Met Trp Gly His Leu Ser Glu Gly Tyr Gly Gln Leu
1 5 10 15
Cys Ser Ile Tyr Leu Lys Leu Leu Arg Thr Lys Met Glu Tyr His
Thr Lys Asn Pro Arg Phe Pro Gly Asn Leu Gln Met Ser Asp Arg
Gln Leu Asp Glu Ala Gly Glu Ser Asp Val Asn Asn Phe Phe Gln
Leu Thr Val Glu Met Phe Asp Tyr Leu Glu Cys Glu Leu Asn Leu
Phe Gln Thr Val Phe Asn Ser Leu Asp Met Ser Arg Ser Val Ser
Val Thr Ala Ala Gly Gln Cys Arg Leu Ala Pro Leu rle Gln Val
100 105
Ile Leu Asp Cys Ser His Leu Tyr Asp Tyr Thr Val Lys Leu Leu
110 115 120
Phe Lys Leu His Ser ~ys Leu Pro Ala Asp Thr Leu Gln Gly His
125 130 135
Arg Asp Arg Phe Met Glu Gln Phe Thr Lys Leu Lys Asp Leu Phe
140 145 150
Tyr Arg Ser Ser Asn Leu Gln Tyr Phe Lys Arg Leu Ile Gln Ile
155 160 165
Pro Gln Leu Pro Glu Asn Pro Pro Asn Phe Leu Arg Ala Ser Ala
170 175 180
Leu Ser Glu His Ile Ser Pro Val Val Val Ile Pro Ala Glu Ala
185 190 195
Ser Ser Pro Asp Ser Glu Pro Val Leu Glu Lys Asp Asp Leu Met
200 205 210
Asp Met Asp Ala Ser Gln Gln Asn Leu Phe ASp Asn Lys Phe Asp
215 220 225
Asp Ile Phe Gly Ser Ser Phe Ser Ser ASp Pro Phe Asn Phe Asn
230 235 240
Ser Gln Asn Gly Val Asn ~ys Asp Glu Lys Asp His Leu Ile Glu
245 250 255
Arg Leu Tyr Arg Glu Ile Ser Gly Leu Lys Ala Gln Leu Glu Asn
2~0 265 270
CA 0223807~ l998-0~-l9
WO97/18825 PCT~S96/18370
_ Z9 _
Met Lys Thr Glu Ser Gln Arg Val Val Leu Gln Leu Lys Gly His
275 280 285
~al Ser Glu Leu Glu Ala Asp Leu Ala Glu Gln Gln HiS Leu Arg
290 295 300
~ln Gln Ala Ala Asp Asp Cys Glu Phe Leu Arg Ala Glu Leu Asp
305 310 315
~lu Leu Arg Arg Gln Arg Glu Asp Thr Glu Lys Ala Gln Arg Ser
320 325 330
~eu Ser Glu Ile Glu Arg Lys Ala Gln Ala Asn Glu Gln Arg Tyr
335 340 345
~er LyS Leu Lys Glu Lys Tyr Ser Glu Leu Val Gln Asn His Ala
350 355 360
~sp Leu Leu Arg Lys Asn Ala Glu Val Thr Lys Gln Val Ser Met
365 370 375
~la Arg Gln Ala Gln Val Asp Leu Glu Arg Glu Lys Lys Glu Leu
380 385 390
~lu Asp Ser Leu Glu Arg Ile Ser Asp Gln Gly Gln Arg Lys Thr
395 400 405
~ln Glu Gln Leu Glu Val Leu Glu Ser Leu Lys Gln Glu Leu Gly
410 415 420
~hr Ser Gln Arg Glu Leu Gln Val Leu Gln Gly Ser Leu Glu Thr
425 430 435
~er Ala Gln Ser Glu Ala Asn Trp Ala Ala Glu Phe Ala Glu Leu
440 445 450
~lu Lys Glu Arg Asp Ser Leu Val Ser Gly Ala Ala His Arg Glu
455 460 465
~lu Glu Leu Ser Ala Leu Arg Lys Glu Leu Gln Asp Thr Gln Leu
