WO2012007740A1 - Neurodegenerative disorders - Google Patents

Neurodegenerative disorders Download PDF

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Publication number
WO2012007740A1
WO2012007740A1 PCT/GB2011/051291 GB2011051291W WO2012007740A1 WO 2012007740 A1 WO2012007740 A1 WO 2012007740A1 GB 2011051291 W GB2011051291 W GB 2011051291W WO 2012007740 A1 WO2012007740 A1 WO 2012007740A1
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Prior art keywords
polypeptide
ntrp
app
seq
fragment
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PCT/GB2011/051291
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French (fr)
Inventor
Paul Charles Richard Hopkins
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King's College London
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Priority to GB1306866.3A priority Critical patent/GB2497502A/en
Publication of WO2012007740A1 publication Critical patent/WO2012007740A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to neurodegenerative disorders, and in particular, to conditions involving the presence of amyloid precursor protein (APP), such as
  • AD Alzheimer's disease
  • the invention relates to novel genes and proteins that are involved in the metabolism of ⁇ -secretase, APP and APP-like proteins.
  • the invention also relates to methods and screening assays for identifying modulators of APP metabolism.
  • the invention also extends to methods for investigating and testing therapies for conditions that are biologically related to apolipoprotein E (apoE), such as neurodegenerative disorders, such as Alzheimer's disease.
  • apoE apolipoprotein E
  • AD Alzheimer's disease
  • APP amyloid precursor protein
  • AD may occur either early or late in life. Pathologically, early- and late-onset AD are highly similar, and are therefore believed to share a common underlying molecular mechanism. Early-onset AD is associated with mutations in one of three genes, APP, presenilin 1, or presenilin 2. Early-onset AD is also associated with duplication of the APP gene itself, and with Down syndrome, a condition where all or part of
  • chromosome 21 (which harbours the APP gene) is duplicated.
  • APP is a single-pass transmembrane protein with both extracellular and intracellular domains. These domains are proteolytically separated by the ⁇ -secretase complex as part of its normal metabolism.
  • the ⁇ -secretase complex minimally contains presenilin 1 or 2 and three other proteins, PEN2, aphl and nicastrin.
  • presenilin 1 or 2 and three other proteins, PEN2, aphl and nicastrin.
  • APP is, however, not a direct substrate for ⁇ -secretase.
  • Processing of APP by ⁇ - secretase requires the prior release of the APP ectodomain by either a- or ⁇ -secretases, which cleave APP proximal to the membrane, releasing secreted APP (sAPP), and a 99- amino-acid C-terminal fragment (CTF) that is a direct substrate for ⁇ -secretase.
  • sAPP secreted APP
  • CTF 99- amino-acid C-terminal fragment
  • Proteolysis of the CTF by ⁇ -secretase produces the APP intracellular domain (AICD) and the ⁇ - amyloid peptide ( ⁇ ), in cases where the initial proteolysis was achieved by ⁇ -secretase. If the initial cleavage was by OC- secretase, a shorter non-amyloidogenic A ⁇ species (p3) is produced. Thus, the sequential proteolysis of APP by ⁇ -secretase and then by ⁇ -secretase is necessary to generate the A ⁇ peptides that accumulate as plaques in AD.
  • ⁇ -secretase may cleave the APP CTF at several sites generating peptides with different C-termini, the most common of which are A ⁇ 1-38, 1-40 and 1-42. Most mutations in the presenilins favour the production of the more amyloidogenic form, A ⁇ 1 -42.
  • AD pathology By manipulating the expression of the endogenous genes for APP, presenilin 1 or 2, and tau, and by expressing normal and variant human genes, various aspects of AD pathology have been recaptured in experimental animals. However, it has emerged that while over- expression of either normal or mutated APP in mice causes amyloid plaque formation and cognitive deficits, it does not cause AD-like neurodegeneration. Mutations in the APP or presenilin genes are relatively uncommon, and are therefore not believed to be causative in most cases of late-onset AD, although the similar pathologies of late- and early-onset AD indicate their involvement. Late-onset AD is, however, linked to variations in the gene for apolipoprotein E (apoE).
  • apoE apolipoprotein E
  • apoE2 Three common polymorphisms of the apoE gene exist that differ by amino acid substitutions at positions 112 and 158, referred to as apoE2, apoE3 and apoE4.
  • ApoE2 contains a cysteine at both positions; apoE3 contains a cysteine at position 112, and an arginine at position 158, while apoE4 contains arginines at both positions. It is now widely accepted that possession of a single copy of the apoE4 allele leads to an earlier onset of "late-onset" AD. However, significant disagreement exists as to how apoE4 leads to an increased susceptibility to AD.
  • apoE The best understood biological role of apoE is as a ligand for lipoprotein receptors. However, it is unclear if this role mediates the impact of apoE on AD. ApoE- knockout mice do not develop frank neurodegeneration, neither do mice expressing the human apoE3 or apoE4 genes. Nevertheless, when crossed with mice expressing mutated forms of human APP, it can be seen that apoE affects the metabolism of APP or its proteolysed products, as evidenced by the fact that they have a reduced development of amyloid plaques. The incomplete understanding of molecular mechanisms linking apoE variants and neurodegeneration presents an obstacle to the discovery of therapies that influence the apoE-modulated development or progression of AD. Elucidating the molecular mechanisms that underlie AD is therefore of significant interest, as is creating animal models that exhibit neurodegeneration in a manner related to apoE.
  • an isolated polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's: 1—16, or a functional variant or functional fragment thereof, wherein the polypeptide, variant or fragment thereof is capable of: (i) influencing amyloid precursor protein (APP) metabolism; (ii) influencing the activity of ⁇ -secretase; (iii) modulating the progression of neurodegeneration; and/ or (iv) binding to apolipoprotein E.
  • APP amyloid precursor protein
  • ⁇ -secretase influencing the activity of ⁇ -secretase
  • modulating the progression of neurodegeneration and/ or (iv) binding to apolipoprotein E.
  • apolipoprotein E apoliprotein E
  • the polypeptide of the first aspect can bind to apoliprotein E (apoE), and can also influence the function of ⁇ -secretase (i.e.
  • the inventor has also found that disruptions of the polypeptide of the first aspect or variants and fragments thereof can lead to neurodegeneration.
  • APP and apoE are both well-established risk factors for neurodegenerative disorders, for example Alzheimer's disease, the inventor believes that the polypeptide of the first aspect also plays an important role in such diseases.
  • the polypeptide according to the invention or functional variant or fragment thereof is capable of influencing amyloid precursor protein (APP) metabolism.
  • the polypeptide, variant or fragment may alter the rate of cleavage of APP by one of the secretases (e.g. ⁇ -secretase, OC-secretase or BACE), for example by increasing the rate of cleavage by ⁇ -secretase.
  • the polypeptide, variant or fragment may also alter the site at which a secretase (e.g. ⁇ -secretase) preferentially cleaves APP or may also regulate the cellular trafficking of APP, or the rate of generation or cellular activity of the APP intracellular domain.
  • the polypeptide, variant or fragment thereof may be capable of increasing the rate of ⁇ -secretase cleavage of APP C-99, or decreasing the trafficking of APP to the cell surface and subsequent cleavage by OC-secretase or BACE by at least 20%, 50%, 100%, 150%, 200%, 250% or at least 300% compared to the amount of APP metabolism which occurs in the absence of the polypeptide, variant or fragment thereof (i.e. the basal level of the polypeptide is its concentration normally found in vivo when no exogenous polypeptide has been provided).
  • the polypeptide according to the invention or functional variant or fragment thereof is capable of influencing the activity of ⁇ -secretase.
  • the polypeptide, variant or fragment may directly or indirectly interact with ⁇ -secretase to alter the preferred cleavage site on ⁇ -secretase substrates (such as APP C-99) and/ or directly interact with the 99-amino acid C-terminal region of APP to increase the cleavage by ⁇ - secretase.
  • the polypeptide, variant or fragment thereof may be capable of increasing the activity of ⁇ -secretase in a manner that increases the production of ⁇ - amyloid by at least 20%, 50%, 100%, 150%, 200%, 250% or at least 300% compared to the amount of ⁇ -amyloid which is produced in the absence of the polypeptide, variant or fragment thereof.
  • the polypeptide according to the invention or functional variant or fragment thereof is capable of modulating the progression of neurodegeneration.
  • the polypeptide, variant or fragment may increase neurodegeneration if it comprises a pathological mutation or if the gene encoding it comprises a pathological mutation (for example, in an intron). It may also increase neurodegeneration if it interacts with an apolipoprotein, for example apoE4 or apoE3 or fragments thereof.
  • the polypeptide, variant or fragment may also protect against neurological damage, for example in cases where APP is over-produced.
  • the polypeptide according to the invention or functional variant or fragment thereof is capable of binding to apoE.
  • the novel polypeptide according to the first aspect binds to apolipoproteins, such as apoE, he believes that the bound conjugate will have utility in neurodegenerative disorder research studies.
  • a conjugate comprising the polypeptide according to the first aspect, and an apolipoprotein.
  • the apolipoprotein present in the conjugate may comprise apolipoprotein E.
  • the apolipoprotein may be apolipoprotein E3 (apoE3) or apolipoprotein E4 (apoE4). It can be seen from Figure 1 A, that in yeast the polypeptide binds to apoE3 more strongly than it does to apoE4.
  • the conjugate comprises the polypeptide according to the first aspect and apoE3.
  • the inventor believes that the polypeptide of the first aspect and the conjugate of the second aspect presents a new model for the role of apoE and/ or ⁇ - secretase in neurodegenerative disorders.
  • polypeptides according to the invention can influence the metabolism of APP and APP-like proteins, alter ⁇ -secretase activity, disrupt the cellular distribution and/ or function of proteins involved in neuronal synaptic transmission, and can also bind to apoE.
  • a polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's: 1-16, or a functional variant or functional fragment thereof, for: (i) influencing amyloid precursor protein (APP) metabolism; (ii) influencing the activity of ⁇ -secretase; (iii) modulating the progression of neurodegeneration; and/ or (iv) binding to apolipoprotein E.
  • APP amyloid precursor protein
  • the use of the third aspect may comprise altering the metabolism of APP or APP-like proteins, wherein the concentration of polypeptide, variant or fragment thereof is increased or decreased relative to basal levels, i.e. the concentration of the polypeptide normally found in vivo when no exogenous polypeptide has been provided.
  • the use of the third aspect may comprise altering the activity of ⁇ -secretase, wherein the concentration of polypeptide, variant or fragment thereof is increased or decreased relative to basal levels.
  • a method of altering the activity of a polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's:l-16, or a functional variant or functional fragment thereof, the method comprising contacting the polypeptide, functional variant or functional fragment thereof, with an apolipoprotein.
  • the apolipoprotein may comprise apolipoprotein E, for example apoE3 or apoE4.
  • the polypeptide, variant or fragment thereof may be contacted with apoE3.
  • the use and method of the invention may be carried out in vitro.
  • a yeast- two-hybrid screen was performed using apoE3 and apoE4 as baits and a human brain cDNA library as a source of targets.
  • the screen yielded a clone (i.e. SEQ ID No.l) that corresponds to residues 421 to 709 of a protein (i.e. SEQ ID No. 3).
  • the inventor used BLAST searches and publicly-available databases to collate sequences having sequence similarity to SEQ ID No.1 , and found that it was a member of a novel family of highly conserved proteins found in a wide variety of animal species, including man, mouse, rat, Orosophila melanogaster and Caenorhabditis elegans.
  • the only published work specifically addressing a protein displaying any similarity to SEQ ID No:l is the human protein known as Tex 28.
  • the function of Tex28 has not been investigated or predicted. Due to the level of sequence identity between the polypeptide of the first aspect and Tex28, the inventor decided to name the polypeptide that he had isolated (i.e.
  • NTRP Novel Tex-28 Related Protein
  • the protein sequence of one embodiment of a human NTRP orthologue (Human TMCC2 421 - 709 ) is provided herein as SEQ ID No:l , as follows:
  • SEQ ID No.l The protein sequence of another embodiment of a human NTRP orthologue (Human TMCC1) is provided herein as SEQ ID No:2, as follows:
  • the protein sequence of another embodiment of a human NTRP (Human TMCC2) is provided herein as SEQ ID No:3, as follows:
  • the protein sequence of another embodiment of a human NTRP (Human TMCC3) is provided herein as SEQ ID No:4, as follows: MPGSDTALTVDRTYSDPGRHHRCKSRVERHDMNTLSLPLNIRRGGSDTNLNFDVPDGILDFHKVKLTADS LKQKILKVTEQIKIEQTSRDGNVAEYLKLVNNADKQQAGRIKQVFEKKNQKSAHSIAQLQKKLEQYHRKL REIEQNGASRSSKDI SKDHLKDIHRSLKDAHVKSRTAPHCMESSKSGMPGVSLTPPVFVFNKSREFANLI RNKFGSADNIAHLKNSLEEFRPEASARAYGGSATIVNKPKYGSDDECSSGTSGSADSNGNQSFGAGGAST LDSQGKLAVILEELREIKDTQAQLAEDIEALKVQFKREYGFI SQTLQEERYRYERLEDQLHDLTDLHQHE TANLKQELASIEEKVAYERSRDIQ
  • the protein sequence of another embodiment of a human NTRP (Human Tex 28) is provided herein as SEQ ID No: 5, as follows: MVLKAEHTRSPSATLPSNVPSCRSLSSSEDGPSGPSSLADGGLAHNLQDSVRHRILYLSEQLRVEKASRD GNTVSYLKLVSKADRHQVPHIQQAFEKVNQRASATIAQIEHRLHQCHQQLQELEEGCRPEGLLLMAESDP ANCEPPSEKALLSEPPEPGGEDGPVNLPHASRPFILESRFQSLQQGTCLETEDVAQQQNLLLQKVKAELE EAKRFHI SLQESYHSLKERSLTDLQLLLESLQEEKCRQALMEEQVNGRLQGQLNEIYNLKHNLACSEERM AYLSYERAKEIWEITETFKSRI SKLEMLQQVTQLEAAEHLQSRPPQMLFKFLSPRLSLATVLLVFVSTLC ACPSSLI SSRLCTCTMLMLIGLGVLAWQRW
  • rat NTRP orthologue (Rat TMCC1) provided herein as SEQ ID No:6, as follows:
  • rat NTRP rat NTRP
  • SEQ ID No:7 The protein sequence of another embodiment of a rat NTRP (Rat TMCC2) is provided herein as SEQ ID No:7, as follows:
  • rat NTRP The protein sequence of another embodiment of a rat NTRP (Rat TMCC3) is provided herein as SEQ ID No: 8, as follows: MPGSDTALTVDRTYSDPGRHHRCKSRVERHDMNTLSLPLNIRRGGSDTNLNFDVPDGILDFHKVKLSADS LRQKILKVTEQIKIEQTSRDGNVAEYLKLVSSADKQQAGRIKQVFEKKNQKSAHSIAQLQKKLEQYHRKL REIEQNGATRSSKDISKDSLKEIQHSLKDAHVKSRTAPHCLESSKSSMPGVSLTPPVFVFNKSREFANLI RNKFGSADNIAHLKNSLEEFRPEASPRAYGGSATIVNKPKYGSDDECSSGTSGSADSNGNQSFGAGGAST LDSQGKLAI ILEELREIKVTQAQLAEDIEALKVQFKREYGFI SQTLQEERYRYERLEDQLHDLTELHQHE TANLKQELASAEEKVAYQA
  • SEQ ID No:8 The protein sequence of another embodiment of a rat NTRP (Rat Tex28) is provided herein as SEQ ID No:9, as follows:
  • mouse NTRP orthologue (Mouse TMCC1) is provided herein as SEQ ID No: 10, as follows:
  • mouse TMCC2 The protein sequence of another embodiment of mouse NTRP (Mouse TMCC2) is provided herein as SEQ ID No:l l, as follows:
  • mouse TMCC3 The protein sequence of another embodiment of mouse NTRP (Mouse TMCC3) provided herein as SEQ ID No: 12, as follows:
  • the protein sequence of another embodiment of a mouse NTRP is provided herein as SEQ ID No: 13, as follows: MVLKVESTKSSSATLPTNLPSYRSLSSFCEDCPSSHTSFSDGELARNVREGVKHRIFYLSEQLRVEKASR DENTMSYLKLVSKADRHQAPHIRKAFERVNQRTSATIAHIERKLYQCHQQLKELEEGCSPTSSVLKVGSG LDSHKQPSGKVSYSKLSKPGGEDSLPINVARSSTLESHLSEMQQRKFSDKKYVAQQQKLLLQKMKEELTE AKKVHASFQVSHQSLKESHMIDVQRILESLQEKKTKQSLMEKQVNDHLQRYLDEICHLKQHLACTEEKMA YLSYERAKEIWDVMEIFKSRITKLETLQQATQLEMMASLRTRPKDFLFRFI SLLLTLTTILLVVVSTLCS CPLPLLSSRLRIFIVFMI IGLGTLAWQKRH
  • the protein sequence of one embodiment of Drosophila me/atiogasterNT V is provided herein as SEQ ID No: 14, as follows:
  • SEQ ID No: 14 The protein sequence of one embodiment of Caenorhabditis elegans NTRP is provided herein as SEQ ID No: 15, as follows:
  • the inventor has created a protein sequence alignment of the NTRP-related sequences (SEQ ID No's 1-15), which is shown in Figure 15 and illustrated schematically in Figure 2.
  • the alignment shows that they are all highly conserved in the C-terminal end, but the N-terminal region varies considerably among family members, though it is conserved in homologues.
  • the inventor has found a consensus sequence that defines the NTRP family, which is shown in Figure 16. To date, no one has appreciated that any of the embodiments of NTRP disclosed herein in any species (i.e. SEQ ID No's 2- 15), including the consensus sequence (i.e. SEQ ID No: 16), plays a pivotal role in APP metabolism or in ⁇ -secretase activity.
  • NTRP protein sequence of one embodiment of NTRP (i.e. a consensus sequence), is provided herein as SEQ ID No: 16, as follows, where X may refer to a variable number and/ or type of amino acids, for example, but not limited to, those found in SEQ ID's No.1 -15:
  • polypeptides having the identified conserved residues as shown SEQ ID No:16, in Figure 16, and schematically in Figure 2 may be capable of: (i) influencing APP metabolism; (ii) influencing the activity of ⁇ -secretase; (iii) modulating the progression of neurodegeneration; and/ or (iv) binding to apoE.
  • an isolated nucleotide sequence encoding the polypeptide, variant or fragment thereof according to the first aspect.
  • SEQ ID No:17 The DNA sequence encoding the polypeptide of SEQ ID No:2 is provided herein SEQ ID No: 17, as follows:
  • SEQ ID No: 17 The DNA sequence encoding the polypeptide of SEQ ID No:l (partially) and SEQ ID No:3 is provided herein as SEQ ID No:18, as follows:
  • SEQ ID No: 18 The DNA sequence encoding the polypeptide of SEQ ID No:4 is provided herein as SEQ ID No: 19, as follows:
  • SEQ ID No:20 The DNA sequence encoding the polypeptide of SEQ ID No:5 is provided herein as SEQ ID No:20, as follows:
  • SEQ ID No:21 The DNA sequence encoding the polypeptide of SEQ ID No:6 is provided herein as SEQ ID No:21, as follows:
  • SEQ ID No:21 The DNA sequence encoding the polypeptide of SEQ ID No:7 is provided herein as SEQ ID No:22, as follows:
  • SEQ ID No:22 The DNA sequence encoding the polypeptide of SEQ ID No:8 is provided herein as SEQ ID No:23, as follows:
  • SEQ ID No:24 The DNA sequence encoding the polypeptide of SEQ ID No:9 is provided herein as SEQ ID No:24, as follows: ATGGTTTTAAAGGTGGAAAGTACCAAGAGCTCAAGTGCAACCTTCCCCCCCAACGTGCCCTCCTACAGGT CTCTGTCTTCCTCCCATGAGGATTGCCCTAGCAGTCACACTAGCTTCTCGGATGGCGAGCTTGCCCGGAA TGTGAGGGAAGGTGTCAAACACCGAATCTTCTACCTCTCAGAGCAGCTGAGAGTGGAGAAGGCCAGTAGG GATGAAAATACCATGAGCTACCTCAAACTGATATCCAAAGCTGACCGGCAC CAGGCCCCACACATCCGGA AGGCCTTTGAAAGGGTGAACCAGCGCACCTCTGCCACTATTGCTCACATAGAACGAAAACTCTATCAGTG TCATCAGCAGCTGAAGGAGTTGGAAGGGCTGCAGTCCCACAAGCTTAGTGCTGAATGTGGACAGTGGG ATGGACAGCCATAAGCAGCCAG
  • SEQ ID No:23 The DNA sequence encoding the polypeptide of SEQ ID No:10 is provided herein as SEQ ID No:25, as follows: GGGGGGGTGGTCGGGGTGGGGATGGACCGGGCGCGGCGGCGGCAGCTGCAGGAGCCGGCGCTGGAGACCG AGCCGCGGCGCTGGCGGAGACGCCGGCTGTCCGTCGTCTCTCCACCGCTTCTCCTAAAAGGACATCAGTG CCCCAAGTACGTGCTTGAGGGAAGCCTCCATCTCTCTCAGCTGCATGCATT GTCACTCTTGAAGCAAATGCCTACCTAATTTGACAGTCTCGGTGTGTTTAAAATTTTTTGAGTTTGCAAA TAAGCTTATTAAGCTTACTGATGGAGCCTTCCGGCAGTGAACAGTTATATGAGGACCCTGATCCTGGAGG CAAATCCCAAGATGCAGAAGCCAGGAGGCAGACAGAGTCAGTCAGAACAGTTATATGAGGACCCTGATCCTGGAGG CAAATCCCAAGATGCAGAAGCCAGGAG
  • SEQ ID No:26 The DNA sequence encoding the polypeptide of SEQ ID No:l l is provided herein as SEQ ID No:26, as follows:
  • SEQ ID No:27 The DNA sequence encoding the polypeptide of SEQ ID No: 12 is provided herein as SEQ ID No:27, as follows:
  • SEQ ID No:29 The DNA sequence encoding the polypeptide of SEQ ID No: 14 is provided herein as SEQ ID No:29, as follows: CCGGCACGGGCAGTGGTCTGGAGGATTCCCGCCTCAGTGGCAACGAGGACTACTACTCGTCCTTCGTCTC TGACGAGTTTGACAGCAGCAAGAAGGTCCATCGCCGCTGCCACGAACGCAGCTCCAGCGTTCAGGCCATC GAC C G AT T G AAC AC G AAG AT C C AAT GC AC C AAGG AG T C C AT C C G AC AGG AG C AAAC T GC C AG AG AC GAT A ATGTCAACGAGTATCTTAAGTTGGCAGCCAGTGCAGACAAGCAGCAGCTGCAGCGCATCAAGGCTGTCTT TGAGAAAAAGAACCAGAAGAGCGCACACAATATCTCGCAGCTGCAGAAAAAGCTGGACAACTACACGAAG CGGGCCAAGGACTTGCAGAATCACCAGTTCCAGACGAAGAGCCAGCACCGTCAACCGC
  • nucleotide sequence of the fifth aspect may comprise a nucleic acid sequence substantially as set out in any one of SEQ ID No's: 17 to 30, or a functional variant or a fragment thereof.
  • a genetic construct comprising the nucleotide sequence of the fifth aspect.
  • Genetic constructs of the invention may be in the form of an expression cassette, which may be suitable for expression of the polypeptide in a host cell.
  • the genetic construct of the invention may be introduced in to a host cell without it being incorporated in a vector.
  • the genetic construct which may be a nucleic acid molecule, may be incorporated within a liposome or a virus particle.
  • a purified nucleic acid molecule e.g. histone-free DNA, or naked DNA
  • suitable means e.g. direct endocytotic uptake.
  • the genetic construct may be introduced directly in to cells of a host subject (e.g. an animal) by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment.
  • genetic constructs of the invention may be introduced directly into a host cell using a particle gun.
  • the genetic construct may be harboured within a recombinant vector, for expression in a suitable host cell.
  • a recombinant vector comprising the genetic construct according to the sixth aspect.
  • the recombinant vector may be a plasmid, cosmid or phage. Such recombinant vectors are highly useful for transforming host cells with the genetic construct of the sixth aspect, and for replicating the expression cassette therein.
  • the skilled technician will appreciate that genetic constructs of the invention may be combined with many types of backbone vector for expression purposes.
  • Recombinant vectors may include a variety of other functional elements including a suitable promoter to initiate gene expression.
  • the recombinant vector may be designed such that it autonomously replicates in the cytosol of the host cell. In this case, elements which induce or regulate DNA replication may be required in the recombinant vector.
  • the recombinant vector may be designed such that it integrates into the genome of a host cell.
  • DNA sequences which favour targeted integration e.g. by homologous recombination are envisaged.
  • the recombinant vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA.
  • the selectable marker gene may be in a different vector to be used simultaneously with vector containing the gene of interest.
  • the vector may also comprise DNA involved with regulating expression of the coding sequence, or for targeting the expressed polypeptide to a certain part of the host cell.
  • a host cell comprising the genetic construct according to the sixth aspect, or the recombinant vector according to the seventh aspect.
  • the host cell may be an animal cell, for example a mouse or rat cell, or a Drosophila cell. It is preferred that the host cell is not a human cell.
  • the cell may be transformed with the genetic construct or the vector according to the invention, using known techniques. Suitable means for introducing the genetic construct into the host cell will depend on the type of cell.
  • transgenic host organism comprising at least one host cell according to the eighth aspect.
  • the genome of the host cell or the transgenic host organism of the invention may comprise a nucleic acid sequence encoding a polypeptide, variant or fragment according to the first aspect.
  • the polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's 1-16, or a functional variant or functional fragment, or its encoding nucleic acid thereof may comprise a pathological mutation.
  • a pathological mutation may be an insertion into an intron that alters the production of normal NTRP, for example the ntrp 1 allele described herein, or another mutation that alters the amino acid composition of the peptide produced.
  • the nucleic acid sequence may be operably linked to a tissue-specific expression control sequence (such as a promoter), which drives expression of the nucleic acid sequence, wherein expression of the nucleic sequence results in the host organism displaying an altered phenotype.
  • the host organism may be a multicellular organism, which is preferably non-human.
  • the host organism may be a mouse, rat or Drosophila.
  • the host may be used in studies of neurodegenerative disorders.
  • the inventor believes that, because APP and apoE are both well-established risk factors for neurodegenerative disorders, modulation of the polypeptides of the invention could be used for treatment of such disease conditions.
  • the inventor has therefore designed a method of screening for a useful therapeutic agent for preventing or treating neurodegenerative disorder, and a method of identifying a test compound's capability to modulate any of the peptides described herein.
  • test compound is a modulator of the polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof.
  • a method of screening for a therapeutic agent useful in the prophylaxis or treatment of a neurodegenerative disorder comprising the steps of:
  • an alteration in either ⁇ -secretase activity or APP metabolism in the presence of the test compound compared to ⁇ -secretase activity or APP metabolism in the absence of the test compound is an indication of the ability of the test compound to modulate the neurodegenerative disorder.
  • test compound may be used as a therapeutic agent useful in the prophylaxis or treatment of the neurodegenerative disorder.
  • the assay system used in step (a) in the assay or methods may be either an in vitro or an in vivo system.
  • the assay system may be a cell-based system comprising cells, which either over-express or under-express a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof, compared to the level of expression that would occur under physiological conditions (i.e. basal expression levels).
  • the corresponding physiological level of expression of the polypeptide of the first aspect can mean the amount of polypeptide that is present under normal physiological conditions in vivo when no test compound (i.e. the modulator) has been added.
  • Over- expression may include:
  • Under-expression may include:
  • the assay system may not express a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof, or may express a polypeptide but which is rendered non- functional.
  • the assay system may comprise the use of a cell which is mutated such that the polypeptide of the first aspect is either not expressed at all, or only minimally expressed compared to basal expression levels.
  • RNAi may be used to reduce or prevent expression of the polypeptide of the first aspect.
  • the assay system may be a non-human animal model.
  • the animal may be a transgenic ape, monkey, mouse, rat, fish, ferret, sheep, dog, cat, worm or Orosophila.
  • APP metabolism and ⁇ -secretase activity may be determined in step (c) using techniques known to the skilled person, for example, but not limited to, those as described in materials and methods.
  • Figures 11 and 14 show how APP metabolism and/ or ⁇ - secretase activity may be determined.
  • APP metabolism involves cleavage of APP into fragments of various sizes.
  • OC-secretases and BACE cleave APP to release the ectodomain and ⁇ -secretase may cleave the APP C-terminal fragment at several sites generating peptides with different C-termini, the most common of which are ⁇ 1 -38, 1-40 and 1 -42. Most mutations in the presenilins favour the production of more amyloidogenic form, ⁇ 1-42.
  • alteration of APP metabolism or ⁇ -secretase activity may involve analysis of cell lysates or cell culture media for the products of APP metabolism by Western blot or ELISA; analysis of living or fixed cells for alterations in the cellular distribution of APP or it's proteolytic products by, for example, density gradient fractionation or immunohistochemistry.
  • APP metabolism may also be assessed using transcription factor reporter assays as described (Cao, X. & Sudhof, T.C. (2001) Science, 293, 115-120).
  • the assay system may comprise apoE2, apoE3 or apoE4 or a fragment thereof.
  • Alteration in the association between apoE and the polypeptide may be determined using known techniques, such as using intrinsic fluorescence methods.
  • a method of treating, ameliorating or preventing a neurodegenerative disorder in a subject comprising administering, to a subject in need of such treatment, a modulator of a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof.
  • the neurodegenerative disorder may be selected from a group consisting of
  • the neurodegenerative disorder is Alzheimer's disease.
  • the modulator may be a positive modulator or a negative modulator of the function of a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof.
  • the modulator may comprise a modifier of ⁇ -secretase, for example LY450139 (hydroxylvaleryl monobenzocaprolactam).
  • LY450139 hydroxylvaleryl monobenzocaprolactam
  • the modulator may comprise a drug that is known to be involved in lipid metabolism, such as a member of the group of drugs known as statins; for example, but not limited to, simvastatin, atorvastatin or lovastatin.
  • the modulator may comprise a drug from the thiazolidinedione class of drugs, for example, but not limited to pioglitazone or rosiglitazone.
  • the modulator may be a negative modulator of the function of a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof.
  • the modulator which may be used may comprise an RNAi or siRNA molecule, which is capable of inhibiting or slowing expression of the polypeptides of the invention, as demonstrated in Drosophila (see Figure 5).
  • the siRNA molecule comprises a nucleotide sequence which is specific for, or complementary to, at least a region of one or more of the nucleic acid sequences substantially as set out in any one of SEQ ID No's: 17 to 30.
  • the siRNA may comprise 15-30 nucleotides in length, preferably 21-25 nucleotides in length, and the skilled person would readily appreciate how to design a suitable siRNA molecule once provided with the sequence of the target DNA molecule.
  • the siRNA molecule comprises a nucleotide sequence which is specific for, or complementary to, at least a region of the nucleic acid sequence encoding the consensus sequence, SEQ ID No.16, or a variant or fragment thereof.
  • Other negative modulators may include agents that inhibit transcription factors normally activating expression of the polypeptide, variants or fragments of the invention, or agents that modify phosphorylation of the polypeptides, variants or fragments of the invention. It will be appreciated that modulator according to the invention may be used in a medicament which may be used in a monotherapy (i.e.
  • modulators according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing Alzheimer's disease.
  • the modulator of the invention may be used in combination with known agents for treating Alzheimer's disease, such as acetylcholinesterase inhibitors.
  • the modulators according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment.
  • the vehicle of medicaments according to the invention should be one which is well- tolerated by the subject to whom it is given, and preferably enables delivery of the agents across the blood-brain barrier.
  • Medicaments comprising modulators according to the invention may be used in a number of ways.
  • oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid.
  • Compositions comprising agents of the invention may be administered by inhalation (e.g. intranasally).
  • Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin, for example, adjacent the brain.
  • Modulators according to the invention may also be incorporated within a slow- or delayed-release device.
  • Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
  • the device may be located at least adjacent the treatment site, e.g. the head.
  • Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
  • medicaments according to the invention may be
  • injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).
  • the amount of the modulator that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the modulator and whether it is being used as a monotherapy or in a combined therapy.
  • the frequency of administration will also be influenced by the half-life of the modulator within the subject being treated.
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical
  • composition composition, the mode of administration, and the advancement of the
  • a daily dose of between 0.001 ug/kg of body weight and lOmg/kg of body weight of the modulator according to the invention may be used for treating, ameliorating, or preventing neurodegenerative disease, depending upon which agent is used. More preferably, the daily dose is between ⁇ . ⁇ -g/kg of body weight and lmg/kg of body weight, more preferably between ⁇ . ⁇ -g/kg and 100 g/kg body weight, and most preferably between approximately and 10 g/kg body weight.
  • the circulating dosage of the modulator may preferably be between about 10 nM/L and 100 nM/L, or between about 25 nM/L and 75 nM/L.
  • the modulator may be administered before, during or after onset of neurodegenerative disease. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the modulator may require administration twice or more times during a day. As an example, modulators may be administered as two (or more depending upon the severity of the neurodegenerative disease being treated) daily doses of between 0.07 g and 700 mg (i.e. assuming a body weight of 70 kg).
  • a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
  • a slow release device may be used to provide optimal doses of modulators according to the invention to a patient without the need to administer repeated doses.
  • an anti- neurodegenerative disease composition comprising a therapeutically effective amount of a modulator of a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof, and optionally a
  • anti-neurodegenerative disease composition can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of a neurodegenerative disorder in a subject, such as Alzheimer's disease.
  • the invention also provides in a fifteenth aspect, a process for making the composition according to the fourteenth aspect, the process comprising combining a therapeutically effective amount of a modulator of a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof, with a pharmaceutically acceptable vehicle.
  • the modulator may be a positive or negative modulator.
  • the modulator is a negative modulator, such as an siRNA or RNAi molecule, which is capable of inhibiting or slowing expression of the polypeptide.
  • the siRNA molecule comprises a nucleotide sequence which is specific for, or complementary to, at least a region of one or more of the nucleic acid sequences substantially as set out in any one of SEQ ID No's: 17 to 30.
  • the siRNA may comprise 15-30 nucleotides in length, preferably 21-25 nucleotides in length.
  • the siRNA molecule may comprise a nucleotide sequence which is specific for, or complementary to, at least a region of the nucleic acid sequence encoding the consensus sequence, SEQ ID No.16, or a variant or fragment thereof.
  • a "subject” may be a vertebrate, mammal, or domestic animal.
  • medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications.
  • livestock e.g. a horse
  • pets e.g. a human
  • the subject is a human being.
  • a “therapeutically effective amount” of agent is any amount which, when administered to a subject, is the amount of drug that is needed to treat the neurodegenerative disorder condition, or produce the desired effect.
  • the therapeutically effective amount of modulator used may be from about 0.001 ng to about 1 mg, and preferably from about 0.01 ng to about 100 ng. It is preferred that the amount of modulator is an amount from about 0.1 ng to about 10 ng, and most preferably from about 0.5 ng to about 5 ng.
  • a "pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
  • a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
  • the vehicle may also be an encapsulating material.
  • the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
  • the active agent i.e. the modulator
  • the vehicle may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain up to 99% of the active agents.
  • Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
  • the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
  • Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active agent according to the invention (the modulator) may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
  • the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.
  • liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil).
  • the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
  • the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
  • the modulator may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • the modulators and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • the agents used according to the invention can also be administered orally either in liquid or solid composition form.
  • Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
  • Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • nucleic acid or peptide or variant, derivative or analogue thereof which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
  • the terms "substantially the amino acid/ nucleotide/ peptide sequence”, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/ nucleotide/ peptide sequence, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino
  • amino acid/ nucleotide/ peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No:3 (i.e. TMCC2) or the nucleotide identified as SEQ ID No: 18, or 40% identity with the polypeptide identified as SEQ ID No:14 (i.e. Drosophila NTRP), and so on.
  • Amino acid/ olynucleotide/ polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • amino acids/ olynucleotide/ polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • amino acids amino acids
  • acid/ polynucleotide/ polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
  • the skilled technician will appreciate how to calculate the percentage identity between two amino acid/ polynucleotide/ polypeptide sequences.
  • an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.
  • the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FAST A, Smith- Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
  • percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs.
  • percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process.
  • ClustalW The popular multiple alignment program ClustalW (Thompson et al, 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention.
  • acid/ polynucleotide/ polypeptide sequences may then be calculated from such an alignment as (N/T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
  • a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No's: 17-30 or their complements under stringent conditions.
  • stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in 0.2x SSC/0.1% SDS at approximately 20-65°C.
  • a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No: 1-16. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will known the nucleotide sequences encoding these amino acids.
  • FIG. 1 shows that one embodiment of a protein in accordance with the invention known as NTRP (TMCC2 421 - 709 ) binds preferentially to ApoE3 compared to ApoE4.
  • Figure 1 A shows the results of a yeast- two-hybrid screen performed using ApoE3 and ApoE4 as baits and a human brain cDNA library as a source of targets.
  • results are represented in a graph showing relative ⁇ -galactosidase activity of yeast co-transformed with ApoE fused to the DNA-binding domain, and NTRP 421-709 fused to the activation domain of Gal4, lamin fused to the activation domain of Gal4 or the Gal4 activation domain alone.
  • Figure 1 B shows the binding of ApoE and NTRP in vitro by comparing the intrinsic tryptophan fluorescence of recombinant ApoE and an NTRP-thioredoxin fusion protein when incubated alone or together.
  • the data are represented in a graph showing the percentage change in fluorescence after co-incubation relative to the sum of fluorescence values when incubated separately.
  • Co-incubation of ApoE3 with an NTRP -Thioredoxin fusion results in an increased fluorescence relative to the sum of individual fluorescence values.
  • Figure 2 shows the shows the structural organization and sequence similarity of embodiments of human and Drosophila NTRP protein.
  • CC represents domains predicted to form coiled-coil structures
  • TM represents regions predicted to form transmembrane domains. The percent similarity for individual conserved domains (indicated by brackets) is shown. The sequences of the region containing the conserved domains are shown in Figure 15.
  • Figure 3 shows the location of an enhancer trap (NTRP-Gal4) and a lethal mutation (NTRP 1 ) in the Drosophila NTRP gene. Coding exons are in-filled boxes while non- coding exons are shown as open boxes. The structure of the Drosophila NTRP gene was obtained from flybase.org. Introns are indicated by lines, the intron indicated by a dashed line is not to scale.
  • Figure 4 shows the expression pattern of NTRP-gal4 expression in adult flies. NTRP- gal4 was used to drive nuclear green fluorescent protein (GFP).
  • Figure 4A shows a horizontal optical section of the optic lobe showing expression in glia (G) and in medulla neurons (mn).
  • Figure 4B shows a vertical section of the central brain showing NTRP expression in glia associated with the mushroom body peduncle (mp), central complex (cc), and other neuropils.
  • Figure 5 shows the effects of region- and cell-specific depletion of NTRP.
  • Figure 5A is a Western blot using antibody 385 that shows suppression of NTRP expression in adult Drosophila heads by RNAi driven by tubulin-Gal4 in the absence of Dcr2, each sample was prepared from 10-30 heads and the equivalent of 3 heads was loaded per lane.
  • Figure 5C shows the result of cell- and region-specific RNAi on total NTRP levels in the head.
  • RNAi was driven in neurons and glia (repo- and elav-gal4), glia only (repo-gal4), neurons only (elav-gal4), or using the ntrp-gal4 driver.
  • Control flies are wi l l 8 without RNAi.
  • NTRP expression in flies was detected by antibody 385 in an immunoblot. Each sample was prepared from 20-30 flies and the equivalent of 3.5 heads was loaded per lane.
  • the data is represented in a graph showing the percentage of larvae with RNAi for NTRP driven by the indicated Gal4 constructs that eclose as adults.
  • Figure 6 shows the phenotype of flies expressing mutant ntrp 1 .
  • Figure 6A shows that mutant ntrp 1 produces a truncated NTRP protein as determined by immunoblot using antibody 385; each lane was loaded with the equivalent of 4 heads prepared from pools of 30-35 heads of each genotype.
  • Figure 7A shows the optic lobe phenotype in heterozygous and homozygous mutant NTRP 1 flies where the retinal axons were detected by indirect immunofluorescence using antibody 24bl0 against chaoptin, and glia are detected using GFP expressed under the glia- specific driver repo-gal4.
  • This optic lobe phenotype was observed in all flies homozygous for ntrp 1 (n>26).
  • Horizontal optical sections through the optic lobes of flies heterozygous for ntrp 1 images 1 and 2 show a normal appearance, however, the optic lobe of flies homozygous for mutant NTRP 1 (images 3 and 4) are severely deformed.
  • Figure 7B shows retinal axons detected by indirect immunofluorescence using antibody 24bl0 in flies with glial or neuron- specific suppression of NTRP expression by RNAi.
  • Glia-specific RNAi image 1 causes an optic lobe defect, whereas neuron- specific RNAi (image 2) does not cause an optic lobe defect; images are representative of 8 examples for glia-specific RNAi and 12 for neuron-specific RNAi
  • Figure 8 shows the results of cell- and region- specific expression of wild-type NTRP in adult flies that are homozygous for the ntrp 1 allele.
  • Figure 8A shows the expression of NTRP in head extracts of homozygous mutant NTRP 1 flies which do not express wild- type NTRP (none), express wild-type NTRP in glia (repo-gal4), neurons (elav-gal4) or in both neurons and glia (elav-gal4 + repo-gal4) as detected by antibody 385 in a western blot; each lane was loaded with the equivalent of 3 heads prepared from a pool of 20-30 heads in each group.
  • Figure 8B shows that expression of wild-type NTRP in either neurons or glia rescues the developmental lethality associated with the ntrp 1 allele.
  • Figure 8C shows the lifespan of homozygous ntrp 1 flies that express wild-type NTRP in glia only, neurons only, or in both neurons and glia.
  • FIG. 8D shows that NTRP may influence the distribution or expression of synaptic proteins. All flies were homozygous ntrp 1 flies where wild-type NTRP is expressed in glia, neurons or both. Images 1-3 are images of the optic lobe obtained from paraffin sections prepared from brains prepared on the day of eclosion and stained with antibody nc82, which recognises the component of pre-synaptic active zones known as bruchpilot.
  • Image 1 shows the optic lobe of a homozygous ntrp 1 fly expressing wild-type NTRP in both neurons and glia
  • image 2 shows the optic lobe of a homozygous ntrp 1 fly expressing wild-type NTRP in neurons only
  • image 3 shows the optic lobe of a homozygous ntrp 1 fly expressing wild-type NTRP in glia only.
  • the distribution pattern of bruchpilot in flies expressing wild-type NTRP in glia only (image 3) is disrupted and patchy (indicated by an arrow in image 3) relative to that obtained in flies expressing wild-type NTRP in neurons and glia (image 1) or in neurons only (image 2).
  • the images labelled 4 to 6 are similarly prepared sections from flies 30 days after eclosion. Flies expressing wild type NTRP in both neurons and glia (image 4) and flies expressing wild-type NTRP in neurons only (image 5), have retained specific staining for bruchpilot, whereas those expressing wild- type NTRP in glia only (image 6) have a granular appearance and have lost specific staining for bruchpilot. Images are representative of data obtained from 10-12 flies in each group.
  • the retina is marked with the letter r
  • the medulla neuropil which receives the retinal axons shown in Figures 4 and 7 is marked with the letter m
  • the lobula complex which receives inputs from the medulla by the letters lc.
  • Figure 9 shows that homozygous NTRP 1 flies (escapees) fail to expand their wings after eclosion.
  • Figure 9A shows a representative photograph of a homozygous mutant NTRP 1 fly and
  • Figure 9B shows a heterozygous NTRP 1 fly; both photographs were taken 5 hours after eclosion.
  • Figure 10 shows that co-expression of NTRP with human APP rescues the deleterious effect of human APP on Drosophila development and health.
  • Figure 1 OA shows that APP-induced developmental lethality is rescued by co-expression of NTRP, but not by depletion of NTRP.
  • developmental lethality the bars represent the percent of young (LI) larvae of the indicated genotypes that eclose as adults.
  • M:F the bars represent the percent of total eclosing flies that are male.
  • Wing M the bars represent the percent of eclosing male flies that have unexpanded wings, similarly for females in the category "Wing F”.
  • Figure 10B shows the expression level of human APP when NTRP is either co-expressed with APP, or depleted in flies.
  • the immunoblot was prepared from the heads of male flies expressing human APP (UAS-APP) that also have RNAi for NTRP (UAS-NTRP IR) or over-express NTRP (UAS-NTRP) driven by elav-Gal4. Each sample was prepared from 30 flies and the equivalent of 3 heads was loaded per lane.
  • APP was detected with antibodies 5A3 and 1 G7 targeted against the ectodomain of APP. Similar results were obtained with antibody A5137, which is targeted at the C-terminus of APP (not shown) .
  • Figure 11 shows aberrant metabolism of a Drosophila APP orthologue (APPL) in mutant NTRP 1 flies.
  • Figure 11 A shows the detection of APPL by antibody dR14 in a western blot. The detected proteins correspond to (in decreasing order of molecular weight), holo-APPL, secreted APPL, and a novel APPL-reactive protein (arrow).
  • APPL Drosophila APP orthologue
  • Figure 11B shows the detection of NTRP by antibody 385 in a western blot from the same samples as in Figure 11 A.
  • each lane is loaded with the equivalent of 4 heads from preparations made from 20-40 heads of flies collected on the day of their eclosion. All flies were isogenic.
  • Figure 12 shows expression patterns of various embodiments of NTRP in mouse brain as determined by in situ hybridisation.
  • Figure 12A shows the expression pattern of NTRP (TMCC2) (image obtained from the Allen Brain Atlas).
  • Figure 12B shows the expression pattern of TMCC1 (an NTRP paralogue) (image obtained from the Allen Brain Atlas).
  • Figure 12C shows the expression pattern of TMCC3 (an NTRP paralogue) (image obtained from the Allen Brain Atlas).
  • Figure 12D shows the expression of NTRP in human brain (TMCC2) (image obtained from Atlas
  • Figure 13 shows the interaction of one embodiment of mammalian NTRP and APP.
  • Figure 13A shows co-migration of APP (indicated by black arrows) and NTRP
  • Figure 14 shows that NTRP and apoE modulate ⁇ -secretase activity towards APP in SHSY5Y cells.
  • NTRP (TMCC2) and apoE interact to increase the amount of ⁇ -amyloid produced; since the production of ⁇ -amyloid is known to depend on ⁇ -secretase, these data demonstrate modification of ⁇ -secretase activity by NTRP and apoE.
  • Figure 14A shows a representative example of the results of co-expressing NTRP (TMCC2), APP C99-GFP and apoE on the production of the APP intracellular domain, represented here as AICD-GFP.
  • NTRP neurotrophic factor
  • APPC99-GFP the APP intracellular domain fused to GFP
  • apoE the APP intracellular domain fused to GFP
  • Tubulin tubulin in cell lysates as assessed by SDS-PAGE and immunoblot.
  • the arrow indicates the presence of a band representing a slower- migrating species of AICD-GFP, indicating altered ⁇ -secretase activity towards the 99 amino acid portion of APP in the presence of NTRP and apoE.
  • Figure 14B shows the amount of ⁇ -40 and ⁇ -42 produced from cells prepared in the same manner as those analyzed in Figure 14A.
  • Figure 14C shows another example of the effect of NTRP and apoE on APP metabolism; in this case by SHSY5Y cells stably over- expressing full-length APP, said cells were cotransfected with NTRP (TMCC2) and apoE isoforms as indicated and the amount of ⁇ 1-40 and ⁇ 1-42 released into the media determined by ELISA; columns represent the levels of ⁇ — amyloid produced under each condition relative to that produced when apoE3 alone was transfected.
  • TMCC2 NTRP
  • apoE isoforms as indicated and the amount of ⁇ 1-40 and ⁇ 1-42 released into the media determined by ELISA
  • Figure 14D shows the expression NTRP (TMCC2), APP, apoE and tubulin in cell lysates of APP-expressing SHSY5Y cells as assessed by SDS-PAGE and immunoblot.
  • NTRP TMCC2
  • APP APP
  • apoE tubulin
  • FIGs 14B and 14C columns show mean relative values obtained from 5 experiments; error bars represent the standard error of the mean.
  • Vector refers to a pcDNA3.1 vector without an insert, included to normalize DNA levels in each transfection.
  • Figure 15 shows an alignment of various embodiments of NTRP sequences (SEQ ID No's: 1-15) and the derivation of a consensus sequence (SEQ ID No.16).
  • Figure 16 shows a consensus sequence for NTRP and related proteins. The standard single letter code for amino acids is used. Dots indicate poor conservation of both sequence and distance between orthologues. "X" indicates relatively poor conservation of sequence, but better conservation of distance.
  • Figure 17 shows the results that may be obtained from a hypothetical test on a drug modifying the activity of the NTRP and apoE on ⁇ -secretase activity and APP metabolism.
  • the potency of a test drug that suppresses ⁇ -amyloid production with or without NTRP and apoE is illustrated; the drug may be more or less potent in the presence of apoE3 or apoE4 and/ or NTRP.
  • the potency of two drugs, X and Y are compared after normalizing to their own baselines. Drug X is more potent in the presence of NTRP and/ or apoE3 or apoE4, whereas the potency of drug Y is decreased. Assays as described in Figures 17A and 17B would allow a more efficient and economical method of discovering drugs for the treatment of neurological disorders.
  • Figure 18 shows ApoE and NTRP interact to modify ⁇ -amyloid production from
  • AbPPswe i.e. the "Swedish” pathological variant of APP (APPswe, K595M/N596L).
  • APPswe i.e. the "Swedish” pathological variant of APP
  • K595M/N596L the "Swedish” pathological variant of APP.
  • A NTRP and apoE significantly increase ⁇ -amyloid production from AbPPswe.
  • B Representative western blots showing no significant effect of the expression of TMCC2 and apoE on the levels of AbPPswe.
  • Abl-40 and A -42 levels were independently normalized to that where only apoE3 is expressed, since apoE is abundant in the brain, and apoE3 represents the most common isoform of apoE.
  • Example 2 describes a functional assay measuring the metabolism of APP or APP-like proteins
  • Example 3 describes a method for screening small molecules, tissue extracts or body fluids.
  • Example 4 describes a method for investigating and testing therapies for conditions involving apoE, including Alzheimer's disease.
  • Yeast-two-hybrid - A commissioned yeast two hybrid screen using apoE isoforms as baits and a human brain cDNA library as a source of targets used the MATCHMAKER system (Clontech, Palo Alto, USA).
  • Flies - UAS-RNAi line 37338, which targets NTRP, and UAS-Dcr2 flies were obtained from the Vienna Drosophila RNAi Centre (Austria); this RNAi line is predicted to not have off-targets; unless otherwise indicated, all experiments using RNAi were performed in the presence of UAS-Dcr2.
  • ntrp 1 flies are the
  • PBac ⁇ RB ⁇ (CG1021)[e01970] line from the Exelixis collection at Harvard University (MA, USA); the reported insertion site was confirmed by inverse PCR.
  • ntrp-Gal4 flies (P ⁇ GawB ⁇ NP6590) were supplied by the Drosophila Genome Resource Centre (Kyoto, Japan).
  • UAS-NTRP flies were made by amplifying the cDNA for NTRP from clone
  • RE27645 obtained from the Berkeley Drosophila Genome Project (CA, USA), using the primers AGCTGGTACCTTACTTCACCACCAGCGATTGCTTG (SEQ ID No.31) and AGGAGGCGGCCGCATGTCCCGGGAGCGAGCCAGCGAGGCAGC (SEQ ID No.32); the PCR product was cut with Notl and Kpnl and ligated to pUAST cut with the same enzymes; the presence of the designed insert was confirmed by sequencing, w 1118 embryos were injected with DNA for UAS-NTRP at Rainbow Transgenic Flies, Inc. (CA, USA). All flies were raised on cornmeal, and when phenotypes were to be analyzed, at a density of 40-50 larvae per 10 ml of food.
  • flies were kept at less than 20 flies per 10 mL vial and transferred to new food twice per week.
  • larval or adults brains were dissected and fixed in 4% paraformaldehyde, 1.6% L-lysine in phosphate-buffered saline for 20 minutes, washed with 1% Triton X-100 in phosphate-buffered saline (PBX) and blocked with 10% normal goat serum in PBX.
  • PBX Triton X-100 in phosphate-buffered saline
  • flies were fixed overnight in Carnoy fixative, processed through 3 2-h incubations in methylbenzoate at room temperature, followed by a 1 :1 mixture of methylbenzoate and paraffin at 65° C and three changes of paraffin at 65° C (45 min each). Analyses were performed on 7 ⁇ sections.
  • cDNA constructs - Human TMCC2 (Acc. No. AB007950, Kazusa, Chiba, Japan) was amplified by PCR and cloned into pcDNA3.1 (Invitrogen), functionally equivalent constructs are now available from Origene Technologies.
  • a plasmid expressing dsRedER was obtained from Clontech, plasmids expressing apoE isoforms have been previously described (Ljungberg et a/., Neuroreport. 2002, May 7; 13(6):867-870);
  • constructs for the expression of APP695 and APP695 fused to the N-terminus of EGFP were gifts of C. Miller (Kings College, London), a plasmid expressing the 99 amino acid C-terminal fragment fused to EGFP with an additional Asp- Ala sequence after the signal peptide sequence as described (Lichtenthaler et a/., FEBS Lett. 1999 Jun 25;453(3):288-92.) was made by cutting APP695 fused to the N-terminus of EGFP with Bgl II and EcoRI and ligation to a cassette consisting of the oligonucleotides
  • Antibodies - Antibody 94 recombinant TMCC2 residues 252 to 648, corresponding to the conserved region (see Figure 15) prior to the predicted transmembrane domain, was produced as described below and injected into rabbits at Harlan Teklad (UK). Antibody 94 was used at a concentration of 1 :1000 in 5% milk, 150 mM NaCl, 50 mM Tris.Cl pH 7.4.
  • Antibody 385 raised against the peptide QSANADVLGSERLQ (SEQ ID No.35) was raised in rabbits at Eurogentec (Belgium) and used at a concentration of 1 : 1000 in 5% milk, 150 mM NaCl, 50 nM Tris.Cl pH 7.4; anti-APPL antibodies dR-14 and dC-12 targeted to the C-terminal region of the APPL ectodomain (communication from Santa Cruz Biotechnology) were obtained from Santa Cruz Biotechnology and used at a concentration of 1:100 in PBS 0.2% fish gelatin, 0.1% Triton X-100; APPL antigen recognition was best preserved when samples were stored at -80°C.
  • Mouse monoclonal antibodies 5 A3 and 1G7 against the APP ectodomain were gifts of S. Soriano.
  • Rabbit polyclonal antibody A5137 directed against the final 20 amino acids of human APP was a gift of C. Miller (Kings College, London).
  • Goat polyclonal anti-apoE was obtained from Calbiochem, and goat anti-presenilin-1 C- terminal fragment (C20) was obtained from Santa Cruz Biotechnologies.
  • Mouse anti- Golgi 58K and rabbit anti-BiP were purchased from Sigma Aldrich.
  • TMCC2 '-related sequences Collected TMCC2-related sequences were clustered based on homology using the ClustalW program (Larkin et al, Bioinformatics. 2007 Nov l;23(21):2947-8; Higgins et al, Gene. 1988 Dec 15;73(l):237-44.237) followed by manual refinement, and a phylogenetic analysis was performed using the service at phylogeny.fr (Dereeper et al, Nucleic Acids Res. 2008 Jul l;36(Web Server issue):W465- 9465). Coiled-coiled domains were predicted by the TMHMM service (Krogh et al, Journal of Molecular Biology, Jan 2001, 305(3):567-580).
  • TMCC2 cDNA encoding TMCC2 (Acc. No. AB007950, Kazusa, Chiba, Japan) from amino acid Met 79 to Arg 640 (adjacent to the first predicted t ansmembrane region) was amplified by PCR and cloned into the pBADThio vector (Invitrogen); the 5' primer contained an in-frame coding sequence for the FLAG epitope (DYKDDDDK) (SEQ ID No.36).
  • TMCC2 79 640 -thioredoxin protein (TMCC2-TrxA) in which the codons for the first exon were replaced by codons for thioredoxin, followed by the FLAG sequence, truncated TMCC2, and at the C-terminus, a six-His tag.
  • Expression was induced by adding arabinose (final concentration, 0.2%) to a log-phase culture of transformed Escherichia coli at 37 °C. Cell pellets were lysed in 6 M guanidine-HCl, 150 mM NaCl, and 20 mM Tris-Cl, pH 8.0, and cleared by centrifugation at 15,000 ⁇ for 1 h.
  • the supernatant was loaded onto a Ni 2+ -chelating sepharose column, and proteins were eluted with a gradient of 0-150 mM imidazole in 6 M guanidine-HCl, 150 mM NaCl, and 20 mM Tris-Cl, pH 8.0.
  • fractions TMCC2-TrxA were identified by SDS-PAGE and staining with Coomassie-blue, readjusted to 6 M guanidine-HCl, 150 mM NaCl, and 20 mM Tris-Cl, pH 8.0, and passed over a Ni 2+ - chelating column as described above.
  • TMCC2-TrxA Fractions containing TMCC2-TrxA were dialyzed against 1 mM HCl and lyophilized. TMCC2-TrxA was dissolved in 6.0 M guanidine HCl, 1.0 M L-arginine, 150 mM NaCl, 200 mM trimethylamine n-oxide, 40 mM ammonium bicarbonate, and 10 mM dithiothreitol.
  • Proteins were refolded by rapid dilution with 10 volumes of 1.0 M L-arginine, 150 mM NaCl, 200 mM trimethylamine n-oxide, 100 mM ammonium bicarbonate, and 10 mM dithiothreitol and dialyzed against 150 mM NaCl, 100 mM ammonium bicarbonate, and 100 mM trimethylamine n-oxide.