470 475 480
~ys Leu Ala Ser Thr Glu Glu Ser Met Cys Gln Leu Ala Lys Asp
485 490 495
~ln Arg Lys Met Leu Leu Val Gly Ser Arg Lys Ala Ala Glu Gln
500 505 510
~al Ile Gln Asp Ala Leu Asn Gln Leu Glu Glu Pro Pro Leu Ile
515 520 525
~er Cys Ala Gly Ser Ala Asp Eis Leu Leu Ser Thr Val Thr Ser
53Q 535 540
CA 0223807~ 1998-0~-19
WO 97/18825 PCT/US96/~8370
- 30 -
Ile Ser Ser Cys Ile Glu Gln Leu Glu Lys Ser Trp Ser Gln Tyr
545 550 555
~eu Ala Cys Pro Glu Asp Ile Ser Gly Leu Leu His Ser Ile Thr
560 565 570
~eu Leu Ala His Leu Thr Ser Asp Ala Ile Ala His Gly Ala Thr
575 580 585
~hr Cys Leu Arg Ala Pro Pro Glu Pro Ala Asp Ser Leu Thr Glu
590 595 600
~la Cys Lys Gln Tyr Gly Arg Glu Thr Leu Ala Tyr Leu Ala Ser
605 610 615
~eu Glu Glu Glu Gly Ser Leu Glu Asn Ala Asp Ser Thr Ala Met
620 625 630
~rg Asn Cys Leu Ser Lys Ile Lys Ala Ile Gly Glu Glu Leu Leu
635 640 645
~ro Arg Gly Leu Asp Ile Lys Gln Glu Glu Leu Gly Asp Leu Val
650 655 660
~sp Lys Glu Met Ala Ala Thr Ser Ala Ala Ile Glu Thr Cys Thr
665 670 675
~la Arg Ile Glu Glu Met Leu Ser Lys Ser Arg Ala Gly Asp Thr
680 685 690
~ly Val Lys Leu Glu Val Asn Glu Arg Ile Leu Arg Cys Cys Thr
695 700 705
~er Leu Met Gln Ala Ile Gln Val Leu Ile Val Ala Ser Lys Asp
710 715 720
~eu Gln Arg Glu Ile Val Glu Ser Gly Arg Gly Thr Ala Ser Pro
725 730 735
~ys Glu Phe Tyr Ala Lys Asn Ser Arg Trp Thr Glu Gly Leu Ile
740 745 750
~er Ala Ser Lys Ala Val Gly Trp Gly Ala Thr yal Met Val Asp
765 770 775
~la Ala Asp Leu Val Val Gln Gly Arg Gly Lys Phe Glu Glu Leu
780 785 7~0
~et Val Cys Ser His Glu Ile Ala Ala Ser Thr Ala Gln Leu Val
795 800 805
~la Ala Ser Lys Val Lys Ala Asp Lys Asp Ser Pro Asn Leu Ala
810 815 820
CA 02238075 1998-05-19
WO97/18825 PCT~S96/18370
- 31 -
Gln Leu Gln Gln Ala Ser Arg Gly Val Asn Gln Ala Thr Ala Gly
~ 825 830 835
~al Val Ala Ser Thr Ile Ser Gly Lys Ser Gln ~le Glu Glu Thr
840 845 850
~sp Asn Met Asp Phe Ser Ser Met Thr Leu Thr Gln Ile Lys Arg
855 860 865
~ln Glu Met Asp Ser Gln Val Arg Val Leu Glu Leu Glu Asn Glu
870 875 880
~eu Gln Lys Glu Arg Gln Lys Leu Gly Glu Leu Arg Lys Lys His
885 890 895
~yr Glu Leu Ala Gly Val Ala Glu Gly Trp Glu Glu Gly Thr Glu
900 905 glO
~la Ser Pro Pro Thr Leu Gln Glu Val Val Thr Glu Lys Glu
915 920 924