  • the change in total fluorescence at 342 nm on co-incubation is expressed as a percent of the sum of the fluorescence intensity measured for each protein alone.
  • Four independent assays using separately purified and prepared samples of TMCC2-TrxA were performed.
  • Recombinant apoE3, apoE4 and thioredoxin were gifts of Yvonne Newhouse and Karl Weisgraber (Gladstone Institute of Neurological Disease, San Francisco, USA) .
  • Two-dimensional Blue-native I SOS PAGE of rat brain extracts - A TMCC2- enriched rat brain fraction was prepared as follows.
  • the TMCC2- containing sucrose-gradient fraction prepared as above was diluted 10-fold with 50 mM imidazole.HCl, pH 7.0, 0.25 M sucrose, 1 mM EDTA and protease inhibitors (Complete, Boehringer Mannheim) and centrifuged at 1,500 g for 5 min., TMCC2 was enriched in the pellet.
  • the pellet was resuspended in 1% Triton X-100, 1% NP-40 and 1% Coomassie blue R-250 in 50 mM imidazole.HCl pH 7.0 and centrifuged at 100,000 g for 20 min. to remove insoluble material. The supernatant was fractionated by electrophoresis on a 4-13% acrylamide gradient gel as described (Wittig et a/. Nat. Protoc. 2006;l (l):418-28.). Gel strips were excised and incubated in 100 mM Tris.Cl pH 6.8, 1% SDS, 10 mM DTT for 30 min. and inserted between the plates of a previously poured SDS-PAGE gel without a stacking gel. A stacking gel was then poured around and under the strip and allowed to polymerize. The gel was run and the proteins transferred to polyvinyl difluoride membranes (Millipore) and analyzed by western blot in a conventional manner.
  • Co-immunoprecipitation - Cells were co-transfected with TMCC2 (NTRP) tagged at the C- terminus with the FLAG sequence (DYKDDDDK) and APP695 using TMCC2 (NTRP) tagged at the C- terminus with the FLAG sequence (DYKDDDDK) and APP695 using TMCC2 (NTRP) tagged at the C- terminus with the FLAG sequence (DYKDDDDK) and APP695 using
  • Lipofectamine2000 (Invitrogen); 24 hr after transfection cells were lysed in phosphate- buffered saline, 1% Triton X-100, protease inhibitors (Complete, Boehringer
  • phosphatase inhibitors (10 mM NaF, 4 mM ⁇ -glycerophosphate). Lysates were centrifuged at 100,000 g to remove insoluble material and incubated overnight with protein A/ G beads (Santa Cruz Biotechnology) previously coupled to M2 anti-FLAG antibody (Sigma Aldrich), antibody A5137 directed against the C- terminus of APP, or no antibody. The beads were washed 6 times in the above buffer over a period of 2 h. Proteins bound to the A5137 anti-APP antibody were eluted with SDS-containing gel loading buffer, and proteins bound to the M2 anti-FLAG antibody were eluted with the FLAG peptide as recommended by the supplier (Sigma Aldrich) . Eluted proteins were separated by SDS-PAGE and transferred to nitrocellulose for analysis by western blot. TMCC2 was detected using the rabbit antibody 94 directed raised against recombinant TMCC2, APP was detected using antibody A5137.
  • SHSY5Y cells were routinely cultured in 42% F12 media (Gibco), 42% EMEM media (Sigma), 15% bovine calf serum, supplemented with glutamate, non-essential amino acids and sodium pyruvate. SHSY5Y cells were seeded into 6-well dishes and cultured for 48 h before being transfected with the constructs indicated.
  • Transfections were achieved using Lipfectamine2000 (Invitrogen), as directed by the manufacturer, using 2 ⁇ Lipofectamine2000 per 1 ⁇ g of plasmid DNA per well for 4 h., when the transfection mixture was replaced with normal culture media. A total of 1 g of each plasmid DNA was used, and empty pcDNA3.1 used to normalize the amount of DNA in each transfection. Cells were incubated in the transfection mixture for 4 hr when it was removed and replaced with fresh Optimem (Invitrogen), media and cells were harvested after 36 hr.
  • Lipfectamine2000 Invitrogen
  • yeast-two-hybrid screen was performed using apoE3 and apoE4 as baits and a human brain cDNA library as a source of targets.
  • the results of the yeast-two-hybrid screen are shown in Figure 1 A.
  • This screen yielded a clone (i.e. SEQ ID NO.l) that has been called NTRP that corresponds to residues 421 to 709 of a protein (i.e. SEQ ID NO. 3).
  • NTRP NTRP-Thioredoxin
  • the increase in fluorescence may be attributed to a change in the local environment of one or more of the tryptophans in apoE3 on binding to NTRP.
  • NTRP family members are provided in Table 1.
  • NTRP proteins in human and Drosophila are highly similar, with conserved regions showing a percent similarity ranging from 41 % to 84% in amino acid sequence and are thus likely to function in a similar manner.
  • NTRP-related sequences showed that they are highly conserved in the C-terminal portion of the molecule (shown in Figure 15), which in the embodiment of NTRP referred to as TMCC2 spans residues 247-709.
  • the N-terminal region varies considerably among orthologues, though it is conserved in homologues.
  • a minimal consensus sequence that defines the NTRP family was derived using the "seaview" software (Galtier et a/., Comput Appl Biosci. 1996 Dec; 12(6):543-8), followed by manual refinement, and is shown in Figure 16.
  • TM transmembrane
  • CC coiled-coil
  • Drosophila NTRP functions in brain development
  • FIG. 3 illustrates schematically where the enhancer trap (NTRP-gal4) is inserted in the gene for NTRP.
  • NTRP-gal4 was expressed in both neurons and glia.
  • experiments in which NTRP level in Drosophila was depleted by either neuron- or glia- specific RNAi also confirmed that NTRP was expressed in both neurons and glia.
  • NTRP expression was efficiently suppressed by RNAi using tubulin-Gal4, which is expressed in all tissues (Figure 5A); tubulin-gal4-driven suppression of NTRP expression was almost completely lethal during development, showing that flies deficient in NTRP would not develop into adults.
  • Figure 5C and Figure 5D a more restricted suppression of NTRP using glia- or neuron-specific methods of suppressing NTRP production showed that glia-specific suppression of NTRP recapitulated the developmental effect of global suppression of NTRP.
  • the inventor identified a recessive pupal-lethal hypomorphic allele of the Drosophila NTRP gene (NTRP 1 ) a piggyBac transposon insertion (see Figure 3) that leads to the production of a truncated peptide, as is shown in Figure 6A, and as indicated by an arrow.
  • NTRP 1 Drosophila NTRP gene
  • Figure 6B similar to the effect of global RNAi for NTRP ( Figure 5B), homozygous NTRP 1 flies died mainly during metamorphosis, and as shown in Figure 6C, homozygous NTRP 1 escapees died within a few days.
  • the DNA encoding this embodiment of NTRP thus contains a pathological mutation.
  • NTRP 1 flies with glia-only expression of wild-type NTRP had a significantly reduced lifespan compared with those expressing wild-type NTRP in both neurons and glia, suggesting that the shorter lifespan was caused by a deficiency of NTRP in neurons.
  • the inventor determined that ntrp 1 flies without wild-type NTRP in neurons had neurodegeneration.
  • images 1 to 3 show paraffin sections of the optic lobe of ntrp 1 flies fixed on the day of eclosion and stained by indirect immunofluorescence for bruchpilot, a pre-synaptic protein.
  • Images 1, 2 and 3 show the optic lobe of ntrp 1 flies expressing wild-type NTRP in neurons and glia, neurons only, or glia only, respectively.
  • Images 4 to 6 show the optic lobe of flies of the same genotype fixed 30 days after eclosion.
  • NTRP neuronal expression of pathogenic variants of NTRP leads to disruption of neuronal function and eventually to neurodegeneration.
  • NTRP interacts genetically with APP-like proteins in Drosophila
  • APP may interact directly with NTRP (see Figure 13; explained more below) and suppress the effect of APP by sequestering it.
  • the inventor next examined preparations from the heads of newly eclosed NTRP 1 flies for the Drosophila homologue of APP, APPL, which is expressed in both neurons and glia. With reference to Figure 11 , this showed that heterozygous and homozygous mutant NTRP 1 flies, but not isogenic control flies, accumulated a ⁇ 50 kDa protein that reacted with antibodies targeted to APPL (indicated by an arrow), indicating aberrant metabolism of APPL.
  • Drosophila NTRP interacts genetically both with human APP, and it's Drosophila orthologue, APPL.
  • the inventor also investigated mammalian APP and NTRP for evidence of interaction.
  • Data available from the Allen Brain Atlas (www.brain-map.org) and from Atlas Antibodies (www.atlasantibodies.com) show expression of NTRP and related genes in mouse and human brains (see Figure 12).
  • Figure 14A is a western blot showing a novel band that the inventor believes represents a ⁇ - secretase product that is generated in the presence of NTRP and apoE (indicated by an arrow). Alteration of ⁇ -secretase activity, and secretion of ⁇ -amyloid was also shown when NTRP and apoE caused an increase in ⁇ -amyloid production from APP-C99-GFP, as shown in Figures 14B and 14C. Since the cleavage of the C99 fragment of APP is known to depend strictly on ⁇ - secretase, this shows that NTRP may modulate ⁇ -secretase activity.
  • NTRP neurotransmission, and to neurodegeneration. Since (i) the Drosophila and mammalian sequences of NTRP are so similar, (ii) Drosophila NTRP interacts with both human APP and Drosophila APPL, (iii) rat NTRP and APP co-migrate on native gels, and (iv) human NTRP and APP co-immunoprecipitate, (v) NTRP interacts with apoE in an isoform-specific manner to alter ⁇ -amyloid production, (vi) NTRP interacts with apoE to alter ⁇ -secretase activity, the inventor believes that embodiments of NTRP and related proteins play an important role in the metabolism of APP and APP-like proteins in humans. Given that APP and APP-like proteins contribute to neurodegenerative disorders, the inventor believes that embodiments of NTRP and related proteins play a role in such diseases, including Alzheimer's disease.
  • Animals with general, cell- or region-specific regulated expression of NTRP, or similar proteins, or which express modified forms of NTRP, or similar proteins, or which have RNAi-mediated regulation of NTRP protein production may therefore be used to model neurodegeneration, to investigate mechanisms in neurodegeneration, or to test drugs for impacts on neuronal function specifically, or brain function generally, or for impacts on diseases such as neurodegeneration.
  • Such animals may also be used to investigate the metabolism of APP and APP-like proteins, the activity of ⁇ -secretase, or to test compounds that may alter the metabolism of such proteins.
  • NTRP can bind ApoE in an isoform-specific manner
  • animals with modified expression of NTRP or related proteins, or with the expression of modified forms of NTRP may be used to investigate and test therapies for conditions which involve apoE, including Alzheimer's disease, perse.
  • NTRP neoplasmic protein
  • APPL APPL
  • APLP1 APLP2
  • APLP2 ⁇ -secretase substrates
  • NTRP is increased or depleted relative to its physiological levels and the cellular distribution of APP assessed by, for example sucrose gradient centrifugation of by measurement of it's normal processing, such as ⁇ -amyloid.
  • Such assays may also use modified forms of NTRP that are partially active, such as NTRP 1 described above.
  • a screen for drugs intended for the treatment of conditions such as Alzheimer's disease may also be assessed by this invention as illustrated in Figure 17.
  • a test compound may be included in a cell-based or in vitro assay that measures the production of APP metabolites, such as ⁇ -amyloid as described above. This compound may already be suspected to inhibit or increase ⁇ -amyloid production or to interact with apoE.
  • NTRP and apoE By incorporating or removing NTRP and apoE, or either alone, in or from the assay an improved assessment of the merits of the compound can be made.
  • the compound may, for example, be either less effective or more effective in the presence of NTRP and/ or apoE.
  • Such compounds could include those aimed at targeting lipid metabolism, such as those known as statins.
  • Such compounds may also be targeted at insulin metabolism, such as rosiglitazone, pioglitazone or drugs with a similar mechanism of action.
  • Such compounds may include those targeted at ⁇ -secretase, such as LY450139 (hydroxylvaleryl monobenzocaprolactam).
  • Such compounds may be those targeted at apoE, such as those described in US patent number US 2006/0073104 Al .
  • Such compounds may also consist of fragments of apoE or closely related peptides.
  • NTRP interacts differentially with the neurodegeneration-risk versus normal versions of two separate classes of protein, apoE and APP.

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Abstract

The invention relates to neurodegenerative disorders, and in particular, to conditions involving the presence of amyloid precursor protein (APP), such as Alzheimer's disease (AD). The invention relates to novel genes and proteins that are involved in the metabolism of γ-secretase, APP and APP-like proteins, and to methods and screening assays for identifying modulators of APP metabolism. The invention extends to methods for investigating and testing therapies for conditions that are biologically related to apolipoprotein E (apoE), such as neurodegenerative disorders, such as Alzheimer's disease.

Description

NEURODEGENERATIVE DISORDERS
The present invention relates to neurodegenerative disorders, and in particular, to conditions involving the presence of amyloid precursor protein (APP), such as
Alzheimer's disease (AD). In particular, the invention relates to novel genes and proteins that are involved in the metabolism of γ-secretase, APP and APP-like proteins. The invention also relates to methods and screening assays for identifying modulators of APP metabolism. The invention also extends to methods for investigating and testing therapies for conditions that are biologically related to apolipoprotein E (apoE), such as neurodegenerative disorders, such as Alzheimer's disease.
Alzheimer's disease (AD) is a degenerative condition of the brain. Pathologically, it is characterized by an accumulation of the amyloid peptide, derived from the amyloid precursor protein (APP), as insoluble plaques, the presence of neurofibrillary tangles composed of hyperphosphorylated tau (a microtubule-binding protein), disruption of neuronal function, and disruption of the cytoskeletal network. Studies indicate that abnormal metabolism of APP, which leads to the production of amyloid plaques, precedes the hyperphosphorylation of tau and cytoskeletal disruptions. Therefore, abnormal metabolism of APP, or amyloid deposition, may be causative in AD.
AD may occur either early or late in life. Pathologically, early- and late-onset AD are highly similar, and are therefore believed to share a common underlying molecular mechanism. Early-onset AD is associated with mutations in one of three genes, APP, presenilin 1, or presenilin 2. Early-onset AD is also associated with duplication of the APP gene itself, and with Down syndrome, a condition where all or part of
chromosome 21 (which harbours the APP gene) is duplicated.
APP is a single-pass transmembrane protein with both extracellular and intracellular domains. These domains are proteolytically separated by the γ-secretase complex as part of its normal metabolism. The γ-secretase complex minimally contains presenilin 1 or 2 and three other proteins, PEN2, aphl and nicastrin. As all of the presenilin mutations linked to AD examined to date have altered proteolytic activity towards APP, altered proteolytic processing of APP by γ-secretase is believed to represent a fundamental molecular process central to the development of AD.
APP is, however, not a direct substrate for γ-secretase. Processing of APP by γ- secretase requires the prior release of the APP ectodomain by either a- or β-secretases, which cleave APP proximal to the membrane, releasing secreted APP (sAPP), and a 99- amino-acid C-terminal fragment (CTF) that is a direct substrate for γ-secretase. Several different proteases are known to accomplish this at different sites, for example by the β-secretase (BACE), or the γ-secretase. Proteolysis of the CTF by γ-secretase produces the APP intracellular domain (AICD) and the β- amyloid peptide (Αβ), in cases where the initial proteolysis was achieved by β-secretase. If the initial cleavage was by OC- secretase, a shorter non-amyloidogenic A β species (p3) is produced. Thus, the sequential proteolysis of APP by β-secretase and then by γ-secretase is necessary to generate the A β peptides that accumulate as plaques in AD. γ-secretase may cleave the APP CTF at several sites generating peptides with different C-termini, the most common of which are A β 1-38, 1-40 and 1-42. Most mutations in the presenilins favour the production of the more amyloidogenic form, A β 1 -42.
The best evidence to date indicates that disruption of the above process leads to AD, although it is not clear how. Various arguments have been whether this is due to an altered rate of production of the various products of APP (sAPP, A β, or AICD), or specifically to an alteration in the ratio of A β species produced.
By manipulating the expression of the endogenous genes for APP, presenilin 1 or 2, and tau, and by expressing normal and variant human genes, various aspects of AD pathology have been recaptured in experimental animals. However, it has emerged that while over- expression of either normal or mutated APP in mice causes amyloid plaque formation and cognitive deficits, it does not cause AD-like neurodegeneration. Mutations in the APP or presenilin genes are relatively uncommon, and are therefore not believed to be causative in most cases of late-onset AD, although the similar pathologies of late- and early-onset AD indicate their involvement. Late-onset AD is, however, linked to variations in the gene for apolipoprotein E (apoE).
Three common polymorphisms of the apoE gene exist that differ by amino acid substitutions at positions 112 and 158, referred to as apoE2, apoE3 and apoE4.
ApoE2 contains a cysteine at both positions; apoE3 contains a cysteine at position 112, and an arginine at position 158, while apoE4 contains arginines at both positions. It is now widely accepted that possession of a single copy of the apoE4 allele leads to an earlier onset of "late-onset" AD. However, significant disagreement exists as to how apoE4 leads to an increased susceptibility to AD.
The best understood biological role of apoE is as a ligand for lipoprotein receptors. However, it is unclear if this role mediates the impact of apoE on AD. ApoE- knockout mice do not develop frank neurodegeneration, neither do mice expressing the human apoE3 or apoE4 genes. Nevertheless, when crossed with mice expressing mutated forms of human APP, it can be seen that apoE affects the metabolism of APP or its proteolysed products, as evidenced by the fact that they have a reduced development of amyloid plaques. The incomplete understanding of molecular mechanisms linking apoE variants and neurodegeneration presents an obstacle to the discovery of therapies that influence the apoE-modulated development or progression of AD. Elucidating the molecular mechanisms that underlie AD is therefore of significant interest, as is creating animal models that exhibit neurodegeneration in a manner related to apoE.
According to a first aspect of the invention, there is provided an isolated polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's: 1—16, or a functional variant or functional fragment thereof, wherein the polypeptide, variant or fragment thereof is capable of: (i) influencing amyloid precursor protein (APP) metabolism; (ii) influencing the activity of γ-secretase; (iii) modulating the progression of neurodegeneration; and/ or (iv) binding to apolipoprotein E. Surprisingly, the inventor has found that the polypeptide of the first aspect can bind to apoliprotein E (apoE), and can also influence the function of γ-secretase (i.e. the presenilin-containing complex involved in cleaving amyloid precursor protein, APP) such that the metabolism of APP and APP-like proteins is affected. Surprisingly, the inventor has also found that disruptions of the polypeptide of the first aspect or variants and fragments thereof can lead to neurodegeneration. Although not wishing to be bound by any hypothesis, since APP and apoE are both well-established risk factors for neurodegenerative disorders, for example Alzheimer's disease, the inventor believes that the polypeptide of the first aspect also plays an important role in such diseases.
Preferably, the polypeptide according to the invention or functional variant or fragment thereof is capable of influencing amyloid precursor protein (APP) metabolism. Thus, the polypeptide, variant or fragment may alter the rate of cleavage of APP by one of the secretases (e.g. γ-secretase, OC-secretase or BACE), for example by increasing the rate of cleavage by γ-secretase. The polypeptide, variant or fragment may also alter the site at which a secretase (e.g. γ-secretase) preferentially cleaves APP or may also regulate the cellular trafficking of APP, or the rate of generation or cellular activity of the APP intracellular domain. It may also alter the rate at which APP reaches the cell surface. Therefore, the polypeptide, variant or fragment thereof may be capable of increasing the rate of γ-secretase cleavage of APP C-99, or decreasing the trafficking of APP to the cell surface and subsequent cleavage by OC-secretase or BACE by at least 20%, 50%, 100%, 150%, 200%, 250% or at least 300% compared to the amount of APP metabolism which occurs in the absence of the polypeptide, variant or fragment thereof (i.e. the basal level of the polypeptide is its concentration normally found in vivo when no exogenous polypeptide has been provided).
Preferably, the polypeptide according to the invention or functional variant or fragment thereof is capable of influencing the activity of γ-secretase. Thus, the polypeptide, variant or fragment may directly or indirectly interact with γ-secretase to alter the preferred cleavage site on γ-secretase substrates (such as APP C-99) and/ or directly interact with the 99-amino acid C-terminal region of APP to increase the cleavage by γ- secretase. Therefore, the polypeptide, variant or fragment thereof may be capable of increasing the activity of γ-secretase in a manner that increases the production of β- amyloid by at least 20%, 50%, 100%, 150%, 200%, 250% or at least 300% compared to the amount of β-amyloid which is produced in the absence of the polypeptide, variant or fragment thereof.
Preferably, the polypeptide according to the invention or functional variant or fragment thereof is capable of modulating the progression of neurodegeneration. Thus, the polypeptide, variant or fragment may increase neurodegeneration if it comprises a pathological mutation or if the gene encoding it comprises a pathological mutation (for example, in an intron). It may also increase neurodegeneration if it interacts with an apolipoprotein, for example apoE4 or apoE3 or fragments thereof. The polypeptide, variant or fragment may also protect against neurological damage, for example in cases where APP is over-produced.
Preferably, the polypeptide according to the invention or functional variant or fragment thereof is capable of binding to apoE. Given that the inventor has demonstrated that the novel polypeptide according to the first aspect binds to apolipoproteins, such as apoE, he believes that the bound conjugate will have utility in neurodegenerative disorder research studies.
Thus, in a second aspect, there is provided a conjugate comprising the polypeptide according to the first aspect, and an apolipoprotein. The apolipoprotein present in the conjugate may comprise apolipoprotein E. For example, the apolipoprotein may be apolipoprotein E3 (apoE3) or apolipoprotein E4 (apoE4). It can be seen from Figure 1 A, that in yeast the polypeptide binds to apoE3 more strongly than it does to apoE4. Thus, preferably the conjugate comprises the polypeptide according to the first aspect and apoE3. Based on the observation that the polypeptide binds differentially to apoE3 and apoE4, and its influence on APP metabolism, the inventor believes that the polypeptide of the first aspect and the conjugate of the second aspect presents a new model for the role of apoE and/ or γ- secretase in neurodegenerative disorders.
As described in the Examples, the inventor has demonstrated that various
embodiments of the polypeptides according to the invention (i.e. SEQ ID No's: 1 to 16) can influence the metabolism of APP and APP-like proteins, alter γ-secretase activity, disrupt the cellular distribution and/ or function of proteins involved in neuronal synaptic transmission, and can also bind to apoE.
Thus, in a third aspect, there is provided use of a polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's: 1-16, or a functional variant or functional fragment thereof, for: (i) influencing amyloid precursor protein (APP) metabolism; (ii) influencing the activity of γ-secretase; (iii) modulating the progression of neurodegeneration; and/ or (iv) binding to apolipoprotein E.
Preferably, the use of the third aspect may comprise altering the metabolism of APP or APP-like proteins, wherein the concentration of polypeptide, variant or fragment thereof is increased or decreased relative to basal levels, i.e. the concentration of the polypeptide normally found in vivo when no exogenous polypeptide has been provided.
Preferably, the use of the third aspect may comprise altering the activity of γ-secretase, wherein the concentration of polypeptide, variant or fragment thereof is increased or decreased relative to basal levels.
In a fourth aspect, there is provided a method of altering the activity of a polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's:l-16, or a functional variant or functional fragment thereof, the method comprising contacting the polypeptide, functional variant or functional fragment thereof, with an apolipoprotein. The apolipoprotein may comprise apolipoprotein E, for example apoE3 or apoE4. Preferably, the polypeptide, variant or fragment thereof may be contacted with apoE3. Preferably, the use and method of the invention may be carried out in vitro. To isolate novel proteins that differentially bind to apoE3 and apoE4, which may lead to the identification of new models for the role of apoE in neurodegenerative disorders, a yeast- two-hybrid screen was performed using apoE3 and apoE4 as baits and a human brain cDNA library as a source of targets. The screen yielded a clone (i.e. SEQ ID No.l) that corresponds to residues 421 to 709 of a protein (i.e. SEQ ID No. 3). The inventor used BLAST searches and publicly-available databases to collate sequences having sequence similarity to SEQ ID No.1 , and found that it was a member of a novel family of highly conserved proteins found in a wide variety of animal species, including man, mouse, rat, Orosophila melanogaster and Caenorhabditis elegans. The only published work specifically addressing a protein displaying any similarity to SEQ ID No:l is the human protein known as Tex 28. However, the function of Tex28 has not been investigated or predicted. Due to the level of sequence identity between the polypeptide of the first aspect and Tex28, the inventor decided to name the polypeptide that he had isolated (i.e. SEQ ID No:l) as "Novel Tex-28 Related Protein" or "NTRP". The various NTRP family members are provided in Table 1 and shown in Example 1 , and, as can be seen, the inventor has determined that the human, rat and mouse genomes contain at least four NTRP orthologues each, whereas the Orosophila melanogaster and Caenorhabditis elegans genomes contain at least one copy each.
The protein sequence of one embodiment of a human NTRP orthologue (Human TMCC2421-709) is provided herein as SEQ ID No:l , as follows:
VSKPREFASLIRNKFGSADNIAHLKDPLEDGPPEEAARALSGSATLVSSPKYGSDDECSSASASSAGAGS NSGAGPGGALGSPKSNALYGAPGNLDALLEELREIKEGQSHLEDSMEDLKTQLQRDYTYMTQCLQEERYR YERLEEQLNDLTELHQNEMTNLKQELASMEEKVAYQSYERARDIQEAVESCLTRVTKLELQQQQQQVVQL EGVENANARALLGKFINVILALMAVLLVFVSTIANFITPLMKTRLRITSTTLLVLVLFLLWKHWDSLTYL LEHVLLPS
SEQ ID No.l The protein sequence of another embodiment of a human NTRP orthologue (Human TMCC1) is provided herein as SEQ ID No:2, as follows:
MEPSGSEQLFEDPDPGGKSQDAEARKQTESEQKLSKMTHNALENINVIGQGLKHLFQHQRRRSSVSPHDV QQIQADPEPEMDLESQNACAEIDGVPTHPTALNRVLQQIRVPPKMKRGTSLHSRRGKPEAPKGSPQINRK SGQEMTAVMQSGRPRSSSTTDAPTSSAMMEIACAAAAAAAACLPGEEGTAERIERLEVSSLAQTSSAVAS STDGS IHTDSVDGTPDPQRTKAAIAHLQQKILKLTEQIKIAQTARDDNVAEYLKLANSADKQQAARIKQV FEKKNQKSAQTILQLQKKLEHYHRKLREVEQNGIPRQPKDVFRDMHQGLKDVGAKVTGFSEGVVDSVKGG FSSFSQATHSAAGAVVSKPREIASLIRNKFGSAD IPNLKDSLEEGQVDDAGKALGVI SNFQSSPKYGSE EDCSSATSGSVGANSTTGGIAVGASSSKTNTLDMQSSGFDALLHEIQEIRETQARLEESFETLKEHYQRD YSLIMQTLQEERYRCERLEEQLNDLTELHQNEILNLKQELASMEEKIAYQSYERARDIQEALEACQTRIS KMELQQQQQQVVQLEGLENATARNLLGKLINILLAVMAVLLVFVSTVANCVVPLMKTRNRTFSTLFLVVF IAFLWKHWDALFSYVERFFSSPR
SEQ ID No.2
The protein sequence of another embodiment of a human NTRP (Human TMCC2) is provided herein as SEQ ID No:3, as follows:
MKRCRSDELQQQQGEEDGAGLEDAASHLPGADLRPGETTGANSAGGPTSDAGAAAAPNPGPRSKPPDLKK IQQLSEGSMFGHGLKHLFHSRRRSREREHQTSQDSQQHQQQQGMSDHDSPDEKERSPEMHRVSYAMSLHD LPARPTAFNRVLQQIRSRPSIKRGASLHSSSGGGSSGSSSRRTKSSSLEPQRGSPHLLRKAPQDSSLAAI LHQHQCRPRSSSTTDTALLLADGSNVYLLAEEAEGIGDKVDKGDLVALSLPAGHGDTDGPI SLDVPDGAP DPQRTKAAIDHLHQKILKITEQIKIEQEARDDNVAEYLKLANNADKQQVSRIKQVFEKKNQKSAQTIAQL HKKLEHYRRRLKEIEQNGPSRQPKDVLRDMQQGLKDVGANVRAGI SGFGGGVVEGVKGSLSGLSQATHTA WSKPREFASLIRNKFGSADNIAHLKDPLEDGPPEEAARALSGSATLVSSPKYGSDDECSSASASSAGAG SNSGAGPGGALGSPKSNALYGAPGNLDALLEELREIKEGQSHLEDSMEDLKTQLQRDYTYMTQCLQEERY RYERLEEQLNDLTELHQNEMTNLKQELASMEEKVAYQSYERARDIQEAVESCLTRVTKLELQQQQQQVVQ LEGVENANARALLGKFINVILALMAVLLVFVSTIANFITPLMKTRLRITSTTLLVLVLFLLWKHWDSLTY LLEHVLLPS SEQ ID No.3
The protein sequence of another embodiment of a human NTRP (Human TMCC3) is provided herein as SEQ ID No:4, as follows: MPGSDTALTVDRTYSDPGRHHRCKSRVERHDMNTLSLPLNIRRGGSDTNLNFDVPDGILDFHKVKLTADS LKQKILKVTEQIKIEQTSRDGNVAEYLKLVNNADKQQAGRIKQVFEKKNQKSAHSIAQLQKKLEQYHRKL REIEQNGASRSSKDI SKDHLKDIHRSLKDAHVKSRTAPHCMESSKSGMPGVSLTPPVFVFNKSREFANLI RNKFGSADNIAHLKNSLEEFRPEASARAYGGSATIVNKPKYGSDDECSSGTSGSADSNGNQSFGAGGAST LDSQGKLAVILEELREIKDTQAQLAEDIEALKVQFKREYGFI SQTLQEERYRYERLEDQLHDLTDLHQHE TANLKQELASIEEKVAYQAYERSRDIQEALESCQTRISKLELHQQEQQALQTDTVNAKVLLGRCINVILA FMTVILVCVSTIAKFVSPMMKSRCHILGTFFAVTLLAIFCKNWDHILCAIERMI IPR
SEQ ID No:4
The protein sequence of another embodiment of a human NTRP (Human Tex 28) is provided herein as SEQ ID No: 5, as follows: MVLKAEHTRSPSATLPSNVPSCRSLSSSEDGPSGPSSLADGGLAHNLQDSVRHRILYLSEQLRVEKASRD GNTVSYLKLVSKADRHQVPHIQQAFEKVNQRASATIAQIEHRLHQCHQQLQELEEGCRPEGLLLMAESDP ANCEPPSEKALLSEPPEPGGEDGPVNLPHASRPFILESRFQSLQQGTCLETEDVAQQQNLLLQKVKAELE EAKRFHI SLQESYHSLKERSLTDLQLLLESLQEEKCRQALMEEQVNGRLQGQLNEIYNLKHNLACSEERM AYLSYERAKEIWEITETFKSRI SKLEMLQQVTQLEAAEHLQSRPPQMLFKFLSPRLSLATVLLVFVSTLC ACPSSLI SSRLCTCTMLMLIGLGVLAWQRWRAIPATDWQEWVPSRCRLYSKDSGPPADGP
SEQ ID No:5
The protein sequence of one embodiment of a rat NTRP orthologue (Rat TMCC1) provided herein as SEQ ID No:6, as follows:
MEPSGSEQLYEDPDPGGRPQDAEARRQAESEQKLSKMTHNALENINVIGQGLKHLFQHQRRRSSVSPHDV QQIQADAEPEVDLDSQNTCAEIDGVSTHPTALNRVLQQIRVPPKMKRGTSLHSRRGKSEAPKGSPQINRK SGQEVASVIQSGRPRSSSTTDAPTSSSVIEIACAAAVCVPGEEATAERLEYFPGPLVAGVKAESQKSKQV NGKQYMLEEEASAWALDYMACKIERLEVSSLAQTSSAVASSTDGS IHTESVDGIPDPQRTKAAIAHLQQK ILKLTEQIKIAQTARDDNVAEYLKLANSADKQQAARIKQVFEKKNQKSAQT ILQLQKKLEHYHRKLREVE QNGIPRQPKDVFRDMHQGLKDVGAKVTGFSEGVVDSVKGGFSSFSQATHSAAGAVVSKPREIASLIRNKF GSAD IPNLKDSLEEGQVDDGGKALGVI SNFQSSPKYGSEEDCSSATSGSVGANSTTGGIAVGASSSKTN TLDMQSSGFDALLHEVQEIRETQTRLEESFETLKEHYQRDYSLIMQTLQEERYRCERLEEQLNDLTELHQ NEILNLKQELASMEEKIAYQSYERARDIQEALEACQTRISKMELQQQQQQVVQLEGLENATARNLLGKLI NILLAVMAVLLVFVSTVANCVVPLMKTRNRTFSTLFLVAFIAFLWKHWDALFSYVDRLFSPPR
SEQ ID No:6
The protein sequence of another embodiment of a rat NTRP (Rat TMCC2) is provided herein as SEQ ID No:7, as follows:
MFGHGLKHLFHSRRRSREREHQASQEAQQQQQHQGLSDQDSPDEKERSPEMHRVSYAVSLHDLPARPTAF NRVLQQIRSRPSIKRGASLHSSGGSGGRRAKSSSLEPQRGSPHLLRKAPQDSSLTAILHQHQGRPRSSST TDTALLLADGSNAYLLAEEAESTGDKGDKGDLVALSLPSGPGHGDTDGPI SLDVPDGAPDPQRTKAAIDH LHQKILKITEQIKIEQEARDDNVAEYLKLANNADKQQVSRIKQVFEKKNQKSAQTIAQLHKKLEQYRRRL KEIEQNGPSRQPKDVLRDMQQGLKDVGANMRAGISGFGGGVVEGVKGSLSGLSQATHTAVVSKPREFASL IRNKFGSADNIAHLKDPMEDGPPEEAARALSGSATLVSSPKYGSDDECSSASASSAGAGSNSGAGPAGAL GSPRSNTLYGAPGNLDTLLEELREIKDGQSHLEDSMEDLKTQLQRDYTYMTQCLQEERYRYERLEEQLND LTELHQNEMTNLKQELASMEEKVAYQSYERARDIQEAVESCLTRVTKLELQQQQQQVVQLEGVENANARA LLGKFINVILALMAVLLVFVSTIANFITPLMKTRLRITSTALLLLVLFLLWKHWDSLTYLLEHVLLPS
SEQ ID No:7
The protein sequence of another embodiment of a rat NTRP (Rat TMCC3) is provided herein as SEQ ID No: 8, as follows: MPGSDTALTVDRTYSDPGRHHRCKSRVERHDMNTLSLPLNIRRGGSDTNLNFDVPDGILDFHKVKLSADS LRQKILKVTEQIKIEQTSRDGNVAEYLKLVSSADKQQAGRIKQVFEKKNQKSAHSIAQLQKKLEQYHRKL REIEQNGATRSSKDISKDSLKEIQHSLKDAHVKSRTAPHCLESSKSSMPGVSLTPPVFVFNKSREFANLI RNKFGSADNIAHLKNSLEEFRPEASPRAYGGSATIVNKPKYGSDDECSSGTSGSADSNGNQSFGAGGAST LDSQGKLAI ILEELREIKVTQAQLAEDIEALKVQFKREYGFI SQTLQEERYRYERLEDQLHDLTELHQHE TANLKQELASAEEKVAYQAYERSRDIQEALESCQTRISKLELHQQEQQTLQTDAVNAKVLLGKCINVVLA FMTVILVCVSTLAKFVSPMMKSRSHILGTFFAVTLLAIFCKNWDHILCAIERI I IPR
SEQ ID No:8 The protein sequence of another embodiment of a rat NTRP (Rat Tex28) is provided herein as SEQ ID No:9, as follows:
MVLKVESTKSSSATFPPNVPSYRSLSSSHEDCPSSHTSFSDGELARNVREGVKHRIFYLSEQLRVEKASR DENTMSYLKLI SKADRHQAPHIRKAFERVNQRTSATIAHIERKLYQCHQQLKELEEGCSPTSLVLNVDSG MDSHKQPGGKILYSKLSKPDGEDSLPINVARSSTLESHLPGMQQRKFSDKKYVAQQQKLLLQKMKEELTE AKKVHASLQLSHQNLKESHMIDVRRILESLQEKKTRQSLMEEHVNDHLQRYLDEICHLKQHLACTEEKMA YLSYERAKEIWDVMETFKNRITKLETLQQATQLEMMASLRTRPKDFFFRFI SLLLTLTTILLVVVSTLCS CPLPLLNSRLRIFIVFMI IGLGTLAWQKRHVI S I IDWQAWVPFKWRSDLKDAKPPSARH
SEQ ID No:9
The protein sequence of one embodiment of a mouse NTRP orthologue (Mouse TMCC1) is provided herein as SEQ ID No: 10, as follows:
MEPSGSEQLYEDPDPGGKSQDAEARRQTESEQKLSKMTHNALENINVIGQGLKHLFQHQRRRSSVSPHDV QQIQTDPEPEVDLDSQNACAEIDGVSTHPTALNRVLQQIRVPPKMKRGTSLHSRRGKSEAPKGSPQINRK SGQEVAAVIQSGRPRSSSTTDAPTSSSVMEIACAAGVCVPGEEATAERIERLEVSSLAQTSSAVASSTDG SIHTESVDGIPDPQRTKAAIAHLQQKILKLTEQIKIAQTARDDNVAEYLKLANSADKQQAARIKQVFEKK NQKSAQTILQLQKKLEHYHRKLREVEQNGIPRQPKDVFRDMHQGLKDVGAKVTGFSEGVVDSVKGGFSSF SQATHSAAGAVVSKPREIASLIRNKFGSAD IPNLKDSLEEGQVDDGGKALGVI SNFQSSPKYGSEEDCS SATSGSVGANSTTGGIAVGASSSKTNTLDMQSSGFDALLHEVQEIRETQARLEDSFETLKEHYQRDYSLI MQTLQEERYRCERLEEQLNDLTELHQNEILNLKQELASMEEKIAYQSYERARDIQEALEACQTRI SKMEL QQQQQQVVQLEGLENATARNLLGKLINILLAVMAVLLVFVSTVANCVVPLMKTRNRTFSTLFLVAFIAFL WKHWDALFSYVDRLFSPPR
SEQ ID No: 10
The protein sequence of another embodiment of mouse NTRP (Mouse TMCC2) is provided herein as SEQ ID No:l l, as follows:
MKRCKSDELQQQQGEEDGAGMEDAACLLPGADLRHGEASSANSAGGPTSDAGAAVAPNPGPRSKPPDLKK IQQLSEGSMFGHGLKHLFHSRRRSREREHQASQEAQQQQQQQGLSDQDSPDEKERSPEMHRVSYAVSLHD LPARPTAFNRVLQQIRSRPSIKRGASLHSSGGSGGRRAKSSSLEPQRGSPHLLRKAPQDSSLAAILHQHQ GRPRSSSTTDTALLLADGSSAYLLAEEAES IGDKGDKGDLVALSLPSGPGHGDSDGPI SLDVPDGAPDPQ RTKAAIEHLHQKILKITEQIKIEQEARDDNVAEYLKLANNADKQQVSRIKQVFEKKNQKSAQTIAQLHKK LEHYRRRLKEIEQNGPSRQPKDVLRDMQQGLKDVGANMRAGI SGFGGGVVEGVKGSLSGLSQATHTAVVS KPREFASLIRNKFGSADNIAHLKDPMEDGPPEEAARALSGSATLVSSPKYGSDDECSSASASSAGAGSNS GAGPGGALGSPRSNTLYGAPGNLDTLLEELREIKEGQSHLEDSMEDLKTQLQRDYTYMTQCLQEERYRYE RLEEQLNDLTELHQNEMTNLKQELASMEEKVAYQSYERARDIQEAVESCLTRVTKLELQQQQQQVVQLEG VENANARALLGKFINVILALMAVLLVFVSTIAN ITPLMKTRLRITSTALLLLVLFLLWKHWASLTYLLE HVLLPS
SEQ ID No: 11
The protein sequence of another embodiment of mouse NTRP (Mouse TMCC3) provided herein as SEQ ID No: 12, as follows:
MPGSDTALTVDRTYSDPGRHHRCKSRVDRHDMNTLSLPLNIRRGGSDTNLNFDVPDGILDFHKVKLNADS LRQKILKVTEQIKIEQTSRDGNVAEYLKLVSSADKQQAGRIKQVFEKKNQKSAHSIAQLQKKLEQYHRKL REIEQNGVTRSSKDISKDSLKEIHHSLKDAHVKSRTAPHCLESSKSSMPGVSLTPPVFVFNKSREFANLI RNKFGSADNIAHLKNSLEEFRPEASPRAYGGSATIVNKPKYGSDDECSSGTSGSADSNGNQSFGAGGTST LDSQGKIAKIMEELREIKVTQTQLAEDIEALKVQFKREYGFI SQTLQEERYRYERLEDQLHDLTELHQHE TANLKQELASAEEKVAYQAYERSRDIQEALESCQTRISKLELHQQEQQTLQTDAVNAKVLLGKCINVVLA FMTVILVCVSTLAKFVSPMMKSRSHILGTFFAVTLLAIFCKNWDHILCAIERI I IPR
SEQ ID No: 12
The protein sequence of another embodiment of a mouse NTRP (Mouse Tex 28) is provided herein as SEQ ID No: 13, as follows: MVLKVESTKSSSATLPTNLPSYRSLSSFCEDCPSSHTSFSDGELARNVREGVKHRIFYLSEQLRVEKASR DENTMSYLKLVSKADRHQAPHIRKAFERVNQRTSATIAHIERKLYQCHQQLKELEEGCSPTSSVLKVGSG LDSHKQPSGKVSYSKLSKPGGEDSLPINVARSSTLESHLSEMQQRKFSDKKYVAQQQKLLLQKMKEELTE AKKVHASFQVSHQSLKESHMIDVQRILESLQEKKTKQSLMEKQVNDHLQRYLDEICHLKQHLACTEEKMA YLSYERAKEIWDVMEIFKSRITKLETLQQATQLEMMASLRTRPKDFLFRFI SLLLTLTTILLVVVSTLCS CPLPLLSSRLRIFIVFMI IGLGTLAWQKRHVI S I IDWQAWVPFKWRQDLKDAKPPSDGH
SEQ ID No: 13
The protein sequence of one embodiment of Drosophila me/atiogasterNT V is provided herein as SEQ ID No: 14, as follows:
MRHNSPVSRERASEAAAATQTAAATAGGATAHSAGGTAGGSAAATTTAGGATSGSGTASANTNSNSSASS STVAAAQAAVYSGGNTVTGSLGSAGVVARGFRSHSPTHRRRSRERQRRTHGSDQGGLLAYSGLVGVNDMT DFLGPQQGGGGGGGGGGGGGGGGGSAGTGSGLEDSRLSGNEDYYSSFVSDEFDSSKKVHRRCHERSSSVQ AIDRLNTKIQCTKES IRQEQTARDDNVNEYLKLAASADKQQLQRIKAVFEKKNQKSAH I SQLQKKLDNY TKRAKDLQNHQFQTKSQHRQPREVLRDVGQGLRNVGGNIRDGITGFSGSVMSKPREFAHLIKNKFGSADN INQMSEAELQGMQSANADVLGSERLQQVPGAGTSTGSGGGGQNNNTGGAGSGTGKFNSDNGSECSSVTSE SIPGGSGKSQSGASQYHIVLKTLLTELAERKAENEKLKERIERLETGQKEFNNLTATLESERYRAEGLEE QINDLTELHQNEIENLKQTIADMEEKVQYQSDERLRDVNEVLENCQTRISKMEHMSQQQYVTVEGIDNSN ARALVVKLINVVLTILQVVLLLVATAAGI IMPFLKTRVRVLTTFLS ICFVIFVIRQWPDVQDIGSGLVRH LKQSLVVK
SEQ ID No: 14 The protein sequence of one embodiment of Caenorhabditis elegans NTRP is provided herein as SEQ ID No: 15, as follows:
MTTDSASLKS SEGTCS S IGDEREKAKIVQKL IE IKDKLRALNEKREADVEKFLS I TRQSE I SRGVGADNP QRARIRNNFERQNRKHAHETEMLQKKL I DYEERLKLVDSGEYEPSPTKSRVFPTGIRKAKGMTETMVNAP IEFAQRVKSAFSADNVNSTQNGTTGAPKTGQSTFFTTRKSADTDEVESNAVHKNRGAKRNS STLPPNLSL TSPDPLSAYKEEDS SDPESRPGSAADETSNVPYHTADNSLYLPPNHPYHSAHAAPSEEGFNAIHEHLNS I LQHLML I DRKYDRLEDDIKKE IKFYAEALEEERFKTTKLEE I LNEAVELQQAE IATLKEQNLMATRVDYQ HNDRFRNVEENMESLQNHLVRIENALMDVRQVKLTSNVWQRVALNAG IVVELLKIALFVVAS I LDLVRP LTGSRNRSAMAFGLVFLAIFFGHHLQKVTYLFGGSTPDVNKTGGPTK
SEQ ID No: 15
The inventor has created a protein sequence alignment of the NTRP-related sequences (SEQ ID No's 1-15), which is shown in Figure 15 and illustrated schematically in Figure 2. The alignment shows that they are all highly conserved in the C-terminal end, but the N-terminal region varies considerably among family members, though it is conserved in homologues. The inventor has found a consensus sequence that defines the NTRP family, which is shown in Figure 16. To date, no one has appreciated that any of the embodiments of NTRP disclosed herein in any species (i.e. SEQ ID No's 2- 15), including the consensus sequence (i.e. SEQ ID No: 16), plays a pivotal role in APP metabolism or in γ-secretase activity.
The protein sequence of one embodiment of NTRP (i.e. a consensus sequence), is provided herein as SEQ ID No: 16, as follows, where X may refer to a variable number and/ or type of amino acids, for example, but not limited to, those found in SEQ ID's No.1 -15:
[X ] RXKXXXXXLXQKI LKLTEQIKIEQAXRDDNVAEYLKLANNA [ X ] DKQQXXRI KQVFEKKNQKS AQX I IQLQKKLEXYHRKLEEXE [ X ] QNG [ X ] PKDV [ X ] R [ X ] RDM . QGLKDVGA [X ] VKGGSTGXXXXXXXXX XXAVSKPREFAXL IRNKFG [ X ] SADNIXXLKSGLEEXXXEX [ X ] KALXXXXXXXXXPKYGSDDECS SXTS GSA [ X ] LXXLLEE IXE IKXXXXXLEE IQLELKQRYQRDYXL IXXTLQEERYRXERLEEQLNDLTELHQNE XXNLKQELASMEEKVAYQSYERARDIQEALEXCQTRXSKXXELQQQQQQQVQLEGXXXNAARXLLGKIXN XXXXXLAMVLWSTXXXXXXAPLMKTRXRXXXXXXXXXXXTVLWKXWDXXXXXXXXXXXXPK
SEQ ID No: 16
From a careful study of the consensus sequence of SEQ ID No: 16, the inventor has determined sections which are believed to be especially conserved between each of the embodiments of NTRP, for example those shown in SEQ ID No:16. Therefore, polypeptides having the identified conserved residues as shown SEQ ID No:16, in Figure 16, and schematically in Figure 2 may be capable of: (i) influencing APP metabolism; (ii) influencing the activity of γ-secretase; (iii) modulating the progression of neurodegeneration; and/ or (iv) binding to apoE.
In a fifth aspect, there is provided an isolated nucleotide sequence encoding the polypeptide, variant or fragment thereof according to the first aspect.
The DNA sequence encoding the polypeptide of SEQ ID No:2 is provided herein SEQ ID No: 17, as follows:
GGCGGCCGCAGTGGAAGGAGCAGGCGCTTGAGCTCGAGCGACGGCGCTGGCGGAGACGCCGGCTGCTCCT CCCCTCCCCGCCGCTTTTCCTAAAAGGATTGTACACCTTAGAAGTGCTTAAGGAAGAGTGATGAAGCTCT GAATCGTGTCCTGCAGCAGATTCGAGTGCCACCCAAGATGAAGAGAGGGACAAGCTTGCATAGTAGGCGG GGCAAGCCAGAGGCCCCAAAGGGAAGTCCCCAAATCAACAGGAAGTCTGGTCAGGAGATGACAGCTGTTA TGCAGTCAGGCCGACCCAGGTCTTCATCCACAACTGATGCACCTACCAGCTCTGCTATGATGGAAATAGC TTGTGCTGCTGCTGCTGCTGCTGCTGCATGTCTACCAGGAGAGGAGGGAACTGCGGAGCGGATCGAACGG TTGGAAGTAAGCAGCCTTGCCCAAACATCCAGTGCAGTGGCCTCCAGTACCGATGGCAGCATCCACACAG ACTCTGTGGATGGAACACCAGACCCTCAGCGCACAAAGGCTGCCATTGCTCACCTGCAGCAGAAGATCCT GAAGCTCACAGAACAAATCAAGATTGCACAAACAGCCCGGGACGACAACGTTGCTGAATACTTGAAGCTT GCCAACAGTGCAGACAAACAGCAGGCTGCCCGCATCAAGCAAGTCTTTGAGAAGAAGAACCAGAAATCTG CCCAAACTATCCTCCAGCTGCAAAAGAAACTTGAGCACTACCACAGGAAGCTCAGAGAGGTAGAGCAGAA TGGGATCCCCCGGCAGCCAAAGGATGTCTTCAGGGACATGCACCAGGGTCTGAAGGATGTAGGAGCAAAG GTGACTGGCTTCAGTGAAGGTGTGGTGGATAGTGTCAAAGGTGGGTTTTCCAGCTTCTCCCAGGCCACCC ATTCAGCAGCAGGCGCTGTAGTCTCAAAGCCCAGAGAGATTGCCTCACTCATTCGGAACAAATTTGGCAG TGCAGACAACATCCCCAACCTGAAGGACTCTTTAGAGGAAGGGCAAGTGGATGATGCGGGGAAGGCTTTG GG AG T G AT T T C AAAC T T T C AG T C T AGC C C AAAAT AT GG T AG T G AAG AAG AT T G T T C T AG T GC C AC T T C AG GCTCAGTGGGAGCCAACAGCACCACAGGGGGCATCGCTGTAGGAGCATCCAGCTCCAAAACAAACACCCT GGACATGCAGAGCTCAGGATTTGATGCACTACTACATGAGATCCAGGAGATCCGGGAAACCCAGGCCAGA CTAGAGGAATCCTTTGAGACTCTCAAGGAACATTATCAGAGGGACTATTCCTTAATAATGCAGACCTTAC AGGAGGAGCGATATAGATGTGAACGATTGGAAGAACAGCTAAATGACCTAACAGAGCTCCACCAGAATGA AATCTTGAACTTGAAGCAGGAACTGGCAAGCATGGAAGAAAAAATCGCGTATCAGTCCTATGAACGGGCC CGGGACATCCAGGAGGCCCTGGAGGCATGCCAGACGCGCATCTCCAAGATGGAGCTGCAGCAGCAGCAGC AGCAGGTGGTGCAGCTAGAAGGGCTGGAGAATGCCACTGCCCGGAACCTTCTGGGCAAACTCATCAACAT CCTCCTGGCTGTCATGGCAGTCCTTTTGGTCTTTGTCTCCACTGTAGCCAACTGTGTGGTCCCCCTCATG AAGACTCGCAACAGGACGTTCAGCACTTTATTCCTTGTGGTTTTTATTGCCTTTCTCTGGAAGCACTGGG ACGCCCTCTTCAGCTATGTGGAACGGTTCTTTTCATCCCCTAGATGATGCTGGCACAGAAGGCATTGTTC CCTACCCTCTGGCGAGTGCATGCAGCAGAGAGTTAGACAGCAACTTACCTACTCTGAAGTTTTCTACAAC AAAAAAAGAGTTGAGTGAATCTGTTTACATTTAGAATAATGTTTTTTTCTTCAAGAGACGCAATTGCAAT AGTATTTTTTAGATTTTATCCAAGAAGTTTTTTGGGCGAAAATCTTGGATCATTTTTATGTAGCATGATT TTCCTTGGGATGCAAATCTTAAAACAGTCCTTTAATATGAACCAACAATCTGGAGCACACCGAAGGGCAA TCTAAATTGTGGCTTGAAGGACTGCACTAAAACCCACTAAAAAGATGCGAAAACCTGATGAGGGCAAACC AGTTAAACCTAACACCCTGCCTTGTCTGGGCTCATCACCTCTCCCTATCCCAGACTAACTTTACTGTGAA ATCCTACCACATTCCATGTCTGAATTTTTGGATTCGGGGTGGATTTTCGTTGTCCGTGGAAGAACACATG GATCTCTCTGGCTTTCTCACCCAAGTTGGCCACTTACGCTAATCCTGGAAGTATGATCACTTTTGAACCT GCCCCTTAACCTTGACGAGGATACAAAAGTGAAAGCATCATCCCCCAAAGGATCACTGCACAGTCCTACT ACAGTATTTTTAAGTAGCCCTCTAAATACTTAATTTTAAGCAAAATCCCTTGGCCGCACTTTTAAGGTTT TTTTATATGTGTATAGTTACCAACCTAAAAATAAAAAATCCGAACAGCATACTTGAAGAATGTAATACTC AAACTCTCAGTGCTTCCTTATGGTTTCTAATAGGATTTTTTATTATTGTTATTATTATTATTGGGTTTTT TTGGACAGGGTTGGGAGGGTCTTTTATTTTTCCTTTGAAATAAAGAAGTGATGTTTTTAAATGAAGAAAT GTGTGGATATTTAAGTGTGCTGCTCCCTCTTGTCTTGAAACAGTTTGAGTAAGAAAGTCTTGCTGTAAAT GCTGCCCTCTGCCGCCTTTGTTTTGAGATGCAGTTTAAACTCCCTCTGGCTGCTGCTGCTGCTTTTTGGT GTCCCGACATACCTACGCCCCCGTTTTATGGGTTTGGCTTAGTTGAAGAGGAAAGGGTTGTGCAAGGAGA GCAGGAGGCTGTTTCCAAAAACCAGTGTAGTAGGATAGGGATTTTTTTTTTTTTTTTTTGCCCCAAGAAA ACGTTCACCCAGTGATCTTGGGCTGGGGTTGTCTTTAGGAAAAGTTGAGAC TATAAGAGTCATAAATAAG TCCTTGTGTTTCCTTAATTTATTTTGTTAACACCCCTAATTACAACCAAAGTGATGATGTGGAGTCTTCT GTCTTCATTTTGGCCCCAGCATTCTTAATTTCAAAGCTTTATTCTGTCTGCCTAAGAGAATCAACCAAAG GTGATTCTCCTAAAGAGCAGTGAAGGAAATGTCAGGTTAGCAGGACCCAAGTTTTGGGTGTGAAATGTTG CCAGCTTCCTATAATGTAAACGGACTTGTTAACCTAACCTAATTATGCTCAGTGGACTTCTATAGATGGT TTTGAAAAATGAACTGAGCTGCCTTCCCCCGTCGCATAACCAGTTCCATCATCCTGGTGGAACTTGAACA TTTAGAGTTTATCTAGAGAGCTTGGTTAATCTTTCCATATTATTTGTAGTATTGGTCACAAATGCTGTTC CCTCTTAGCCTCATTCTGTGCAACCAAGTGCATATAAGATGCCCTGAAAAGAGTAACAAAGTATGCTTTG CCTGTTTCCACTTACCAGGAAATTCCTTCAGAACTAGATTAGCATTGCCCTGCCTGTCTGAAAGGACAGT TTACCTAATGGTGCCAGCCTCCTTTTGCTTTGGCAAGCTGGATTTCTCAGAGCCAGCATGTTGTTTCCAT AACTACTTTGATATTTTAACTCAGGTACTCCAGTCTTCACCCCAACCTCAGCTGATTGTAGTACACCTGC TAGCTCTGTTGCCCCCTCAAAACTGCACCCAGAGCAGGGCCACAAGGGTGCTTTTTTTCTTTAAAAAAAA AAAAATTAGAACCAATTCATGTTCATGCCAAAAACAAATTGTCCCCAAGCCTATATGTATTAAAATGTTA ACTTTGCCTAAAAATATTGCAGTGACTTTTTAGGCAGGAGTGCCAAAGGACACTATGAACTTTTTGAACT GACAGTTTCTCCTAACTTTCTGCTTTAGCGTAATTGCTCAGAGTAGAGAGCCCCCACAAAGTTATTTAAA AGATGCCCTAGCAGCAATCCACCAGTTTTTCTAAGCTAGAACCTTTGAGTCCCCCAAACTGCCTGAAGAC TTAAGTTTTGTGGGCACTGGAAGTCACTTTGATAGATGGATTGAAACTGTTCCTATTTGCCCTGGGACGG TTTCTATCTATCAAAGGAAGGTTTTCACCTGTAGAAAGCCCCCTGCCTCCAGCCAAATAGTCCCATGCTG ACTTTCTATCTTCCTTTCTCAAACTGTCTTAGGAAGGACCTTCAGTGCAGATCAGGTGCAGTAATGGCTT TCTTGTCCCTTAATTATTCACCAGACCCAGAAGTTGTACGCATTTAATGCTGTTTGTAACCATGCATCTG TTTTCATTCTTTGCTGTACCTTTTGCTGCCCATCCTGTTACTTTTGAGTTTCTTTCATTGTGGTTGTTCT TGGGTTCTTTTGTCTTGTCAGAGCTCTTCTATAACCTCGCTCTAATGGCTTAACAGTTGTTCTGGGTGGA AAC G T C C C C T C AT T T G AAT GC T C C T C T AAAAAAAAAAG AAAAG AAAAC T G T AT T C AT T C C C T T T AAAAT G AAACATTCCTGGTTTATTTGTCCATGCCTCTAGCCTGGGTGAGTGAAGCTGGCTGTGTTGGCCTGGGTGA TTGTTCAGGTTGTAGAATGCGCATTTTACATTGTTTATGATAATTGAAGGGTACTTCTGTCTGCTCCAAT TTCCATTCCTTGGTATACTCAGTATGTCCTAGTAAGGAAGGCTTTCCACTCTACTGGCTCCTTAAGAAGC AAAAGTAGGTTAAATTTTATACTTCACTGACTAGGGTCTTCTCTCTCCCCTGTTCTGAATGGAGTAAAAG TCTGATGCCAAGACAAATATTGGGAGCGAGCCTTCCTCACTAGCCATGTCCAAATAATGAGCGTATTTTC ATGTGGTCTTCACTGCATTTGGTTTTGTTTCTGATTTCATGTTCCTTTGAGGTACAGTCAGATGAAAATG CTGAGTTCTGAGAGAGTTCCAATGAGGAGCTGCCTTTCAGCTTTGGAAAATATGCAACTAAAACAAAACC AAACATTATTGTAATCTGACACAGGCAAAATTATGGTTCCCACACCCCAACCCCAAATGAAACCTGGGAT TTTGAATGTGGCTCTAAGAGTTAACATTGCTGTCTGTATGTCGTGTATGCTAGGTGATATCCTCAGTAGG GATTGACTACTAGACTGTGTGTTTTATCAAAGTGTGTAAGAATAAAAACTCACTTGCACGAATTGAAACG TAAAGAAATGACACTTGTGAACGTGTGAACGTTAACACTGTAGTTGATAGATCTTAAGCTGCTAATTGTT GAGAGAGATTTAAATTTATGTTCTGTCTGGTCGCTGCAGATTCTCAGCAGCACTTTTACTGTATATAGAA AT T G AAAT AAAG AC C T C AGC T AT T AAAAAAAAAAAA
SEQ ID No: 17 The DNA sequence encoding the polypeptide of SEQ ID No:l (partially) and SEQ ID No:3 is provided herein as SEQ ID No:18, as follows:
CCGCTCCCGGGCGGAGGCAGCCGGGCGGGGTAGGTTGCGCGCTCGCCGCGGGCTCGGGCCGCGGTCGCGG CTTTGCGGCAGGCTGCGCGTCAGGCGGGGAGCGGGGCGCGCGGGCCGGGGAGGGGGCCGGGCGCGCTGCG GGCTCCGCGGCCGGACCATGCGGGGCAGGGGCCGGTTGCAGGGCCGGGGGCTGCAGCCGGCGCCGATGGC GGCCGACTAGGACCTGCCCGGCCGGCTGCCCCGCGCCCCGCCTCGCCCCGCAGCCCGGCCGGCCGGGAGG GATGCGCTGTGCCGCCCAGCTCCTCTCCGTCCTGCCCATGCCCTGAAGCAGAAAGTTTGGGGGCCGGGGG TTGTCTCCCTTCTCCCTCCTGCAATGACTGCCCAAGGACTCTTGCTGCCCAGCCTCGACTGTGACCTGTC TTCGCTCCCCAGGTCGAAATGAACTATTCCAAGCTATAACCAAGGCTCCCCCTTCTCGCCCCTCCCTCAC CCGCCTTTAAGAATTTTTTTTTTAATTCAAGAAATTGTGGTCTGCCATCTCCCCTCCTTGTTAATAATTT AGACCCCAGGCCTCATATGAATATAAGAGGGGGTGCGGTCTTCCCCAAGACGGCGCGCTGGAAGGACAGA TTCCCCTTGCCGACCCACATACACCATGAAGAGGTGCAGATCGGACGAGCTGCAGCAACAACAGGGCGAG GAGGATGGAGCTGGGCTGGAAGATGCCGCTTCCCACCTGCCGGGCGCGGACCTCCGGCCTGGGGAGACCA CGGGTGCTAACTCTGCTGGCGGGCCAACTTCAGACGCCGGCGCTGCCGCGGCGCCCAACCCAGGTCCCCG AAGCAAGCCTCCTGATTTAAAGAAAATCCAGCAGCTGTCAGAGGGCTCCATGTTTGGCCACGGTCTGAAG CACCTGTTCCACAGCCGCCGTCGGTCTCGGGAAAGGGAGCACCAGACGTCTCAGGATTCCCAGCAGCATC AGCAGCAGCAGGGTATGTCCGACCATGACTCCCCAGATGAGAAGGAGCGCTCTCCGGAGATGCATCGCGT CTCCTACGCCATGTCCCTGCACGACCTGCCCGCCCGGCCCACCGCCTTCAACCGCGTGCTGCAGCAGATC CGCTCCCGGCCCTCCATCAAGCGGGGCGCCAGCCTGCACAGCAGCAGTGGGGGCGGCAGCAGCGGGAGCA GCAGCCGGCGCACCAAGAGTAGCTCCCTGGAGCCCCAGCGTGGCAGCCCTCACCTGCTGCGCAAGGCCCC CCAGGACAGCAGCCTGGCCGCCATCCTGCACCAGCACCAGTGCCGTCCCCGCTCTTCCTCCACCACCGAC ACTGCTCTGCTGCTGGCCGACGGCAGCAACGTGTACCTCCTGGCTGAGGAGGCCGAAGGCATCGGGGACA AGGTCGATAAGGGAGACCTGGTGGCCCTGAGCCTCCCCGCCGGCCATGGTGACACCGACGGCCCCATCAG CCTGGACGTGCCCGATGGGGCACCGGACCCCCAGCGGACCAAGGCCGCCATTGACCACCTGCACCAGAAG ATCCTGAAGATCACCGAGCAGATCAAGATTGAGCAGGAGGCTCGCGACGACAATGTGGCAGAGTATCTGA AACTGGCCAACAACGCGGACAAGCAGCAGGTGTCACGCATCAAGCAAGTGTTCGAGAAGAAGAACCAGAA GTCAGCCCAGACCATCGCCCAGCTGCACAAGAAGCTGGAGCACTACCGCCGGCGCCTGAAGGAGATTGAG CAGAACGGGCCCTCGCGGCAGCCCAAGGACGTGCTGCGGGACATGCAGCAGGGGCTGAAGGACGTGGGCG CCAACGTGCGCGCAGGCATCAGCGGCTTTGGGGGCGGCGTGGTGGAGGGCGTCAAGGGCAGCCTCTCTGG CCTCTCACAGGCCACCCACACCGCCGTGGTGTCCAAGCCCCGGGAGTTTGCCAGCCTCATCCGCAACAAG TTTGGCAGTGCTGACAACATCGCCCACCTGAAGGACCCCCTGGAAGATGGGCCCCCTGAGGAGGCAGCCC GGGCACTGAGCGGCAGTGCCACACTCGTCTCCAGCCCCAAGTATGGCAGCGATGATGAGTGCTCCAGCGC CAGCGCCAGCTCAGCCGGGGCAGGCAGCAACTCTGGGGCTGGGCCTGGTGGGGCGCTGGGGAGCCCTAAG TCCAATGCACTGTATGGTGCTCCTGGAAACCTGGATGCTCTGCTGGAAGAGCTACGGGAGATCAAGGAGG GACAGTCTCACCTGGAGGACTCCATGGAAGACCTGAAGACTCAGCTGCAGAGGGACTACACCTACATGAC CCAGTGCCTGCAGGAGGAGCGCTACAGGTACGAGCGGCTGGAGGAGCAGCTCAACGACCTGACTGAGCTT CATCAGAACGAGATGACGAACCTGAAGCAGGAGCTGGCCAGCATGGAGGAGAAGGTGGCCTACCAGTCCT ATGAGAGGGCACGGGACATCCAGGAGGCCGTGGAGTCCTGCCTGACCCGGGTCACCAAGCTGGAGCTGCA GCAGCAACAGCAGCAGGTGGTACAGCTGGAGGGCGTGGAGAATGCCAACGCGCGGGCGCTGCTGGGCAAG TTCATCAACGTGATCCTGGCGCTCATGGCCGTGCTGCTGGTGTTCGTGTCCACCATCGCCAACTTCATCA CGCCCCTCATGAAGACACGCCTGCGCATCACCAGCACCACCCTCCTGGTCCTCGTCCTGTTCCTCCTCTG GAAGCACTGGGACTCCCTCACCTACCTCCTGGAGCACGTGTTGCTGCCCAGCTGAGTGGCCAGCCACACC AACCCTGTGCTCTCTGGCCCCCAGCTGGCCACACTTCTCCAGGAGGGACCCTTGGACTTCTTTGTGTGTC CAGTTTGGCCTCCTGCCCAAACTGTCCATTCCAGCAGCTCCTGCCCCCTTCTCTGTACTTGCTTCTGTCT GACACCTTCTCCCTGTTGGCCTGAAGGGAGCTTAGAATGCAGCCCTACCTGGAGATAGTGCGGGCACCTG TGGCCAAGTGGAGCAGAGGTGGACATGGGGTTGGATTGTTTTGATTATTTATAGTTACACAAGGACTTCT CCCAGCTGACCCTCAGGATGCCCCAAGTCAGGAAGACCATTAAGAATAGGAGGAGAGGGCTCTGCCTCAA CTTTCCTAGGAAAGAGCCCACCTCGGAGATAGCTACGGTTTTCTCTGGTGGAGATGGTGAGGATGAAGGC TGGAGAGTGAGGGAGGAGGCTCTGCTGGCCGCAGAGAACACAGGGATGGGAGGGTCCCTAGCCTTCGGGC ACCTCCAGGGCCAGAGAGCAGGCTCAGAGCAGCTAGTGTGGAGCTCAGCATCCCCACCCCACCCCTCCTC CCTGTAGAGCTGATTTGAGGCCTCCTTCTGGGGCTGGGCTCTGCAGGCCAGGTGGGTGTGGCCTGTGTTT TCCCTTCTGTTCTTTCTGCCTGTACTGGATCTGTTATTTTCAGGGAAACAGGCCCCAGGGCCCCCCTGAG CCTCACCCTAAGCCCTTAGGCCTCTGAGAGTGCTGTTGGGTTCTATTTATTTATTTATTTGTTCCTTTGT TCCCTACCCGTGCCCCCAGTGTCTTCCCTGCTGAGTACCAGGAGAGGTCCTGCCCCATCCTCTCTCTGAA GCCAGGGCCCTTCCATTCCATTTAGCCTTTGGATCATCCTGGCTGGGAGAAGTGGGACCGAGCCACCCAG CCCCACTATCCCCAAGCAGCCCTACAGCCGGGATGGGAGGCACGTGGCCTCTCTTTTATCCGTCTATTTA TTTTGTAAGTGTATTCGTGTGGAGGAGGTTGTTGCTTTATTTTTTTAAGGCTCTGGAGTGTTGTGTATGG TTTCTTTTCACATCCCAGCCTCCCATGGGCACTTCTAAGAAGAGAGGGGATTTCTTGGAAAAGGAGAGAG GAATCCCCTAGAGCAGGGAAAGCAGTGCCTGCCAGCTGTTGTGCACCTTCCTGAGAAATAAATATCCTCT AAAT T T T C AAAC C AAAAAAAAAAAAAAAAAA
SEQ ID No: 18 The DNA sequence encoding the polypeptide of SEQ ID No:4 is provided herein as SEQ ID No: 19, as follows:
AGCTGCAGCAGCCGCGCCGCCCCCTCGCCCGCGGCTCCCAGGGTGATCCCGAGCACAGCCGGTGGCTCGC GGCGCGGCAGCCCCAGAAGACGGGAAAGTTCGCGGCCGGAGCGCGGAGATGCCGGGCAGCGACACGGCGC TCACCGTGGACCGGACCTACTCGGACCCCGGCCGGCACCACCGCTGCAAGAGCCGGGTAGAACGTCATGA CATGAATACCTTAAGCCTGCCCCTGAACATACGCCGAGGGGGGTCAGACAC CAACCTCAACTTTGATGTC CCGGATGGCATCCTGGACTTCCACAAGGTCAAACTCACTGCAGACAGCCTGAAGCAAAAAATTCTAAAGG TAACAGAGCAGATAAAAATTGAGCAAACATCGCGCGATGGGAATGTTGCGGAGTATCTGAAACTAGTGAA CAACGCAGACAAGCAGCAGGCGGGACGTATCAAGCAAGTCTTTGAGAAGAAGAATCAGAAATCAGCTCAC TCCATCGCCCAGCTGCAGAAGAAGTTAGAGCAGTATCATCGAAAGCTCAGAGAGATCGAGCAGAATGGAG CCTCTAGGAGCTCAAAGGACATTTCCAAAGACCACCTGAAGGATATACATCGCTCTTTGAAAGATGCCCA CGTGAAATCTCGAACTGCCCCCCATTGCATGGAGAGCAGCAAATCGGGCATGCCAGGGGTCTCACTTACT CCACCTGTGTTCGTTTTCAATAAGTCCAGAGAGTTTGCCAACCTGATCCGGAATAAGTTTGGCAGCGCCG ACAACATTGCTCACTTGAAAAATTCCTTAGAGGAGTTTAGGCCAGAGGCGAGTGCCAGGGCCTACGGGGG CAGCGCTACCATCGTGAACAAACCCAAGTATGGCAGTGATGATGAATGTTCGAGTGGCACGTCAGGCTCG GCCGACAGTAACGGAAACCAGTCGTTTGGGGCTGGTGGAGCCAGCACACTGGACAGCCAGGGCAAGCTCG CCGTGATCCTGGAGGAACTGAGGGAGATCAAGGATACCCAAGCTCAGCTGGCTGAGGACATCGAGGCACT GAAGGTGCAGTTTAAGAGAGAATATGGTTTTATTTCTCAGACCCTGCAAGAGGAAAGATACAGGTATGAG CGACTGGAGGACCAGCTGCATGACCTGACGGACCTGCATCAGCATGAGACAGCCAACCTGAAGCAGGAGC TGGCCAGCATTGAGGAGAAGGTGGCCTACCAGGCCTACGAGCGCTCGCGGGACATCCAGGAAGCCTTGGA ATCCTGCCAGACTCGCATTTCTAAGCTGGAGCTCCACCAGCAAGAGCAGCAAGCTCTGCAGACAGACACC GTGAATGCTAAAGTTCTCCTGGGGAGGTGCATCAACGTGATCCTGGCCTTCATGACTGTCATCTTAGTGT GTGTGTCCACCATCGCGAAGTTCGTCTCACCCATGATGAAGAGTCGCTGCCACATTCTTGGCACCTTCTT TGCCGTGACTCTTCTTGCTATATTTTGTAAAAACTGGGACCATATCCTGTGTGCCATAGAAAGGATGATA ATACCAAGATGAAGCCACTGGTTCCTGCCTTCAAGTTCTTTCAAGTTTTTATTTTAAAGAAAACTCTGTG CATACTACCAAATTTTACAGTGAATGATTGTGCGGACTCGTGTGTAAGAAAAACTAGGACTGTGTGGTGT AAATAACTACAATTCTCTTAACTCCGTAGCAGTTGCCAACTCAGTCCTTGTACTTCGTTAACACGAATCT GTTTCAGAGCTCTCCTACCTTGCTCACTGCCTTAATCAGACCGATTTCCTGCCCACCTGACCAGCCCAGC GTGGTAAACCTCTGTATATTGAGACCTTGGCATAATTGGTGATCCTGAAGAAAGAGGTCTCTCTCCTAAG TCTCTGTCAGAATTGAGCTTCACAATTGCTAATGGTTGTTTTCTGTGAGTCCTATAAAAAGCAAGGATAT GCATGATTCAGGGAATGAAGAATCACAGGCTTGGGCAGTGTTAAACACTGTGGCCTATGGTCCCCGTGTG ATCCACCCTGCTTCTCTCCAGGGGACCATAGGTCCCGTCATGTACTCAGTGTCCACAGCAGTCAGTCGTG TATGACCCTGTAACGTGGAAATCTTATCACACACCTGTTATCCAACAAGTCTACCTGAGGGGTTTTGTTA CACTTTAAATGGGAAGGCATAGGGATTTATGAATGGGGCTTTCACCTTCTCATACCCAGGCAACCAACAC CTGATTTTGTCTCAACTGGCTAGCAAATGCCCAGCCTTCAGAGTGTGCAGGAATGTTTTCAAATCCCTCA TCAGACTGTGACTTTAACATTAATTTGGAATCCTGTGAGCACTACTCTGAAGGTTTGTGTTTTGGCAAAT CTTTTTTCTTTTTTGAGACAGGGCTCTGCTAAATATTGCTCAGGCTGGTTGCAAACTCCTTGCTTCAAGG GATCCTCCCACCTCAGCCTCCCAAGCAGCCGGGACTGCAGGCACAAGCCACCATGCCTGGCTGTTTTTTG GCAAATCTTGATTGTGATAAGCCCCCCTGGAGGATATGATTCACTTTATGTGATTCATCTTATTCACAGG TCTGTGAGGGACTGCAAAGCTTACTCAGGAAATGAAAACAAATGATGGTCATGTTGCAGTTTTTTCCTTG AAGGACAACCGAACCATAGCCTCTAAAGTTCAAGTGCACTGAGGTGTCGGAACGCTGAAAGCATGAGGAA ACGAGGACGTAGGGTGTGACTGAATGGTGGCTAGATTAGTGGGAGCAGTTCACCTGGATGAAGATTGAGA GCATCGTCTTT G AG AAG T G AAAG AC T AGC AAG AAT AAAAT AAAT TAAG TCCAGTGTTT GAGCCAAGGT T G CCACCTGTCTCTTAACATCTCACTGAACATAAGTCCTGAGGTATTAGGACGACCATACTGCCTCTGAGCT GAAAACATTCAAAAGTTCACATCCCTGTTTGGGGGATACCATTCACCGCCTTCAGCCCAGATGATACTTT CCTTTAAATCTGTGTCTCTGTGTGTATAACAAAGAGGAAGATGGAAACAATGTTCATGGAAACTGCTGTT GAGCCCCTTGTCCCACCACTCCCGCCATCTGCTGCAGGCAGGAAGGCATGTGAGTGTACGTTTTCTTCCA GGAGACATCAGGTCCCCCTGGATTCAAATTAAGTGCAATATTTTGCAAACAGCTCTTCTTAGGGAAATCT C C T G AAGG AAAAAAAT G T G AC AG AAT GTTCCATAGTCT G AG AG AAT GG AAT C G T T G AGC AT T T AG T AC AA GTCCAGTGTGTGTGAGCGGGACTTAGGCAGCTCAAGCTTGCTTTTTTTTTTAAGCGTACAATTGAGTGGT TTTAGTAAATTCACAAACTTGTTCAACCATCACCACTATCTAATTCCAGACTCACGCTTTTTTAAACAAT AAATGTCATTTCATGAAATCTTTGGTGATAAAGTATTTTGGATTCAGAGAAGAGCTCCCTTACCAGTCCC ACCCTGATCTCATGGCTGTCTCTCCTTTCATTGTCAGACTCCCCCTGGTCTACCGCGTTGATGTGTATAC ACTGATCTTTCAAGTCTGGGAGACAGATAAGGAGGCCAGGTGCAAGGCAGGGAGGCAGAGAGAATGTTGT GCTTCCTTTAGCTTTTGTATTTCGATGGCCAGCATTACCCTTTACCTGTGGGCATCAGACTCAGCGTGGG CTGAGTGCTGAGTGTAACTTACACTCCTAAATCAAGCTGGGGCCTGGGTGGGCCCCTCTTGGTATCTGTG AATCTTTCCAAGCACCACTTCGGACACACCAGGGATTGAGTGCTGCTGTTAGTTTAGAGAAGGAGAGATG TCTAACCCTTGAGGTGAAGGGCTCTGGGAGGGTCCAAGAAGACGTAGGCTTCATTTTCACACCAGCCCAC ACCATTCCAGTGCTCAGCCTAGCAAATGTGCTTTAATGCACACTTCTCAGACCTGTGATCCGTGTATCTT CTCCCCAGTGACAGAAGTAGAGAAGAGAATGGAAAGCAGCACACTCCGTCCCCTCTAGTCTGGAGCTGTT AACAGAATCTGCTAGAAACTAGCTTTATTCTAACATACCGTAGGATCTAAATCCTCCTACCTGGATCATG AATTCCTTTGAAATAATTCATATTTTCATTGACTCTCACTAAATGTCAAATAACCTTGTTTTCACTTGGA TAGGCTCAACCTACCTGGCATATTTATTTTGCAGTCTTGTTGAAAGTTCATGAAACTTTGTACTTTTTAA T AAG AT GAT AC AC T C GAAGGAAAC TTTTAATCTCTGCAGTTTATTCTCTCT T AAGG AAT AAAC AC T C C C A CTGTTTGTTCTCTTCAATGTGTAAGGAGATTAAATGACATTTTAGAAATATTACAATTAAAAATAGTGAT GTAGCTGTAACATATGCTGGAATTGGATATTTAATTTATGTTTGTGTCAAC TATAATCCTTTCCCCACCC CTTTCATTTATGGTAAACATCTTGGGCAAACCCAAAGATGGAAAGTGCTTGTTGGGTGGGTAAGCACCAC CTGGTCTCTCAGCAAACACTCCTGAGTGGTTGAAGATGCTGGACATTGGATTCTAGCACTGGGTTTATCT GGTGACATAGTCTCCTGTGGGTCTTGAGTTGGTTATTTCAAGCTCAAACTCTGAATATGATTAAACCAGA AC AC C C C AC C C C C AAC T GC C AAAAAAAAAAAAAAAAAAT GG AAAT C C AT AAT G AAAC AGC AAAAAAG T AC TCCGTTGCTGGGTTTGGACAATGAACATGGGATTAGTCCCAAGTTTGTAGCTTGGAACGGTAGCATTTTT GCCTGCGTGATCCTTGTCAGCTATTCACAGAAGGAAATCTTCCGAAACTCCGTCTTTCATTTCAGCCGGT GAGGCTGTTATCCTTCTCTGTCAATTAGCATTCATGTGGTTTTCGCTCTTTCCAGCTCTGTCACTCTTGT TTTCATTTAAATATTGCCCCACTCTGTGTTTATTGCCTTGCCATTTCTCTAGCATATCAGGTTTAATTAT GGGCTCAGTAATAATGAGGAACTAGCTTCCCTCAGGCAACTAATTACTTTTGTCTTTTTTGGTCGGATTG TACCAAATTATTTTGCATTGAATGGGAATGTTTTTGCAGTGAATCCAGATATAGTTGTATTGGTTTGGAA AAACATTTAAATATATTTATTCATATGAACATGTGTGATTAAACAACCTATTTTACATATTTCTGTAATT CTGAGGAGTGGGCTGGGGGTAGGAAGAAATGGCACCACTTAAAATTCAGGGCTTATATGAAGAGGTGTTT TAAGGTTAGCACATGCACAAAGGAACGAGTTGGTTTAAAAAGATAAATCACTGCAAAGAATGAAATTGGC TTATTCACATCAAAACTAGATAAGATGCTAAAAAAAAAAGATATGAAACAGAACTAACCTATAGTTTCCT GAAATCAGTACAGTTTAATTTATAAGAAGCTAGAAAGTAATGCACCTTGATTGTTTTAGGAATGATTTAT GTGTTGCAATTTTAATTTATTTAAAGCATGTCTACTGTGTTTGTCCTAAGAGAAATATTTCAACAAAACG TGCTCTGTGTTTAAGATATGTTTAGGCAGTAGTTAGCAACTCTGAAAGTAGAAACTGGAAATGTTTATTG TGAGGCTTGTTGCAGAATTTCCATTTTGTGAGTTACTACTTAGTTTCATGTCAGCCTAAAATTGTAAATT CCCTGTAGATCTTCACCCCATTGTGGTGTCATCAATGAATCCAAAGCAGGTGCCATTATTTTTTTAAATA AACACTTGATGTTAGCTTTGGTGTTAAATAAAGAGGTAGATTTCTTAAATTTTAAAA
SEQ ID No: 19
The DNA sequence encoding the polypeptide of SEQ ID No:5 is provided herein as SEQ ID No:20, as follows:
TGGTGCCAGCACTAGCCCCCATGTCGGTCTCAGAGAACCTTCTCCCCACCTCTGAGTTATTCTCTCAGTG TATCGAAGATATCAGTCAACTATCTTCTGGATTGCATTGATGCTATTGAGAAGGCAGCCTGCAGTCTAAA AGTCATGGTTTTAAAGGCGGAACACACCAGGAGCCCCAGCGCAACCCTCCCCTCCAATGTGCCTTCATGC CGGTCCCTGTCATCCAGCGAAGACGGCCCCAGTGGCCCTTCCAGCCTCGCAGATGGAGGCCTAGCCCACA ACTTACAGGATAGTGTCAGGCACCGCATCCTCTACCTCTCAGAGCAGCTGAGAGTGGAGAAGGCCAGTCG GGATGGCAACACTGTGAGCTACCTCAAGCTGGTATCCAAAGCAGACCGGCACCAGGTGCCGCACATCCAG CAGGCCTTTGAGAAGGTGAACCAGCGCGCCTCTGCCACCATCGCCCAGATCGAGCACAGGCTCCACCAGT GTCACCAGCAGCTCCAGGAGCTGGAGGAAGGCTGCAGGCCCGAGGGCTTACTGCTGATGGCAGAAAGCGA CCCAGCCAACTGCGAGCCACCCAGTGAGAAGGCCCTGCTTTCAGAGCCCCCCGAGCCAGGTGGGGAAGAC GGGCCGGTCAACCTGCCTCATGCCAGCAGGCCCTTCATCTTGGAGAGTCGCTTCCAGAGCTTACAGCAGG GGACGTGCTTAGAGACAGAGGATGTGGCCCAGCAACAAAACCTGCTGTTGCAGAAGGTAAAGGCAGAGCT GGAAGAAGCCAAGAGGTTCCACATCAGCCTCCAGGAGTCCTATCACAGCCTAAAGGAGAGGTCTCTGACT GACCTGCAGCTGTTGCTGGAGTCCCTTCAGGAGGAGAAGTGTAGGCAAGCATTGATGGAAGAACAGGTGA ATGGTCGCCTGCAGGGACAGCTGAATGAGATTTACAACCTCAAACACAATCTGGCCTGCAGCGAAGAGAG AATGGCCTATCTATCCTATGAGAGAGCCAAGGAAATATGGGAGATCACGGAGACCTTCAAGAGCCGAATA TCCAAGCTGGAGATGCTACAGCAAGTCACCCAACTGGAGGCAGCGGAGCACCTCCAAAGCCGTCCCCCGC AGATGTTGTTCAAGTTCCTGAGTCCGCGCCTCTCACTGGCAACCGTCCTCTTGGTCTTTGTCTCCACCTT GTGTGCCTGCCCCTCGTCACTGATCAGCTCACGCCTGTGCACCTGCACCATGCTGATGCTGATCGGGCTT GGGGTCCTGGCCTGGCAGAGGTGGCGCGCCATCCCTGCCACAGACTGGCAGGAATGGGTCCCCTCCAGGT GTAGACTGTACTCCAAGGACTCTGGGCCTCCAGCAGATGGACCTTAAGGGGCCAGGAGGGCCACCTGCCT TAGCTTGCTAGCTCCCTCCCTCCTCCTGGGTGCTGAGGGCATCCAGCAAGCCCCTCCACAGCTCTTGCTT GCCGATTATGTAACCACCAGCCTGGTGAAATGGATATAGACGCCCACCTGCCTCA
SEQ ID No:20
The DNA sequence encoding the polypeptide of SEQ ID No:6 is provided herein as SEQ ID No:21, as follows:
ATGGAGCCTTCCGGCAGTGAGCAGTTGTATGAGGACCCTGACCCTGGAGGCAGACCCCAAGATGCAGAAG CCAGGAGGCAGGCAGAGTCAGAACAAAAGTTATCTAAAATGACCCACAATGCTCTGGAGAACATTAACGT GATTGGCCAAGGCTTGAAGCATCTCTTCCAGCACCAGCGCAGGCGGTCATCAGTGTCTCCACACGATGTG CAGCAGATTCAGGCAGATGCAGAGCCTGAAGTGGATCTGGACAGTCAGAACACATGTGCTGAGATTGATG GTGTCTCCACCCATCCCACAGCTCTGAATCGTGTTCTCCAGCAGATCCGAGTGCCGCCCAAGATGAAGAG AGGAACAAGCTTGCATAGTAGGCGGGGCAAATCAGAAGCCCCAAAGGGAAGTCCCCAGATCAACAGGAAA TCTGGCCAAGAGGTAGCATCTGTTATACAATCAGGTCGACCTAGGTCTTCATCCACAACTGATGCTCCTA CCAGCTCTTCTGTGATAGAAATAGCTTGTGCTGCTGCTGTGTGTGTACCTGGAGAGGAGGCAACTGCAGA ACGGATCGAGCGTTTGGAAGTAAGCAGCCTCGCCCAGACTTCCAGTGCCGTGGCCTCCAGCACCGATGGC AGCATCCACACAGAGTCTGTGGATGGAATACCAGATCCTCAGCGCACAAAAGCTGCCATTGCACACCTGC AGCAGAAGATCCTAAAGCTCACAGAACAGATCAAAATTGCACAGACAGCCCGAGACGACAACGTTGCTGA ATACTTGAAGCTTGCCAACAGTGCAGACAAGCAGCAGGCTGCTCGCATCAAACAGGTGTTTGAGAAGAAA AACCAGAAATCTGCACAGACTATTCTCCAGCTGCAAAAGAAGCTTGAGCATTACCACCGAAAGCTCCGTG AGGTGGAACAGAATGGGATCCCCCGGCAGCCTAAGGATGTCTTCAGGGACATGCACCAGGGTCTGAAGGA TGTGGGTGCAAAGGTGACTGGCTTCAGTGAAGGTGTAGTGGACAGTGTCAAAGGTGGATTTTCCAGCTTC TCCCAGGCTACCCATTCAGCAGCAGGGGCTGTCGTCTCCAAACCCAGAGAGATTGCCTCACTGATTCGGA ACAAATTTGGTAGTGCAGACAATATCCCTAACCTGAAAGACTCATTGGAGGAAGGACAAGTGGATGACGG GGGAAAGGCTTTAGGGGTGATTTCAAACTTTCAGTCAAGTCCAAAATACGGTAGTGAGGAAGATTGTTCT AGTGCCACTTCAGGCTCAGTAGGAGCCAACAGTACCACTGGGGGCATTGCTGTAGGAGCGTCCAGCTCCA AAACAAACACCCTAGACATGCAGAGCTCAGGGTTTGATGCACTGCTGCATGAGGTCCAGGAGATCCGGGA AACCCAGACCAGACTGGAGGAGTCCTTCGAGACCCTCAAGGAGCACTACCAGAGGGACTACTCCTTAATA ATGCAGACCTTACAGGAAGAGCGATATAGATGTGAACGACTAGAAGAGCAACTGAATGACCTGACAGAGC TCCACCAGAATGAGATCCTGAACTTAAAGCAGGAGTTGGCCAGCATGGAAGAGAAAATCGCCTATCAGTC ATATGAACGGGCCCGGGACATCCAGGAGGCCCTGGAGGCCTGTCAAACCCGCATTTCCAAGATGGAGCTG CAGCAGCAACAGCAGCAGGTGGTGCAACTGGAAGGGCTGGAGAATGCCACTGCCCGAAACCTTCTGGGCA AACTCATCAATATCCTCCTGGCTGTCATGGCAGTCCTCTTGGTCTTTGTGTCCACGGTAGCCAACTGTGT GGTCCCCCTCATGAAGACACGCAACAGGACGTTCAGCACTTTATTCCTTGTGGCTTTCATTGCCTTTCTT TGGAAGCACTGGGATGCCCTCTTCAGCTACGTGGACCGACTCTTCTCACCTCCCAGATGATGCTGGCACT TCTCACCGTCTGGCGAGTGCATGCCAAGAGAGCTAGACAGCAGCATTACCCACTCTGAAGTTTTCTACAA GAG AG T T GAG T GAAT CTGTTTACATT T AG AAT AAT GTTTTTTTCTT C AAG AG AC GC AAT TGCAATAGTAT TTTTTAGATTTTATCCAAGAAGTTTTTTGGGCAAAAATCTTGGATCATTTTTATGTAGCATGATTTTCCT TGGGATGCAAATCTGAAACAGTCCTTTAACATGAACAATCTGCAACACACTGAAGGGCATTCTGAATTGA TGCTTGGAGTACTGCACTAAACCCACTAAACAGATGCAAAAACCTGACCAGGCCCAACACCCACCCTGCC TGGGCTCCTCTCCTCATCCAGACTAACTCTACTGTGAAGTCCCACCACATTCCAGTTTGGAATTTTAGAT TCAGGGTGGATTTCCCCTGCCGTGGAAGAACACAGGCATCTCCCTAGCTTTCTCATGCTAACCCTGAAAT ATGATCACTTCTGAACCTGCTCCTTTCTTAACTGCCAGGATTGGAGGCAAGATCATCAAGCCCATAGGAT GACCATGCAATCCTGTTCAGTATTGTGAAGTAGCCCTTAATCCTAACTTTTAAGCAAAATCCCTTGGCCG CACTTTTAAGGTTTGGTTGTTTGTTTTTTTATATATATATGTGTATAGTTACCAACTTAAAAATAAAAAA T C C G AAC AAC AT AC T T G AAG AAT G T AA
SEQ ID No:21 The DNA sequence encoding the polypeptide of SEQ ID No:7 is provided herein as SEQ ID No:22, as follows:
ATGAAGAGGTGCAAATCGGATGAGCTGCAGCAACAGCAAGGCGAGGAGGATGGGGCTGGGATGGAAGACG CCGCTTGCCTTCTGCCAGGCGCGGACCTCCGGCATGGGGAGGCCTCGAGTGCTAACTCCGCTGGCGGGCC AACTTCAGATGCTGGCGCTGCGGTGGCACCCAACCCGGGTCCCCGAAGCAAGCCTCCTGATTTAAAGAAA ATCCAGCAGCTCTCAGAGGGCTCCATGTTTGGCCACGGCCTGAAGCACCTGTTTCACAGCCGCCGCAGGT CACGGGAGAGGGAGCACCAGGCGTCTCAGGAGGCCCAGCAACAGCAGCAACATCAGGGCCTATCGGATCA GGACTCCCCAGATGAGAAGGAGCGCTCCCCGGAGATGCACCGCGTCTCCTATGCTGTGTCCCTGCACGAC CTGCCCGCGCGACCTACTGCTTTCAACCGGGTGTTACAGCAAATCCGTTCCAGGCCCTCCATCAAGCGGG GCGCCAGCCTGCACAGCAGTGGCGGGAGCGGTGGCCGCCGTGCCAAGAGCAGCTCCCTGGAGCCGCAGCG TGGCAGCCCTCACCTGCTTCGTAAGGCCCCCCAGGACAGCAGCCTGACCGCCATCCTGCACCAGCACCAG GGCCGACCCCGGTCCTCCTCTACCACCGATACCGCCTTGCTCCTGGCTGACGGCAGCAATGCGTACCTCC TGGCTGAGGAGGCAGAGAGCACTGGGGACAAGGGTGACAAGGGAGATCTTGTGGCCCTGAGCCTCCCCTC TGGCCCTGGCCATGGTGACACTGATGGACCCATCAGCCTGGATGTGCCAGATGGAGCGCCAGACCCCCAG CGGACTAAGGCTGCCATTGACCACCTGCACCAAAAGATCCTGAAGATCACTGAGCAGATCAAGATCGAGC AGGAGGCGCGGGATGATAACGTGGCTGAGTACCTGAAGCTGGCCAACAATGCGGACAAGCAGCAGGTGTC GCGCATCAAGCAGGTGTTTGAAAAGAAGAACCAGAAGTCAGCCCAGACCATCGCCCAGCTGCACAAGAAA CTGGAGCAGTACCGTCGGCGCCTGAAGGAGATCGAGCAGAATGGGCCCTCGAGGCAGCCCAAGGATGTGC TACGTGATATGCAGCAAGGGCTGAAGGATGTAGGCGCCAACATGCGTGCCGGCATCAGTGGCTTCGGGGG TGGCGTGGTGGAGGGCGTCAAGGGCAGTCTCTCTGGCCTCTCGCAAGCCACCCACACTGCAGTGGTGTCC AAGCCCCGGGAGTTCGCCAGCCTCATTCGCAACAAGTTCGGCAGTGCGGACAACATCGCCCACCTCAAGG ACCCTATGGAAGATGGACCCCCTGAAGAGGCAGCCCGGGCGCTGAGTGGCAGTGCCACATTGGTTTCCAG CCCCAAGTACGGCAGTGACGATGAATGCTCGAGTGCCAGTGCCAGCTCAGCTGGAGCAGGCAGCAACTCC GGGGCTGGGCCAGCAGGCGCACTGGGGAGCCCTAGATCCAACACTCTTTATGGTGCCCCTGGAAATCTGG ACACTCTGCTGGAGGAGCTTAGGGAGATAAAGGATGGCCAGTCACACCTGGAGGACTCCATGGAGGACTT GAAGACTCAGCTGCAGAGGGACTACACTTACATGACCCAGTGCCTGCAAGAAGAGCGCTACAGGTATGAG AGGCTGGAGGAGCAGCTGAACGACCTGACTGAGCTTCACCAGAATGAGATGACCAACCTAAAGCAGGAGC TTGCCAGCATGGAGGAGAAGGTGGCCTACCAGTCCTACGAGAGGGCCCGGGATATTCAGGAGGCCGTGGA GTCTTGCCTGACCCGTGTCACCAAGCTGGAGCTGCAGCAGCAGCAGCAGCAGGTGGTGCAGTTAGAAGGT GTAGAGAACGCCAACGCGCGCGCTCTGCTGGGCAAGTTCATCAACGTGATCCTGGCACTCATGGCAGTGC TGCTGGTGTTTGTGTCCACCATTGCCAACTTCATCACGCCCCTCATGAAGACGCGCCTCCGTATCACCAG CACCGCCCTCCTGCTCCTCGTCCTCTTCCTACTCTGGAAGCACTGGGACTCGCTCACCTACCTCCTGGAG CATGTGCTGCTGCCCAGCTGAGCGGCTGCTGTCCACCCTGTGCTCTCTGGATTCCACTGGCCACACGCAT CTCCAGGAGGGCCCAGGCCTCCTGAGTCCCTCGGGGTCTGTCATGTGACCTCTTTGGCCCCTAACTGTCC CTTCTCTGCACTGCTTCTGTCTGGCACCATCTCCCTTTCAGCCTGAAGGTTGCAGAGAGGGTGCTCTGGG CTGGAGGGAGCAGGGACACTCACAGCCAAATGGAGCAGCCGGGAGACACCGGGGCTGAATTATTTGAATT ATTTGCAGTCACATGGGACTTTTCTGGGCTGACCGGAGGATGCTCTAGTCATGAAAGCCACTAAGATAGA AGGCCAGTCTCGATTTCCCTAGGGAAGAGCCCATCCTAGAGGCAGCCCAGTTTCCTCCCATGGCAACAGT CTGGATATAAGCCAGTGGGCGGCAGTGTTGAGCCCAAAGGGTGCAGAAAGGGGAGGGTCCCCAGGCTTGG GGCCAGACAGCAGGCTTGGCACAGCCACTGGGAGCTCAGCCTCCCCTCCCCCTGCTTCTCTTCCCACTTG AACTGGTTTGGGACTGGTTTCTGGAGGCTGGGTGGGGTGTGGCTTTGGGTTTGTGTTTTCTCTGCTCATC TTTCTGCCTGTCACTGGGTGTGCTGTTTTTCTGAAAAAAACAGGCCCCAAGGGGCCCTGAGCCTCCAGCC CAAACCCTTAGACCTCTGGGATCACATTGAGTTCTATTTATTTATTTATTTATTTACTTATTTATTTATT TGTTCCTATCCTTTCCACGCTTCTGCCCGGCTGGGCTCCAGGGAGAGGACCCTACCCGTCCTCCCTCTGA AGCCCGGGCTTATTCTTGGTTCATCACACCCCAGACCACGTCTCCGGTGTTCCTGGCTGGAAGAAGGGGC GTGTGGCCATGCAGCCCGCTCTCCCAAGCCACCCCAGCAGAAGGGTTGGAGGGGGCATGGCCTCTTTTAT ATATATCTATTTATTTTACGTGTGTGTCTTTGTGAGGAAGAGGATGTTTGTCATTTCCTTACGCTCTGGA GTGTGTGTTCCAATTCTGCACAGCCCAGCCTCCCCAGGAGCCCTTCCAAGCACAGTGGGGATTTCTCAGA ATAGGGGAGAGGAACCCATGAAGCAGGAGAAGCAGTGCCTGCCAGCCATGGTGGACCTTCCTAAGAAATA AATATCCTCTATGTTTTCAAACCATCA
SEQ ID No:22 The DNA sequence encoding the polypeptide of SEQ ID No:8 is provided herein as SEQ ID No:23, as follows:
CCAGCGGCGGCGGCGCAGGGCCGCGGGGTGTCGGCAGCAGCTGCAGCCGCCCGCGCCACCCCCCCTCGGC CGCGGCTCCGGGGCGAGGGTGAGCGGAGCACGGCGGGCAGCTGCCACCCGGCGCCGAGTGGAGCGGCGGC CCCCGAGAACGCAAAGTTGTCAGTCTAAGCGCAGACATGCCGGGCAGCGACACGGCGCTCACTGTGGACC GGACCTACTCCGACCCCGGCCGGCACCACCGCTGCAAGAGCCGGGTAGAACGCCATGACATGAATACCCT CAGCCTGCCCCTGAATATCCGCCGAGGAGGGTCAGACACCAACCTCAACTTCGACGTCCCAGATGGTATC CTGGATTTCCACAAGGTCAAACTCAGCGCAGACAGCCTGAGACAGAAAATCTTAAAGGTGACGGAGCAGA TAAAAATCGAGCAGACGTCCCGAGATGGGAACGTTGCCGAGTATCTGAAACTGGTCAGCAGTGCTGACAA GCAGCAGGCCGGGCGCATCAAGCAGGTCTTTGAGAAGAAGAACCAGAAGTCAGCCCACTCCATCGCCCAG CTGCAGAAGAAGTTGGAGCAGTATCACAGGAAACTCAGGGAGATTGAACAGAATGGAGCGACCAGAAGCT CGAAGGACATTTCTAAAGACAGCCTGAAGGAAATACAACACTCTCTGAAGGATGCCCACGTGAAATCTCG AACTGCTCCCCACTGCCTAGAGAGCAGTAAGTCGAGCATGCCAGGGGTATCCCTCACGCCCCCCGTGTTT GTCTTCAATAAGTCCAGAGAGTTTGCCAACTTGATCCGGAATAAGTTTGGCAGTGCAGACAACATCGCTC ACTTGAAGAATTCCCTAGAAGAGTTCAGGCCCGAAGCCAGCCCCAGGGCTTACGGCGGAAGCGCTACCAT CGTGAACAAGCCCAAGTACGGCAGCGATGACGAGTGTTCTAGTGGTACATCCGGCTCAGCTGACAGCAAT GGGAACCAGTCCTTTGGGGCTGGTGGAGCCAGCACCCTGGACAGCCAGGGAAAACTTGCCATAATCCTAG AGGAACTGAGGGAGATCAAGGTTACCCAAGCCCAGCTGGCAGAGGACATCGAGGCGTTGAAGGTGCAGTT TAAGAGAGAATATGGCTTTATCTCTCAGACTCTGCAAGAGGAGAGGTACAGGTATGAGCGACTTGAAGAC CAGCTCCATGACCTGACAGAGCTGCACCAGCATGAGACAGCTAACCTGAAGCAGGAGCTAGCCAGCGCCG AGGAGAAGGTGGCCTACCAGGCCTATGAGCGCTCAAGGGACATCCAGGAAGCCTTGGAGTCCTGCCAGAC ACGAATCTCCAAGCTGGAGCTGCATCAACAGGAGCAGCAGACCCTGCAGACAGATGCCGTGAATGCCAAG GTCCTGCTGGGGAAGTGCATCAACGTGGTCCTGGCCTTCATGACTGTCATCCTGGTATGCGTGTCCACCC TGGCCAAGTTCGTCTCGCCCATGATGAAGAGTCGCTCCCACATCCTGGGCACCTTCTTTGCTGTGACTCT TCTCGCAATATTCTGTAAAAACTGGGACCACATCTTGTGTGCTATAGAAAGGATAATCATCCCGAGATGA GCCACCGGCTCCCACCTTCACATCCTTCCAAGTTTTTATTTTGAAGAAAACTCTGTGCATACATACTACC AAAATTTCTGAATGAACGGCTGTGCCAAAGACGGCGAGAAGGACCCAGAGCTGTGTGGTGTAAATACGTA GAGTCTCTTAACCCAGTAGCAGTTGCCAACACAGTCCCTGTATGTGGTTCACGTGAAACTGTCCCAGAGT CCTCCTCACATGGCTCACTGCCTTAATCAGAGCAGATCTCCAGCCCACTGTCGAGCTCGGTATGGTAAAG CCCTGCTGATTGAGCGCCATGACTGCTCACAGCCGTGTTCTGAGTTCCCCTGGTCAGCAAGAATATGGTA GGATTTGGGGGAAGTGAGCAAACACAGGTACTTGGCAGGGTTGAGCTCCTGGGGGCCTATAGAAGCTCAG TGAGTCTCTCTGTGATCTACCCCACTTGTCCACAGGTGGCTCTGTGTCCTACTGTGTACCCAGCGTTCAC AGCGGTCAATGGTGTGTGTCCTCATAACATGGAAATCTTCTACCTGCTCATCCAGCGAAAGTCTGCCCGA CTAGTAGCATTGCACTTTACAAGGTAGACACTAGGATTTACAGCCGGGCTCATGCCCAGGGCACCGCACA CCTGATTTCATCTTGCGTGCTTAGCAGTGCTCTGCCCTCAGAGTGTGTGGCTGAGCTGTTGTCAAACTGT AGCTTCGGTAACTCGGGGTCCTTGTGAGCGTCCCTGGGGAGGGCCATGCTCTGGCAACTGTTAACTGTGA TAAATGCCCTTAGAGGATGGGATGCACTTTATGTGAATCACTTTACTCACAGGTCTGTGGGGATCGGATT GCAAAGCTTGCTCAGGAAATGAAAATGAATGGCGTTCTTGTTGCCCGGTTTTCTTTTTCTCAGAGACAGC AAAATCAAACTTCATAAAGCTTCTGTGCACGAGGCAGGACACTGGAGGCCTAAGAGGGATGTGAACTTTT AGGTCTAATCAAATGGTGGCTAGATTAGTGAGAGCCCTTTACCCAGATGAGAGCAGGAAAGGTCCCCTTA GGGACGTGAAAGGCAGCAGGAATAAAATTAAGTCCTGCGCTGGAACTGAGCTCCTGCTGTCTCTTAAGGC TCAGACAAGCCCTGAGCTCACAGGATAAACGCACCAACAGTGAGCAGAAAGAGTACAAGAGCTGCTGTGT TTTATTTGATGGCTGCCATTATACT G AC AAAC CTAGTTTTCTGTGTACACTAC C AAG AGG AG AG AG AT T T CATTGGATGCTGTCAGCCAGCCAGCCAGGACTCTGGTCCTCCCACCCCACCTGCTGAAGGTGTAAAAATA CTCTTAAATACTTCCAGAAGAACCTCTCTTGGATTTAAGGATCCTCACTTAGGGGCATTCCCTGGGGGGG CGGGGCGTTGGGGGTGGCAGCATGTAGCAATTTAGTACAAGTCCAGGTTATATGAGTGGGACCCACACAG CTCCAACCTTTGCCCCTCCCCCATTCCCAACTTCCAAGTCAATAATTCGGCAAATTCAGTAAATTCAGTA ATTTTAGTAAATTAAGTATTTAACCATCACCCCTAACCAATTTCAGAGCCACACTTTTCAAAAAATAATA AATAGAACTTTATGAAATACAAAGGATTTTGAAATCAGGGACGGGGGAATCCCCAGCCCCTCTCCTACCC ACGGCATTGTCCCCACACAGGAGACAGAGGTTTCTAATCCAAGAAAGGGAGAGCCATGATCTTCCCTTAT TCGTGTACTTTAATGGCCTGCTCCCTTACCTGTGGCACCAGACTCCATCTGCCTGCACCCGGAAACAAGC TGGGCCCTGCTGACCTCCCGTTCCTATTTGTAAATATTTCCACGTGGCACTTAGAGCGTGCCGGGGGGCC TGGGATACCCATGAATGCTGCTATCGGGGTTTCAGGATAAGTTTGAATACAGCCTTAGAGGCTTAGCTAG GGCTCTGGGAGAACCCACAAAGGGGTTCTGTTCTCATGTCAGCACCCGCCCCACCCCCACACAGCCTAAC AGGACGGCTCTCGATTGACACTTGTCAGACCTACAAGCTGTGTTTCTTCTTCCTGATGAGAAAACCCAAA AC AGC AC AC T C AAC AC C C T C C AG AC C GAGCT T T GG AC AG AAT C T G T TAG AG AC CAGCACCATTGT G AAC T AGTCGGGGTGGGGGCATCCAAGTCATCATAGGTTCCTCCTAAAGAACTTAGATTCCCACCGACTCCCATT AAATGTCAAATCACCTTCTTGGCCTGCGTACATGCAGCTCATGTGGCATAGTTGTTCCGGTCTTATGGAA AATTTGTGAGACTTTTTATTTTTTTAAATAAGATTGTATAGTTCAAGGAGACTTTGATTCCTCCGTTCTT TATTCTCTCAAGGGCTATGACCCCTCTCTCGCTCTCTTGAACACATAACTTACACACTGAAGAAATGAAT TATATAAATACACAATTAGAGGTAGCAATGGAGCTGCGAGGTGCCCTGGAATTGGAATAGATTTGTAATC GAGTCAGCCCATCTTGTTTTGCCCTTTACGTTGGGGGTATTCCTCGTGTTGGGGGCTAGTTACAGATGTG TGAGCATGTTCCCTGCTAGCTTAGGAACACTGGTGAGGGATTCTAGATGCTGGACTTTGGATCTCTAGTG ACGATATCATGCTCAATCATCAGAAGAGTGATTACAGAGGCCAGAACAGAACGCTAAGATACTAAGAATA ATAAATATTAAAAAGGAAAAAGCAGCCGCTCACAGTGAAACAAGCGGTGACTTCAAGTGCTCTCTCTGAT TTGGACTATGAAGGCAGGATTCCCCATTACAAGCTCCTGGCTTGTGACTTAGTTTTCCGGTGTTCTTGTC TTGTCTCCTGGCGTTCATTCACATACTCTGTAGCTCTTGACTTTCTTTAAGCATCTTTAAGAAGTGCCTG GCCACTTCTCTAACACAGGGTGGAGCTGTGGTGAGGGAGCTTGTCCTCCCGGCCCCCCTCCCTGCCACCC AGCAACTAATTACTTGGTTCTTTTGGGTTGAATTTTAAATACCAAATTACTTTTGTGTTGAATGGCAATG TTATCGTGGTGGTGTTGTTAATGTTTTGTTCAGCTGTCCAATCCAGACCTAATTGTCTGGGTTTTGAAGA AACACATGTACTCACTGATAAGGAGGTGTATGATTAAACAACACATTTTGCATGTTTTCTTAATTCTGAC CTTGGGCTGGGGCATGAATATGGGCTTATTTGGAGAGATGGTTCAATGTTAGCAGGTAACTTTTAAGGTG T C T AAAAAAAG AC AAAT C AC AG AAAAC AAT T AG AT T GGC C T AT T T G T AT C AAAAAT AC AG AGC T T T AAAA ACGTGTCATAATGGGAACTGGGTAACTTGTTAGAAATAAAGCCAAAATATAATTTTCTGTCATGGATATA ATCCTGTGCGTAAAGAGTCTGGAAGTGAGGTGGAACACCTGGGGTGTCTGAGGGTGGTTTACATGTTGCT GTTCTCATATATTTAAAGCATGTCTGCCATGTTTGTTCTAAAAGAAACATTTCAACTAAACGTACCATGT GTTTGTTTCAGATGCGCTGGGGCAATAGCAGCAATTCTGACTGGACCGTCAGGAGTGCTAATTGTAACTT CTGTTGCAGAATTCTTGTGGGCTGCTACATAGTTTTGTATCTGCCTAAAGTTGTAATTTCTCTGTAGATC TTCCCCATAGCGGTGTCGTCAAGGAATTCAAAGCAGGTGCCATTATTTTTTAAATAATTGATGTTTGCTT T GG T G T T AAAT AAAAG AAC TAG AT T T C SEQ ID No:23
The DNA sequence encoding the polypeptide of SEQ ID No:9 is provided herein as SEQ ID No:24, as follows: ATGGTTTTAAAGGTGGAAAGTACCAAGAGCTCAAGTGCAACCTTCCCCCCCAACGTGCCCTCCTACAGGT CTCTGTCTTCCTCCCATGAGGATTGCCCTAGCAGTCACACTAGCTTCTCGGATGGCGAGCTTGCCCGGAA TGTGAGGGAAGGTGTCAAACACCGAATCTTCTACCTCTCAGAGCAGCTGAGAGTGGAGAAGGCCAGTAGG GATGAAAATACCATGAGCTACCTCAAACTGATATCCAAAGCTGACCGGCAC CAGGCCCCACACATCCGGA AGGCCTTTGAAAGGGTGAACCAGCGCACCTCTGCCACTATTGCTCACATAGAACGAAAACTCTATCAGTG TCATCAGCAGCTGAAGGAGTTGGAAGAGGGCTGCAGTCCCACAAGCTTAGTGCTGAATGTGGACAGTGGG ATGGACAGCCATAAGCAGCCAGGTGGGAAGATTTTATATTCCAAGTTGTCCAAGCCAGATGGGGAAGATA GCCTGCCCATCAACGTTGCTAGGTCCTCTACTCTAGAGAGTCACTTGCCAGGCATGCAGCAGAGGAAATT T T C AG AC AAAAAG TATGTGGCC C AGC AAC AAAAAC T AC T T T T GC AG AAG AT G AAAG AAG AAC T G AC AG AA GCCAAGAAGGTTCATGCTAGCCTTCAGCTCTCCCATCAAAACCTCAAGGAAAGTCATATGATTGATGTGC GGAGGATACTGGAGTCACTCCAGGAAAAGAAAACCAGACAATCACTGATGGAAGAACATGTGAATGATCA CTTGCAGAGATACCTGGATGAGATTTGCCACCTCAAACAGCATCTGGCATGCACTGAAGAGAAAATGGCC TATTTGTCCTATGAAAGAGCCAAGGAAATATGGGATGTAATGGAGACTTTCAAGAACAGAATAACAAAGC TAGAAACTCTACAGCAAGCTACCCAACTGGAGATGATGGCCAGCCTCAGAACCCGCCCCAAGGACTTTTT CTTCAGATTCATAAGCCTGCTTCTCACTCTGACCACTATCCTCTTGGTCGTGGTCTCTACATTGTGTTCT TGCCCTTTGCCCTTGCTCAACTCACGCCTGCGCATCTTCATTGTATTTATGATCATTGGGCTTGGGACCT TGGCCTGGCAGAAGCGGCATGTCATCTCTATTATTGACTGGCAGGCCTGGGTCCCCTTTAAGTGGAGATC AG AC T T AAAGG AT GC T AAAC C T C C AT C AGC TAG AC AC T G A
SEQ ID No:23 The DNA sequence encoding the polypeptide of SEQ ID No:10 is provided herein as SEQ ID No:25, as follows: GGGGGGGTGGTCGGGGTGGGGATGGACCGGGCGCGGCGGCGGCAGCTGCAGGAGCCGGCGCTGGAGACCG AGCCGCGGCGCTGGCGGAGACGCCGGCTGTCCGTCGTCTCTCCACCGCTTCTCCTAAAAGGACATCAGTG CCCCAAGTACGTGCTTGAGGGAGAGCAATGAAGGTAGGCATGAAGCCTCCATCTCTCAGCTGCATGCATT GTCACTCTTGAAGCAAATGCCTACCTAATTTGACAGTCTCGGTGTGTTTAAAATTTTTTGAGTTTGCAAA TAAGCTTATTAAGCTTACTGATGGAGCCTTCCGGCAGTGAACAGTTATATGAGGACCCTGATCCTGGAGG CAAATCCCAAGATGCAGAAGCCAGGAGGCAGACAGAGTCAGAACAAAAGTTATCTAAAATGACCCACAAT GCTCTGGAGAACATTAATGTGATTGGCCAAGGCTTGAAGCATCTCTTCCAGCACCAGCGCAGGAGGTCTT CAGTGTCTCCACACGATGTGCAGCAGATTCAGACAGATCCAGAGCCTGAAGTGGATCTGGACAGTCAGAA CGCATGTGCTGAGATTGATGGTGTCTCCACCCATCCCACAGCTCTGAATCGTGTTCTCCAGCAGATCCGA GTGCCACCCAAGATGAAGAGAGGGACAAGCTTGCATAGTAGGCGGGGCAAATCAGAAGCCCCAAAGGGAA GTCCCCAAATCAACAGGAAATCTGGCCAAGAGGTGGCAGCTGTAATACAGTCAGGTCGACCCAGGTCTTC ATCCACAACTGATGCTCCTACCAGCTCTTCTGTGATGGAAATAGCTTGTGCTGCTGGTGTGTGTGTACCT GGAGAGGAGGCAACTGCAGAACGGATCGAGCGTTTGGAAGTAAGCAGCCTCGCCCAGACTTCCAGTGCCG TGGCCTCCAGCACTGATGGCAGCATCCACACAGAGTCTGTGGATGGAATACCAGATCCTCAGCGTACAAA AGCTGCCATTGCACACCTGCAGCAGAAGATCCTAAAGCTCACAGAACAGATCAAGATTGCACAGACAGCC CGAGACGACAACGTTGCTGAGTACTTGAAGCTTGCCAACAGTGCAGACAAGCAGCAGGCTGCTCGAATCA AACAGGTGTTTGAGAAGAAAAACCAGAAATCTGCGCAGACCATTCTCCAGCTGCAAAAGAAGCTTGAGCA TTACCACCGAAAGCTCCGTGAGGTGGAACAGAATGGGATTCCCCGACAGCCCAAGGATGTCTTCAGGGAC ATGCACCAGGGTCTGAAGGATGTGGGTGCAAAGGTGACTGGCTTCAGTGAAGGTGTAGTGGACAGCGTCA AAGGTGGATTTTCCAGCTTCTCCCAGGCTACCCATTCAGCAGCAGGGGCTGTCGTCTCCAAACCCAGAGA GATTGCCTCACTGATT C GG AAC AAAT T T GG T AG T GC AG AC AAT AT C C C T AAC C T G AAAG AC T C AT TAG AG GAAGGACAAGTGGATGATGGGGGAAAGGCTTTAGGGGTGATTTCAAACTTTCAGTCAAGTCCAAAATATG GTAGTGAGGAAGATTGTTCTAGTGCCACTTCAGGCTCAGTAGGAGCCAACAGTACCACTGGGGGCATTGC TGTGGGAGCATCCAGCTCCAAAACCAACACCCTCGACATGCAGAGCTCAGGGTTTGATGCACTGCTGCAT GAGGTCCAGGAGATCCGGGAAACCCAGGCCAGACTGGAGGACTCCTTCGAGACCCTCAAGGAGCATTATC AGAGGGACTACTCCTTAATAATGCAGACCTTACAGGAAGAGCGGTATAGATGTGAGCGACTGGAAGAGCA GCTGAATGACCTGACAGAGCTGCACCAGAATGAGATCCTGAACTTAAAGCAGGAGTTGGCCAGCATGGAA GAGAAAATCGCCTATCAGTCATATGAACGGGCCCGGGATATCCAGGAGGCTCTGGAGGCCTGTCAAACCC GCATTTCCAAGATGGAGCTGCAGCAGCAACAGCAGCAGGTGGTGCAACTGGAAGGGCTGGAGAATGCCAC TGCCCGAAACCTTCTGGGCAAACTCATCAATATCCTCCTTGCTGTCATGGCAGTTCTCTTGGTCTTTGTG TCAACAGTAGCCAACTGTGTGGTCCCCCTCATGAAGACACGCAACAGGACGTTCAGCACTTTATTCCTAG TGGCTTTCATTGCCTTTCTTTGGAAGCACTGGGATGCCCTCTTTAGCTACGTGGACCGGCTCTTCTCACC CCCCAGATGATGCTGGCATGGAACGCAGTGCTCTTCACCGTCTGGCGAGTGCATGCCAAGAGAGCTAGAC AGC AGC AT T AC C C AC T C T GAAG T T T T C T AC AAG AG AG T T GAG T G AAT CTGTTTACATTTAGGATAATGTT TTTTTCTTCAAAAGACGCAATTGCAATAGTATTTTTTAGATTTTATCCAAGAAGTTTTTTGGGCAAAAAT CTTGGATCATTTTTATGTAGCATGATTTTCCTTGGGATGCAAATCTGAAACAGTCCTTTAACATGAACAA TCTGCAGCACACTGAAGGGCATTCTGAATTGAAGCTTGGAGAACTGCACTAAACCCACTAAACAGATGCA AAAACCTGACCAGGCCCAACACCCACCCTGCCTGGCTCCTCTCCTCATCCAGACTAACTCTACTGTGAAG TCCCACCACATTCCAGGTTGGAATTTTAGATTCAGGGTGGATTTTCCCTGCCGTGGAAGAACACAGGCAT CTCCCTAGCTTTCTCACGCTAACCCTGAAGTATGATCACTTCTGAACCTGCTCCTTTCTTAACTGCCAGG ATTGGAAGCAAGAGCATCAAGCCCATAGGATGACTGCGCAATCCTGTTCAGTATTGTGAAGTAGCCCTTA ATCCTAACTTTTAAGCAAAATCCCTTGGCCGCACTTTTAAGGTTTGGTTGTTTTTTATATATATATGTGT AT AG T T AC C AAC T T AAAAAT AAAAAAT C C G AAC AAC AT AC T T G AAG AAT G T AAT AC T C AAAC T C T C AG T G CTTCCTTATAATTTCTAATAGAATTTTTTATTATTGTTATTATTATTGGGGTTTTTTTGGTTGGTTGGTT GGTTGGTCGGGTTTTTTGGGGGGTGAAGGGGACTGGGTTGGGAGGGTCTTTTATTTTTTTCCTCTGAAAT AAAGCAGTGCTGTTTTCAAATGAAGCCGTGTGGATATTTCAGCGTGCTGCTCCCTGTTGTCTGGAAACAG TTGAGTTGGAGCCTGTTGCCAATGCTGCCCTCTGCCACCCTGTGGCTGCCCTGGCTCTCTGGTCTCTATC ACCCACACAGCACCATCTCCCCGGCTTGGCTTGGTTAGAATGGTGAGGGGTTTACAGGGTAAGGGGACAA GACTACACACACTAGCGTGGTAGGACTAGATTTTTGTTTTCCCCACCAAGTAAATGTTCACCTGTGACCT TGGGCTGGGATTCTCTTTAGGAAAAAAGGCTACTACTATGTTATGAGAGTCAAAAATAACTCCTTGTGTT TGCTTAATTTATTGTTTAACACCCCCTAACTGCCGAAACCAAAGTGATACGGAGTCGTCTTGTCTGCATT TTGGCCTCAGCATCATTAACTTCAAAGCTTTATTCTGTCTGCCTGAGAGGATCCATGGAAGATGATTCTC CCAAAAAGCAGAAAAAGAAATGTCAGGTTAGAAGGACCCAAGTTCTAGGTGCACACTTTTGCAAGCTTCC TACAAGCCCAGACTGACTTACACTAACCTAACTGCTCAGTGGCTGTCTAGAGACACTTTAGGAAGATAAA CTGAGCTTGATCCTCATCATATAGCCAGTTCCATCACTCAGGTAGTGCCTGAATCAACCTTCAGAGCCTG ATTAGTCACATATCATTGGAGGTCAAACGCCATTTCCAGTGGCCTCTCTGTGTGCCCACATGATGCAAAG GGGTCATGTACTATGCCCTGGACAAAGAAGTATGTTTTGCCTATGTCCCCATACCAGGACCTTCTTTCAG AACGAGGGTAGTATTACCCTATCTGTTGGAAAGGATGGTTTACATAGCAGTTCCAGCCTCCTTTTGCTTT GGCCAGTTGGCCTTCTCACTTTGATAGTTTAACTCAGGTACCCAGTCTTCACTCCATCCCCAGGTGCTTG TTCACCTCATTAATGAGCACCTAGAGCAAGGACCAAAAGAAGGAAAACTAATTAAAACCAATGCATATTC ATGCCAAAACAAGTGGTCCCCAAGCCTGTTTAAAAGGTTAACTTGGCCCGAAAATATTGCAGCAGCTTCA TGCAAGAATTGCCACAGGACACTATGGCCTTGCTGGGCTAGCATTTGCTGCTGTAATCTAGTTGCTCTGG TATAGAAGAGAACCCCATGGGGCTGTGGGAGAGACAATTCAGTGGCAGCCCACTGGCTTCTCTACCCATT CCACAGGAACCCTATTCCTACCCCCACCCCTGCCCACAAATGCCACTGTGATCCACAGCTGAAGTTGTTT CTAATTGCCTAAAGGCAGTCTCTGTGTCTGGAGCTTCAAAGAGACAGGCAGACCTGCACTAATTCCCTTC TTCCTCTAGCAGACTGCCTTGGGAAGGACCTTCAGTCCAGGACAGACCCTGTCATGGCTTCTTGCTGTCT TACCCTTCTCACTCACCAGGCCCACAGTCGCCTGCACTCAGTGCTGTTTGTAACACATCTGTTATTCTGC CTACCTGTACTTTTTGCTGCCCATCCCTGTTGCCTTTGAGTTTCTTGGAATTGTGGCTGTCCTCGGAATC TCTTTTCTATTCACAGCCCTTCTATAGCCTTGCTCTGATGGTTTTAGCACTTGATCTGAGAGAAGCTTGC CC AT AAT T T AAT GC T T C T T T T C C T T AAAAAAT AAAAAAAAAAAT C AAAAAAAC C C AC T AAAG T G AAGC AT TTCTTGATTCCCTTTTCCATGCTGGTGCCTAGCCCGGGTGAGTGAGGCTGGCTGTGTTGGCCTGGGTGAC TTGTGGTTGTAGAGTTGGCCATCATTCTGCACTGTTGATAGCTGTCTGCTCCAGTTTCTGGTCTTTGGTA TACTCAGTGTGGCCCGGTGATGAAGGCTTTCTACTCCTTAACAGGTACAGTTGGATCCATCGCTGATGGT GTTTCTTTAGCCCCATCCCAATTGGCCTAAAACATTTGTCACCAGAACAGTGGGGAAAAGCCTTCCTTTC CAGCCATGTCTGACAGTGAGCATGCTTTGGTGTGGTCTTCACTGCTTTTGGTTTTGCTTCTGATCTTGTG TTCTAAGGTACTCAGTGAATCCTGGATTCTGAGTAAGTTCCAGGGTTCTGGCCTTGAAGCCTTGGACTAC ACACATTTCAAACAAAACCAAATGCTGTCGTCGACAAGCCAGCACAGGCGAAGTGATGGTTCCCACAGCA CCCCCAATAAAAACTGGGATTTTGAATGTGGCTCTACAAGTTAAACATTTCTGTGTGTTGTGTATGCTAG GTGATGCCCT C AG AAGGG AC T G AC T AC TAG AC T G TAAG T G T G T T T T AT AC AAG T G T G T AAG AC T AAG AC T C AC G T GC AC G AAT C G AAG AG T AAAG AG AC AAT AC T T G T G AAC G T G T G AAC G T T AAC AC TGTAGTTGCTAG ATCTTAAGCTGCTAATTGTCGAGAGAGAATTCAGTTGTGTGCTGTCTGGCTGCTGTGCCTTCTCAGCAGC ACTTTTGCTG T AC AT AC AAT GG AAAT AAAG AC C T C AGC T AG AAAC C T GC
SEQ ID No:25
The DNA sequence encoding the polypeptide of SEQ ID No:l l is provided herein as SEQ ID No:26, as follows:
TTTAAACCCCAGGTCTTTTGAGTGCAAGAGGGGGGTGCGGTCTCTCCCCAAGATAGCACGCTAGAAGGAC AGATTCCCCTTGCCGACCCACTTACACCATGAAGAGGTGCAAATCGGACGAGCTGCAGCAACAGCAGGGC GAGGAGGATGGGGCTGGGATGGAAGACGCTGCTTGCCTTCTGCCAGGCGCGGACCTCCGGCATGGGGAGG CCTCGAGTGCTAACTCCGCTGGCGGGCCAACTTCAGATGCTGGCGCTGCGGTGGCACCCAACCCGGGTCC CCGAAGCAAGCCTCCTGATTTAAAGAAAATCCAGCAGCTCTCAGAGGGCTCCATGTTTGGCCACGGCCTG AAGCACCTGTTTCACAGCCGCCGCAGGTCACGGGAGAGGGAGCACCAGGCGTCTCAGGAGGCCCAGCAGC AGCAGCAACAGCAGGGCCTATCCGATCAGGACTCCCCAGATGAGAAGGAACGCTCCCCGGAGATGCACCG CGTCTCCTATGCTGTGTCCCTGCACGACCTGCCTGCCCGACCTACTGCTTTCAACCGGGTGTTACAGCAA ATCCGCTCCAGGCCCTCCATCAAGCGGGGCGCCAGCCTGCACAGCAGTGGCGGGAGCGGTGGTCGCCGTG CCAAGAGCAGCTCCCTGGAGCCACAGCGTGGCAGTCCTCACCTGCTTCGTAAGGCCCCCCAGGACAGCAG CCTGGCCGCCATCCTGCACCAGCACCAGGGCCGACCCCGTTCCTCCTCTACCACCGACACCGCCTTGCTC CTGGCCGATGGCAGCAGTGCGTACCTCCTGGCTGAGGAAGCAGAGAGCATCGGGGACAAGGGTGACAAGG GAGATCTTGTGGCCCTGAGCCTCCCCTCTGGCCCTGGCCATGGTGACTCTGATGGACCCATCAGCCTGGA TGTGCCAGATGGAGCACCGGACCCCCAGCGAACTAAGGCTGCCATCGAACACCTGCACCAAAAGATCTTG AAGATCACTGAGCAGATCAAGATCGAGCAGGAGGCGCGGGATGATAACGTGGCAGAGTACCTGAAGCTGG CCAACAATGCGGACAAGCAGCAGGTGTCGCGCATCAAGCAGGTGTTTGAAAAGAAGAACCAGAAGTCCGC CCAGACCATCGCCCAGCTGCACAAAAAACTGGAGCACTACCGTCGGCGCCTGAAGGAAATCGAGCAGAAT GGGCCCTCGCGGCAGCCCAAGGATGTGCTACGCGATATGCAGCAAGGGCTGAAGGATGTGGGCGCCAACA TGCGTGCCGGCATTAGTGGCTTCGGGGGTGGTGTGGTGGAGGGTGTCAAGGGCAGTCTTTCCGGCCTCTC GCAAGCTACCCACACTGCGGTGGTGTCCAAGCCCCGGGAGTTCGCCAGCCTCATTCGCAACAAGTTCGGC AGTGCCGACAACATCGCCCACCTGAAGGACCCCATGGAAGATGGACCCCCTGAAGAGGCAGCCCGGGCGC TGAGTGGCAGTGCCACACTGGTGTCCAGCCCCAAGTACGGCAGTGACGATGAATGCTCGAGTGCCAGTGC CAGCTCAGCTGGGGCAGGCAGCAACTCTGGGGCTGGGCCAGGAGGTGCACTGGGAAGCCCGAGATCCAAC ACTCTTTATGGTGCCCCTGGAAATCTGGACACTCTGCTGGAGGAACTGCGGGAGATAAAGGAGGGCCAGT CACACCTGGAGGACTCCATGGAGGACTTGAAGACTCAGCTGCAGAGGGACTACACCTACATGACCCAGTG CCTGCAAGAAGAGCGCTACAGGTATGAGAGGCTGGAGGAACAGCTGAACGACCTGACTGAGCTTCACCAG AATGAAATGACCAACCTAAAGCAGGAGCTGGCCAGCATGGAGGAGAAGGTGGCCTACCAGTCCTACGAGA GGGCCCGGGATATCCAGGAGGCCGTGGAGTCCTGCCTGACCCGAGTCACCAAGCTGGAACTGCAGCAGCA GCAGCAGCAAGTGGTGCAGTTAGAAGGTGTGGAGAACGCCAACGCGCGCGCCCTGCTGGGCAAGTTCATC AACGTGATCCTGGCACTCATGGCCGTGCTGCTGGTGTTTGTGTCCACCATTGCCAACTTCATCACCCCCC TCATGAAGACGCGCCTCCGGATCACCAGCACCGCCCTCCTGCTCCTCGTCCTCTTCCTACTGTGGAAGCA CTGGGCCTCCCTCACCTACCTCCTGGAGCATGTGCTGCTGCCCAGCTGAGCGGCTGCCGCCCCACCCTGT GCTCTCCGGATCCCACTAGCCACAGGCATCTCCAGGAGGGCCCGGGGCTCCTGCGTCCCTCGGGGTCTGT CATGTGACCTGTTTGGCCCCTAACTGTCCCTTCTTTTCACTGCTTCTGTCTGACACCATCTCCCTTTCGG CCTGAAGGTTTCAGAGAGGGTGCTCTGGGCTGGAGGGAGCAGGGACACTCTCAGCCAAATGGAGCAGTGG GGAGACACCAGGGCTGCATTATTTGAATTATTTTGCAGCCACATGGGACTTTTCTGGGTTGACAAGAGGA TGCTCTAGTCATGTAAGCCACTAAGATAGAAGGCCAGTCTCAGTTTCCCTAGGGAAGAGCCCATCCTGGA GGCAGCCACAGTTTCCTCCCATGGAGATGGTCTGGATAAAAGCCAGTGGGAGGCCCTGCTGAGCCTAGAG AGTGCAGAAAGGGGAGGGTCCCCAGCCTTGGGGCCAGAGGGCAGGCTTGGCACAGCCACTGGGAGCTCAG CCTCCCCTCCTCCTGCTTCTCCTTCCACTTGAGCTGGTTTGAGACTGGTTTCTGGAGGCTGTGGGGTGTA ACTTTGGGTTTGTATTTTTTCTGCTCATCTTTCTGCCTGTCACCGGGTGTGCTGTTTTTCTGAAAAAAAA ACAGGCCCCAAGGGCCCTGAGCCTCCAGCCCAAACCCTTAGACCTCTGGGATCACGTTGAGTTCTATTTA TTTATTTACTTATTTATTTATTTGTACCCACCCTTCCCTCACTTCTGCCCAGCTGGGCTCCAGAGAGAGG ACCCTGCCCATCCTCCTTTTGAAGCCAGGACTTATTCTTGGTTCATCATACTCCAGACCATGTATCCTCT GGTGTCCTGGCTGGAAGAAGGGGCGTGTGGCCATGCAGCCCGCTCTCCCAAGCTGTCCCAGCAAATGGGC AGGAGGGGCATGGCCTCTTTTATATATATATATCTATTTATTTTATATGTGTGTTTGTGAGGAAGAGGAT ATTTGTCATTTCCTTAAGGCTCTGGAGTGTTGTGTTCGATTCTGCACAGCCCAGCCTCCCCGAGGGCCTT TCCAAGCACAGTGGGGATTTCTTAGAACAGGAGAGAGGAATCCATGAAGCAGGAGAAGCAGTGCCTGCCA GC T G T GG T GG AC C T T C C T AAGAAAT AAAT AT C C T T T AT G T T T T C AAAC C AAAAAAAAAAAAAAAAAAAAA AAAAAAAAA
SEQ ID No:26
The DNA sequence encoding the polypeptide of SEQ ID No: 12 is provided herein as SEQ ID No:27, as follows:
GGGCGGCGGCGCAGGGCCGCGGGGTGTCGGCAGCAGCTGCAGCGGCCCGCGCCACCCCCTCGGCCGCGGC TCCGGGGCGAGGGTGAGCGGAGCACGGCGAGCAGCTGCCACCCGGCGCCGAGTGGAGCGGCAGCCCCGGA GGACGCAAAGTTGTCAGTCTAAGCGCAGACATGCCGGGCAGCGACACGGCGCTCACCGTGGACCGGACCT ACTCAGACCCCGGCCGGCACCACCGCTGCAAGAGCCGGGTGGACCGCCATGACATGAATACCCTCAGCCT ACCCCTGAACATACGCCGAGGGGGATCAGACACCAACCTCAACTTCGATGTCCCCGATGGCATCCTGGAT TTCCACAAGGTCAAACTCAATGCGGACAGCCTGAGACAAAAAATCTTAAAGGTGACGGAGCAGATAAAGA TCGAGCAGACGTCCCGAGATGGGAACGTTGCAGAGTATCTGAAACTGGTCAGCAGTGCAGACAAGCAGCA GGCTGGACGCATCAAGCAGGTCTTTGAGAAGAAAAACCAGAAGTCAGCCCACTCCATCGCCCAACTTCAG AAGAAGTTGGAGCAGTATCACAGAAAGCTCAGGGAGATCGAACAGAATGGCGTGACCAGGAGCTCAAAGG ACATCTCTAAAGACAGCCTGAAGGAAATACACCACTCTCTGAAGGATGCCCACGTGAAATCTCGAACTGC TCCCCACTGCCTGGAGAGCAGTAAATCTAGCATGCCAGGGGTATCCCTCACGCCCCCCGTGTTTGTCTTC AATAAGTCCAGAGAGTTTGCCAACTTGATCCGGAATAAGTTTGGCAGTGCAGATAACATTGCTCACTTGA AGAATTCCCTGGAAGAGTTCAGGCCCGAAGCCAGCCCCAGGGCTTACGGCGGAAGCGCTACCATCGTGAA CAAACCTAAGTACGGCAGCGATGACGAATGTTCTAGTGGCACATCCGGCTCAGCGGACAGCAATGGGAAC CAGTCCTTTGGGGCTGGTGGGACCAGCACCCTGGACAGCCAGGGGAAGATTGCCAAGATCATGGAGGAAC TGAGGGAGATCAAGGTTACCCAAACCCAGCTGGCGGAAGACATCGAGGCACTGAAGGTGCAGTTTAAGAG AGAATATGGCTTTATCTCTCAGACTCTGCAAGAGGAGAGATACAGGTATGAGCGACTTGAAGACCAGCTC CATGACCTGACAGAGCTGCACCAGCATGAGACAGCCAATCTGAAGCAGGAATTGGCCAGCGCTGAGGAGA AGGTGGCCTACCAGGCCTATGAGCGCTCAAGGGACATCCAGGAAGCCTTGGAATCTTGCCAGACCCGCAT CTCTAAGCTCGAGCTGCATCAGCAGGAACAGCAGACCCTGCAGACGGATGCCGTGAACGCCAAGGTCCTG CTGGGGAAGTGCATCAACGTGGTTCTGGCCTTCATGACGGTCATCCTGGTGTGTGTGTCCACCCTGGCCA AGTTTGTCTCGCCCATGATGAAGAGCCGCTCCCACATCCTTGGCACCTTCTTTGCTGTGACTCTTCTCGC CATATTCTGTAAAAACTGGGACCATATCTTGTGTGCCATAGAAAGGATAATCATCCCGAGATGAGCCGCT GCTTCCTGCCTTCACATCCTCTTAAAGATTTTATTTTGAAGAAAACCCTGTGCATACTACCAAAATTTCT GAATGGACGGCTGTGCCAAAGACGGCGCGAGAGGGACCAGAGCTACGTGCGGTGTAAATAAGTAGAGACT CTTAACCCAGTAGCAGATGCCAACACTGTCCCTGTATGTGGTTAACGTGAAACTGTCCCAGAGTCCTCCT CAAGGGGCTCACTGCCTTAATCAGAGCCGATCTCGAGCCCACCTGTTGAGCTCAGCACAGCAAAGCCCTT GTTCTTTGGGCGCCATGATTGCTAACTGCTGTGTTCTGAGTTCCCCCTGAC CAGCAAGAATATGGCTAGG ACTTGGGGAAGTGAGCAAACAGGAACTGGCAGGGTTGAGCTCCTGGGGGCCTATGGAAGCTCAGTGAGTC TCTCTGTGATCTCTCCTACTTGTCCATGGGTGGCTCTGTGCCCTACTATGTACCCAGTGTTCACAGCGGT CAATGGTGTATGTCCTCATAACGTGGGAATCTTCTACCTGCTTATCCAGCCGTAGTAGTGCACTTTACAA GGTAGACACTGGGATTTACAGCCAGGCTTTCCTCTTATGCCCAGAGCACCACACACCTGATTCCATCTTG CGTGGTTAGCAGTGCTCTGCCCTCAGAGCATTCGGCTGGGCTGTTGTCAAACTGTAGCTTGGGTAACTCA GGGTCCTGCGAGCATCCCTGGGGAGGCAGTGCGTAGGCGACTGTTAACTGTGATAAATGCCCTGAGACGA CAGGATGCACTTTACGTGATTCACTTTACTTGCGGGTCTGTGGGGATCTGACTGCAAAGCTTGTTCAGGA AATGAAAACGAATGACGTTTGTGTTACCCAGCTTTCTTTTCTCAGAGACAGCAAAATCAAACTTCATAAC TCAAGTGTGTGTGTGTGCTGAGGCAGGACACTGGGGGCCTAAGAGGGATGTGAACTTTTAGGTCTAATCA AATGGTGGCAAGAGTAGTGGGAGCTCTTGACCCAGATGAGAGCAGGAGGGTCCCTTAGGGACGCAAAGGC CATCAGGAATAAAATTAAGTCCTGCGCTGGAGTGGAGTTGCTGCTGTCTGTCTCTTAGGTACACAGATAA GCTCTGAGGTCACAGGAGAAATGCACTGCCCCATGAGCAGAGAGCCTGCAAGAGCTGCTGTGTCTTGTTC GATGGCTGCCATTTACTGACAAACCTGGTTTTCTGTGTACGCCACCAAGAGGAGAGAGAAGATTCTTATT GTGTCCTGTCAGCCAGCCAGTCAGGTCTCTGGTCCTCCCCACCCCCAACCCCCTCCCTGCTGAAGGTGTA AAAGATACTCTTAAATGCTTTCTTCCAGAAGAACCTCTCTTAGATTTAACTATCCTCACTTGCAAATGGA TGTCCTTAGGGACACTCCACCCCACCCCACCCCCGGGGCACATTGGGGATGGTGGCATGTAGCAAGTTAG TACAAGGCCAGGTTAGGTGAGTGGGACCCACACAGTTCTAACCTTTGCCCCTCACCCACTCCAACTTCTA AGTCAATAATTCATAGTTTTAGTAAATTCACAAAGTATTTAACCATCACCCCTAACCAATTCCAGAGCCA TGCTTTTCCAAAATAATAATAATAATAATAAATTTAATTTTATAAAATACAAAGGAGTTTGAAATCAGGG ATGGGGAAGTTCCCAGTACCACCCCTATCTATGGCTTTGTTCCTACACGGGAGACAGAGGTTTCTAGTCC AAAAAAAAAAAAAAAAAAGGGAGGAAGAGAGAGACATGATCTTCTTTTATTCTTGTATTTTACTGGCCAG CTCCCTTACCTGTAGGCCCCAGACTCCATCTGCCTGTACCCTGAAGCAAGCTGGGCTCTGCTGGGCTCCC ATTCCTATCTGTGAATATTTCCAAGTGGCACTTAGGGCGTGCCAGGGCCTGGGATGCCCGTGAATGCCGC TAGCAGGTTTCAGAAAGAGTTTGAATGCAACCTTAAGAGACTTAGCTAGGGCTCTGAGAGAATCTGGGAA GAGGTTCTGTTCTCACGCCAGCACCTGCCCCACCCCTACACTGCCTAACCGGACCACTCTTGATTGACGC TTGCCAGACCTGCAATCTGTGTTTCTTCTCCCTGCTGAGAAAGTCCAAAACAGCACACTCCACACCCTTC AGACTGAGCTTTGGACAGAATCTGTTAGAAGCCTCCAGCACCATTGTGAATTAATGGAGGTAGGGGTGGG GCCTCCAAATCTTGGATTCCTTCTAAAGAGCTTAGATGCTCATTGCTTCCCATTAGATGTCAGATCACCT TCTTTGCCTGGGCATGTGCAGCTCTATGGCATACTTGTTTCGGTCTCACGGAAAGTTTGTGAAGCTTTTG ATATTTTTAGCAAGAGTGTATAGTTCAAGGAGACTTTGACTCAACAGTTCTTCATTCTCTCAAGGGCTAT AACCCCACTCTCACTCTCTTGAACATGTAACATACATACTGAAGAAATGAGTTCTATAAATATACAATTG GCGGTAGCAATAGAGCTGTGAGGTACCCTGGAATTGGAATCGATTTGTAATTGAGTCAGCGCATCTTGTT TTGCACTTTATGTTGGGAGTAGACATCTCGTGCATCCCTCGTGTTGAGAGCCAGTTGGAGGTTAGTGAGC ACGTTCCCTGTTCGCCTAGGAACACTGGTGAGAGCTTCTAGATGCTGGACGCTGCGTCTCTGGTTATCAT G AC C GAT CAT C AG AAG AG T G AT T AC GG AGG AC AG AAC AG AAC T T T AAAAT AC T AAG AAT AAT AAAT AT T A AAAAAAAAAAAAAAAAGTAAGCAGCCACTCAGTGAAGCAAGTGGTGACCTCAAGTGCTGTCGCTGACTTT GGACTGTGAAGGCAGGATTTCCCATCTCAAGCTCCTGGCTTGTGGCTTAGTAGTGTTCAGGTGTTCTTGT GTTGTCTGTTGGTGTTCATTCACATTAATACTCTGTAGCTCTTGACTTTATCTAAGCAATGCCCTCAATG AGTTCTTACTGCCTGACTGCCTGGCCACTTCTCTAACATGGGGGGGGGGGGGGGCAGGCGGATGAGGGAG CTCTTCCCCCCAACTCCCTCTCCCAATAATTAGTTACTTTGGCTCGTTTGGGTTGAATTTTAAATACCAA ATTACTTTTGTGTTGAATGGCAGTATATCTATCATAGGGTTGTTGTTGTTATTGTTGTTTTGTTCTGATG TTGAATCCAGACCTAATTGCCTGGGTTTGGAAAAACAAACGTACTCATTGATAAGAACGTGTGTGATCAA ACAACCTATTTTGGACGTTTTCTTAATTCTGACCTTGGGCTGGGATGTGAGCACGGGCTTATGCAGGGAG ATGGTTCAAAGCTAGCAGGTAACCTTTAAGGGGCCTAAAAAGACAAATCACAGAAAACAATTAGATTGGC CTGTTTGTATCAAAAATAAAGAGCTTTAAAAACGTGTCATTGATGGGAATTGGGTAATTTGTTATAAATA AAGCCAAATTATCATTTTCTGCCATGGATACAGTTCTCTGCTTAAAGAGTCTGGAAGTGATGTGTAATTC CTTGGGAGTCTGAGGGAGATTTACATGTTGCTGTCCGCATATATTTAAAGCATGTCTATTGTGTTTGTCC TAAAAGAAACATTTCAACTAACCGTGCTATGTGTTTCAGATACGCAGGGGCAATAGCAGCGATTCTGATG TAACATCAGGAATGCTGACTGTAACTTCTGTTGGAGAATTCCCATCTCATGGGAAGCTACATAGTTTTGT ATCTGCCTAAAATTGTAATTTCTCTGTAGATCTTCCCCACGGCGGTGTTATTAATGAATTCAAAGCAGGT GCCATTATTTTTTAAACAATTGATGTTTGCTTTGGTGTTAAATAAAAGAACTAGATTTCTTAAATTTT SEQ ID No:27 The DNA sequence encoding the polypeptide of SEQ ID No:13 is provided herein as SEQ ID No:28, as follows:
TTATTGATGCTATTAAGAATCAGATTGCTGTCTAAAAGTCATGGTTTTAAAGGTGGAAAGTACCAAGAGC TCAAGTGCAACCCTCCCCACCAACCTGCCCTCTTACAGGTCTCTGTCTTCCTTCTGTGAGGATTGCCCTA GTAGTCACACTAGCTTCTCAGATGGTGAGCTTGCCCGCAATGTGAGGGAAGGTGTCAAGCATCGAATCTT CTACCTCTCAGAGCAGTTGAGAGTGGAGAAGGCCAGTAGGGATGAAAATAC CATGAGCTACCTCAAACTG GTATCCAAAGCTGACCGGCACCAGGCCCCACACATCCGGAAGGCCTTTGAGAGGGTGAACCAGCGCACCT CTGCCACTATTGCTCACATTGAACGAAAACTCTATCAGTGTCATCAGCAGCTGAAGGAGCTGGAAGAGGG CTGCAGTCCCACAAGCTCAGTGCTGAAAGTGGGCAGTGGCTTGGACAGCCATAAGCAGCCAAGTGGGAAA GTTTCATATTCCAAATTGTCCAAGCCAGGTGGGGAAGATAGCCTGCCCATCAACGTTGCTAGGTCCTCTA CTCTAGAGAGTCACTTATCAGAAATGCAACAGAGGAAATTCTCAGACAAAAAGTATGTGGCCCAGCAACA AAAGCTGCTTTTGCAGAAGATGAAGGAAGAACTGACAGAAGCCAAGAAGGTCCATGCTAGCTTTCAGGTA TCCCATCAAAGCCTCAAAGAAAGTCATATGATTGATGTGCAGAGGATACTGGAGTCACTCCAGGAAAAGA AAACCAAACAATCACTGATGGAAAAACAGGTGAATGATCACTTGCAGAGATACCTGGATGAGATTTGCCA CCTCAAACAGCATCTGGCATGCACTGAAGAGAAAATGGCCTATTTGTCCTATGAAAGAGCCAAGGAAATA TGGGATGTAATGGAGATTTTCAAGAGCAGAATAACCAAGCTAGAAACTCTACAGCAAGCTACCCAACTGG AGATGATGGCCAGCCTCAGAACCCGTCCCAAGGACTTTTTGTTCAGATTCATAAGCCTGCTTCTCACTCT GACCACTATCCTCTTGGTCGTGGTCTCTACATTGTGTTCTTGCCCCTTGCCCTTGCTCAGCTCACGCCTG CGCATCTTCATTGTATTTATGATCATTGGGCTTGGGACTTTGGCCTGGCAGAAGCGCCATGTCATCTCTA TTATTGACTGGCAGGCCTGGGTCCCCTTTAAGTGGAGACAAGACTTAAAGGATGCTAAGCCTCCATCAGA TGGACACTGAGGGGCAAGAAGTGCCATTCACCCGAATTTCTTCCTTTCAGTTGTTGAGGGCATCCAGTAA GACCTCTTTAGTAAGCTACTTTGTTGCTCTCATTTTCTTAATTCTGTAACTGCAAGCCCAGTGAGATACA TTGCAGATGCCAACTTGCCT SEQ ID No:28
The DNA sequence encoding the polypeptide of SEQ ID No: 14 is provided herein as SEQ ID No:29, as follows: CCGGCACGGGCAGTGGTCTGGAGGATTCCCGCCTCAGTGGCAACGAGGACTACTACTCGTCCTTCGTCTC TGACGAGTTTGACAGCAGCAAGAAGGTCCATCGCCGCTGCCACGAACGCAGCTCCAGCGTTCAGGCCATC GAC C G AT T G AAC AC G AAG AT C C AAT GC AC C AAGG AG T C C AT C C G AC AGG AG C AAAC T GC C AG AG AC GAT A ATGTCAACGAGTATCTTAAGTTGGCAGCCAGTGCAGACAAGCAGCAGCTGCAGCGCATCAAGGCTGTCTT TGAGAAAAAGAACCAGAAGAGCGCACACAATATCTCGCAGCTGCAGAAAAAGCTGGACAACTACACGAAG CGGGCCAAGGACTTGCAGAATCACCAGTTCCAGACGAAGAGCCAGCACCGTCAACCGCGCGAGGTGCTGC GGGATGTCGGCCAGGGTCTGCGCAACGTGGGAGGCAACATCCGCGATGGCATCACCGGCTTCTCCGGTTC GGTGATGTCCAAGCCACGGGAGTTTGCGCACTTGATCAAGAACAAGTTTGGCAGCGCCGACAACATCAAC CAGATGAGCGAGGCCGAACTACAGGGCATGCAGTCTGCCAATGCCGATGTTTTGGGCAGCGAGCGTTTGC AGCAAGTGCCTGGAGCCGGAACCTCGACGGGCTCAGGCGGCGGCGGGCAGAACAACAACACCGGCGGAGC GGGAAGCGGGACGGGAAAGTTCAACAGTGACAATGGCAGCGAATGCAGCAGCGTAACGAGCGAAAGCATA CCGGGAGGGTCTGGTAAAAGCCAGTCGGGAGCCAGCCAATACCACATAGTGCTCAAGACATTGCTAACAG AGCTGGCCGAGCGAAAGGCCGAAAACGAGAAGCTCAAGGAGCGCATCGAACGACTCGAGACGGGCCAAAA GGAATTCAACAATCTGACCGCCACACTCGAAAGTGAACGCTATCGTGCCGAGGGACTCGAGGAGCAAATC AAT GAC T T G AC GG AAT T AC AT C AG AAT G AAAT T G AG AAT C T G AAAC AAAC G AT T GC C GAC AT GG AGG AG A AAG T AC AAT AC C AAAGC G AT G AAAG AC T AC G T GAC G T C AAC G AAG T GC T C G AG AAC T G T C AAAC G AGG AT ATCAAAAATGGAGCACATGTCGCAGCAACAATATGTCACCGTCGAGGGCATTGATAATTCGAATGCGCGG GCCTTGGTTGTGAAACTCATCAATGTGGTATTAACGATACTGCAAGTGGTGCTCCTGCTTGTGGCCACCG CTGCAGGCATCATAATGCCTTTTCTCAAAACGAGGGTTCGCGTTCTCACAACGTTTTTGTCTATTTGTTT CGTCATCTTTGTGATACGACAGTGGCCGGATGTCCAGGACATTGGATCCGGCCTGGTGCGACATCTCAAG CAATCGCTGGTGGTGAAGTAAACTACGTGGGTTTGGTGGCCACCGTCTCATCTACCGGGTAGTTGACACG TTCTTTAGTTATGTATCCTAAGTTCTAAAGTGCATATGTTGTATATAGCTTATCGATAACGATTATAACA ATAATTTTTAACTGAAATTGTATCAATTATCAACACGAACACACCCGAGAAACTGTACAAAATATTCATA T AC C C T AT C AC AC AC AC AC C T AAT C AT C AC AC C AC AC T C T C GC AT C AAAT C T AGC AAAAT T G AT T T AAAC T AT G T AAT AAC T T G AAAAT GC T G AC AT G T C T C C G T GC AT T AC T C AC T AT AT T T G AAC AT AC AT AAT T T AT GTATTACCTATATAAAATATTTACATTACTATAAAATAATTGAAAATATATAAAAAGAAAGTTTGTATAA AAAAAAAAAAAAAAAA
SEQ ID No:29
The DNA sequence encoding the polypeptide of SEQ ID No:15 is provided herein as
SEQ ID No:30, as follows:
ATGACAACAGACTCGGCGAGTCTAAAATCTTCCGAGGGAACATGCAGTTCGATTGGAGACGAGCGAGAGA AGGCGAAGATTGTGCAGAAGCTAATTGAGATCAAAGACAAGCTACGGGCGTTGAACGAGAAACGTGAGGC TGACGTCGAAAAGTTTCTCTCCATAACGCGACAATCAGAAATCTCTCGCGGAGTTGGAGCAGATAACCCT CAGAGAGCCCGTATTCGCAACAATTTTGAGAGGCAGAACAGAAAACATGCACATGAGACTGAAATGCTTC AGAAGAAACTTATCGATTATGAGGAGCGCTTGAAATTGGTTGACAGTGGAGAATATGAGCCCAGTCCGAC GAAATCGCGAGTTTTCCCGACTGGAATCAGAAAAGCAAAAGGAATGACTGAAACAATGGTGAATGCTCCA ATCGAGTTTGCGCAACGTGTCAAATCAGCTTTTTCGGCGGATAATGTGAACAGCACGCAAAACGGAACAA CAGGAGCTCCGAAAACTGGTCAGTCTACATTTTTCACCACTCGCAAAAGTGCCGACACGGATGAAGTGGA GAGCAATGCCGTACATAAGAATCGTGGGGCAAAACGTAATAGTTCGACTCTTCCGCCAAACTTATCGCTT ACAAGTCCTGACCCTCTCAGTGCATACAAGGAAGAAGACAGTTCTGATCCAGAATCTCGCCCGGGAAGTG CTGCTGATGAGACATCAAATGTGCCGTATCACACTGCCGACAATAGCCTTTACCTGCCTCCAAATCACCC CTATCACTCTGCTCATGCTGCTCCCAGCGAAGAAGGATTTAATGCCATCCATGAGCATCTCAACAGTATC CTGCAACACTTGATGCTTATTGATAGAAAATACGACAGACTGGAGGATGATATCAAAAAGGAGATCAAAT TCTACGCGGAGGCTTTGGAAGAGGAACGATTCAAGACGACGAAGCTCGAAGAAATTCTGAACGAGGCTGT TGAGTTGCAGCAAGCTGAGATTGCGACACTGAAGGAGCAAAACCTGATGGCGACGCGTGTGGATTATCAG CACAATGACCGGTTCAGGAATGTTGAGGAGAACATGGAGTCACTGCAGAATCATCTTGTGAGAATCGAGA ATGCATTGATGGACGTCAGACAAGTCAAGCTGACGAGTAATGTGTGGCAACGTGTTGCTCTCAATGCAGG AAATATTGTCGTAGAGCTGCTGAAAATTGCACTGTTTGTCGTCGCGTCGATCCTTGATTTGGTTCGCCCT TTGACTGGTTCCAGAAATCGCTCTGCAATGGCATTTGGACTCGTTTTCCTGGCCATTTTCTTTGGGCATC ATCTTCAAAAAGTGACCTACCTGTTTGGAGGCAGCACTCCGGATGTAAACAAGACTGGCGGGCCCACCAA GTAA
SEQ ID No:30
Accordingly, the nucleotide sequence of the fifth aspect may comprise a nucleic acid sequence substantially as set out in any one of SEQ ID No's: 17 to 30, or a functional variant or a fragment thereof.
In a sixth aspect, there is provided a genetic construct comprising the nucleotide sequence of the fifth aspect. Genetic constructs of the invention may be in the form of an expression cassette, which may be suitable for expression of the polypeptide in a host cell. The genetic construct of the invention may be introduced in to a host cell without it being incorporated in a vector. For instance, the genetic construct, which may be a nucleic acid molecule, may be incorporated within a liposome or a virus particle. Alternatively, a purified nucleic acid molecule (e.g. histone-free DNA, or naked DNA) may be inserted directly into a host cell by suitable means, e.g. direct endocytotic uptake. The genetic construct may be introduced directly in to cells of a host subject (e.g. an animal) by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. Alternatively, genetic constructs of the invention may be introduced directly into a host cell using a particle gun. Alternatively, the genetic construct may be harboured within a recombinant vector, for expression in a suitable host cell. In a seventh aspect, there is provided a recombinant vector comprising the genetic construct according to the sixth aspect.
The recombinant vector may be a plasmid, cosmid or phage. Such recombinant vectors are highly useful for transforming host cells with the genetic construct of the sixth aspect, and for replicating the expression cassette therein. The skilled technician will appreciate that genetic constructs of the invention may be combined with many types of backbone vector for expression purposes. Recombinant vectors may include a variety of other functional elements including a suitable promoter to initiate gene expression. For instance, the recombinant vector may be designed such that it autonomously replicates in the cytosol of the host cell. In this case, elements which induce or regulate DNA replication may be required in the recombinant vector.
Alternatively, the recombinant vector may be designed such that it integrates into the genome of a host cell. In this case, DNA sequences which favour targeted integration (e.g. by homologous recombination) are envisaged.
The recombinant vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA. Alternatively, the selectable marker gene may be in a different vector to be used simultaneously with vector containing the gene of interest. The vector may also comprise DNA involved with regulating expression of the coding sequence, or for targeting the expressed polypeptide to a certain part of the host cell. In an eighth aspect, there is provided a host cell comprising the genetic construct according to the sixth aspect, or the recombinant vector according to the seventh aspect.
The host cell may be an animal cell, for example a mouse or rat cell, or a Drosophila cell. It is preferred that the host cell is not a human cell. The cell may be transformed with the genetic construct or the vector according to the invention, using known techniques. Suitable means for introducing the genetic construct into the host cell will depend on the type of cell.
In a ninth aspect, there is provided a transgenic host organism comprising at least one host cell according to the eighth aspect.
The genome of the host cell or the transgenic host organism of the invention may comprise a nucleic acid sequence encoding a polypeptide, variant or fragment according to the first aspect. The polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's 1-16, or a functional variant or functional fragment, or its encoding nucleic acid thereof may comprise a pathological mutation. For example, a pathological mutation may be an insertion into an intron that alters the production of normal NTRP, for example the ntrp1 allele described herein, or another mutation that alters the amino acid composition of the peptide produced. The nucleic acid sequence may be operably linked to a tissue-specific expression control sequence (such as a promoter), which drives expression of the nucleic acid sequence, wherein expression of the nucleic sequence results in the host organism displaying an altered phenotype.
The host organism may be a multicellular organism, which is preferably non-human. For example, the host organism may be a mouse, rat or Drosophila. The host may be used in studies of neurodegenerative disorders. Although not wishing to be bound by any hypothesis, the inventor believes that, because APP and apoE are both well-established risk factors for neurodegenerative disorders, modulation of the polypeptides of the invention could be used for treatment of such disease conditions. The inventor has therefore designed a method of screening for a useful therapeutic agent for preventing or treating neurodegenerative disorder, and a method of identifying a test compound's capability to modulate any of the peptides described herein.
Therefore, in a tenth aspect, there is provided an assay for screening for a compound that is a modulator of a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof, the assay comprising the steps of:
(a) providing an assay system in which a polypeptide selected from the group
consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof is either over-expressed or under-expressed compared to the
corresponding physiological level of expression;
(b) introducing a test compound into the assay system; and
(c) determining either γ-secretase activity or APP metabolism,
wherein an alteration in either γ-secretase activity or APP metabolism in the presence of the test compound compared to γ-secretase activity or APP metabolism in the absence of the test compound indicates that the test compound is a modulator of the polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof.
In an eleventh aspect, there is provided a method of screening for a therapeutic agent useful in the prophylaxis or treatment of a neurodegenerative disorder, the method comprising the steps of:
(a) providing an assay system in which a polypeptide selected from the group
consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof is either over-expressed or under-expressed compared to the
corresponding physiological level of expression; (b) introducing a test compound into the assay system; and
(c) determining either γ-secretase activity or APP metabolism,
wherein an alteration in either γ-secretase activity or APP metabolism in the presence of the test compound compared to γ-secretase activity or APP metabolism in the absence of the test compound is an indication of the ability of the test compound to modulate the neurodegenerative disorder.
In a further aspect, there is provided an assay for screening for a compound that is a modulator of apolipoprotein E (apoE), the assay comprising the steps of:
(a) providing an assay system comprising apoE or a fragment thereof, and in which a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof is either over-expressed or under-expressed compared to the corresponding physiological level of expression;
(b) introducing a test compound into the assay system; and
(c) determining either γ-secretase activity or APP metabolism,
wherein an alteration in either γ-secretase activity or APP metabolism in the presence of the test compound compared to γ-secretase activity or APP metabolism in the absence of the test compound indicates that the test compound is a modulator of apoE.
In another aspect, there is provided an assay for screening for a compound that is a modulator of the binding between apolipoprotein E (apoE) and a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof, the assay comprising the steps of:
(a) contacting apoE and a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof;
(b) introducing a test compound; and
(c) determining the impact of the test compound on the binding between apoE and the polypeptide, variant or fragment thereof,
wherein an alteration in the structure of apoE and/ or the polypeptide, variant or fragment thereof, or in the association therebetween, indicates that the test compound is a modulator of the interaction between apoE and the polypeptide, variant or fragment.
The test compound may be used as a therapeutic agent useful in the prophylaxis or treatment of the neurodegenerative disorder.
The assay system used in step (a) in the assay or methods may be either an in vitro or an in vivo system. The assay system may be a cell-based system comprising cells, which either over-express or under-express a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof, compared to the level of expression that would occur under physiological conditions (i.e. basal expression levels). Hence, the corresponding physiological level of expression of the polypeptide of the first aspect can mean the amount of polypeptide that is present under normal physiological conditions in vivo when no test compound (i.e. the modulator) has been added.
Over- expression may include:
(i) increasing, promoting or augmenting transcription, translation or expression of the polypeptide of the first aspect;
(ii) increasing synthesis or release of the polypeptide from intracellular stores; or
(iii) decreasing the rate of degradation of the polypeptide.
It will be appreciated that in each of cases (i) to (iii), the result is that the concentration of the polypeptide will be higher than would be the case under normal physiological conditions.
Under-expression may include:
(i) decreasing, preventing or attenuating transcription, translation or expression of the polypeptide of the first aspect;
(ii) inhibiting synthesis or release of the polypeptide from intracellular stores; or
(iii) increasing the rate of degradation of the polypeptide. It will be appreciated that in each of cases (i) to (iii), the result is that the concentration of the polypeptide will be lower than would be the case under physiological conditions.
The assay system may not express a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof, or may express a polypeptide but which is rendered non- functional. For example, the assay system may comprise the use of a cell which is mutated such that the polypeptide of the first aspect is either not expressed at all, or only minimally expressed compared to basal expression levels. For example, RNAi may be used to reduce or prevent expression of the polypeptide of the first aspect.
The assay system may be a non-human animal model. For example, the animal may be a transgenic ape, monkey, mouse, rat, fish, ferret, sheep, dog, cat, worm or Orosophila. APP metabolism and γ-secretase activity may be determined in step (c) using techniques known to the skilled person, for example, but not limited to, those as described in materials and methods. Figures 11 and 14 show how APP metabolism and/ or γ- secretase activity may be determined. APP metabolism involves cleavage of APP into fragments of various sizes. For example, OC-secretases and BACE cleave APP to release the ectodomain and γ-secretase may cleave the APP C-terminal fragment at several sites generating peptides with different C-termini, the most common of which are Αβ1 -38, 1-40 and 1 -42. Most mutations in the presenilins favour the production of more amyloidogenic form, Αβ1-42. Therefore, alteration of APP metabolism or γ-secretase activity, as assessed by step (c), may involve analysis of cell lysates or cell culture media for the products of APP metabolism by Western blot or ELISA; analysis of living or fixed cells for alterations in the cellular distribution of APP or it's proteolytic products by, for example, density gradient fractionation or immunohistochemistry. APP metabolism may also be assessed using transcription factor reporter assays as described (Cao, X. & Sudhof, T.C. (2001) Science, 293, 115-120). The assay system may comprise apoE2, apoE3 or apoE4 or a fragment thereof.
Alteration in the association between apoE and the polypeptide may be determined using known techniques, such as using intrinsic fluorescence methods. In a twelfth aspect, there is provided a modulator of a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof, for use in treating, ameliorating, or preventing a neurodegenerative disorder.
In a thirteenth aspect, there is provided a method of treating, ameliorating or preventing a neurodegenerative disorder in a subject, the method comprising administering, to a subject in need of such treatment, a modulator of a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof. The neurodegenerative disorder may be selected from a group consisting of
Alzheimer's disease, Parkinson's disease, Huntington's disease, Spinocerebellar type 1, type 2, and type 3, and Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia or Schizophrenia. Preferably, the neurodegenerative disorder is Alzheimer's disease. The modulator may be a positive modulator or a negative modulator of the function of a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof.
In one embodiment, the modulator may comprise a modifier of γ-secretase, for example LY450139 (hydroxylvaleryl monobenzocaprolactam). In another
embodiment, the modulator may comprise a drug that is known to be involved in lipid metabolism, such as a member of the group of drugs known as statins; for example, but not limited to, simvastatin, atorvastatin or lovastatin. In another embodiment, the modulator may comprise a drug from the thiazolidinedione class of drugs, for example, but not limited to pioglitazone or rosiglitazone. One hypothesis for the causative agent in Alzheimer's disease is β-amyloid, i.e. excess production of β-amyloid is thought to lead to Alzheimer's disease. The inventor has demonstrated that, under certain circumstances (see Figure 14), the conjugate of the polypeptide of the first aspect and apoE4 leads to increased amyloid production, compared to the combination of the polypeptide of the first aspect and apoE3. Thus, the inventor believes that reducing the activity of the polypeptide of the first aspect or the concentration thereof will reduce the likelihood that Alzheimer's disease would develop in a subject. It follows therefore that, in one embodiment, the modulator may be a negative modulator of the function of a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof. Accordingly, the modulator which may be used may comprise an RNAi or siRNA molecule, which is capable of inhibiting or slowing expression of the polypeptides of the invention, as demonstrated in Drosophila (see Figure 5). Preferably, the siRNA molecule comprises a nucleotide sequence which is specific for, or complementary to, at least a region of one or more of the nucleic acid sequences substantially as set out in any one of SEQ ID No's: 17 to 30. The siRNA may comprise 15-30 nucleotides in length, preferably 21-25 nucleotides in length, and the skilled person would readily appreciate how to design a suitable siRNA molecule once provided with the sequence of the target DNA molecule. It may be preferred that the siRNA molecule comprises a nucleotide sequence which is specific for, or complementary to, at least a region of the nucleic acid sequence encoding the consensus sequence, SEQ ID No.16, or a variant or fragment thereof. Other negative modulators may include agents that inhibit transcription factors normally activating expression of the polypeptide, variants or fragments of the invention, or agents that modify phosphorylation of the polypeptides, variants or fragments of the invention. It will be appreciated that modulator according to the invention may be used in a medicament which may be used in a monotherapy (i.e. use of a modulator of a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof), for treating, ameliorating, or preventing neurodegenerative disorder, such as Alzheimer's disease. Alternatively, modulators according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing Alzheimer's disease. For example, the modulator of the invention may be used in combination with known agents for treating Alzheimer's disease, such as acetylcholinesterase inhibitors.
The modulators according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well- tolerated by the subject to whom it is given, and preferably enables delivery of the agents across the blood-brain barrier.
Medicaments comprising modulators according to the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin, for example, adjacent the brain.
Modulators according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site, e.g. the head. Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection). In a preferred embodiment, medicaments according to the invention may be
administered to a subject by injection into the blood stream or directly into a site requiring treatment. For example, the medicament may be injected at least adjacent the brain. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).
It will be appreciated that the amount of the modulator that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the modulator and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the modulator within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical
composition, the mode of administration, and the advancement of the
neurodegenerative disease. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration. Generally, a daily dose of between 0.001 ug/kg of body weight and lOmg/kg of body weight of the modulator according to the invention may be used for treating, ameliorating, or preventing neurodegenerative disease, depending upon which agent is used. More preferably, the daily dose is between Ο.ΟΙμ-g/kg of body weight and lmg/kg of body weight, more preferably between Ο.ΐμ-g/kg and 100 g/kg body weight, and most preferably between approximately
Figure imgf000038_0001
and 10 g/kg body weight.
The circulating dosage of the modulator may preferably be between about 10 nM/L and 100 nM/L, or between about 25 nM/L and 75 nM/L. The modulator may be administered before, during or after onset of neurodegenerative disease. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the modulator may require administration twice or more times during a day. As an example, modulators may be administered as two (or more depending upon the severity of the neurodegenerative disease being treated) daily doses of between 0.07 g and 700 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of modulators according to the invention to a patient without the need to administer repeated doses.
Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the modulators according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration). The inventor believes that he is the first to suggest an anti-neurodegenerative disease compositions, based on the use of a modulator of the polypeptides of the invention.
Hence, in a fourteenth aspect of the invention, there is provided an anti- neurodegenerative disease composition comprising a therapeutically effective amount of a modulator of a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof, and optionally a
pharmaceutically acceptable vehicle. The term "anti-neurodegenerative disease composition" can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of a neurodegenerative disorder in a subject, such as Alzheimer's disease.
The invention also provides in a fifteenth aspect, a process for making the composition according to the fourteenth aspect, the process comprising combining a therapeutically effective amount of a modulator of a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof, with a pharmaceutically acceptable vehicle.
The modulator may be a positive or negative modulator. Preferably, the modulator is a negative modulator, such as an siRNA or RNAi molecule, which is capable of inhibiting or slowing expression of the polypeptide. Preferably, the siRNA molecule comprises a nucleotide sequence which is specific for, or complementary to, at least a region of one or more of the nucleic acid sequences substantially as set out in any one of SEQ ID No's: 17 to 30. The siRNA may comprise 15-30 nucleotides in length, preferably 21-25 nucleotides in length. The siRNA molecule may comprise a nucleotide sequence which is specific for, or complementary to, at least a region of the nucleic acid sequence encoding the consensus sequence, SEQ ID No.16, or a variant or fragment thereof.
A "subject" may be a vertebrate, mammal, or domestic animal. Hence, medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. Most preferably, the subject is a human being.
A "therapeutically effective amount" of agent is any amount which, when administered to a subject, is the amount of drug that is needed to treat the neurodegenerative disorder condition, or produce the desired effect.
For example, the therapeutically effective amount of modulator used may be from about 0.001 ng to about 1 mg, and preferably from about 0.01 ng to about 100 ng. It is preferred that the amount of modulator is an amount from about 0.1 ng to about 10 ng, and most preferably from about 0.5 ng to about 5 ng.
A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions. In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent (i.e. the modulator) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent according to the invention (the modulator) may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The modulator may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. The modulators and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The agents used according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms "substantially the amino acid/ nucleotide/ peptide sequence", "functional variant" and "functional fragment", can be a sequence that has at least 40% sequence identity with the amino
acid/ nucleotide/ peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No:3 (i.e. TMCC2) or the nucleotide identified as SEQ ID No: 18, or 40% identity with the polypeptide identified as SEQ ID No:14 (i.e. Drosophila NTRP), and so on. Amino acid/ olynucleotide/ polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino
acid/ polynucleotide/ polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
It will be appreciated that the invention extends to polypeptides having substantially the pattern of residues identified in SEQ ID No:16.
The skilled technician will appreciate how to calculate the percentage identity between two amino acid/ polynucleotide/ polypeptide sequences. In order to calculate the percentage identity between two amino acid/ polynucleotide/ polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FAST A, Smith- Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al, 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.
Preferably, calculation of percentage identities between two amino
acid/ polynucleotide/ polypeptide sequences may then be calculated from such an alignment as (N/T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N/T)*100.
Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No's: 17-30 or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in 0.2x SSC/0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No: 1-16. Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will known the nucleotide sequences encoding these amino acids.
All of the features described herein (including any accompanying claims, abstract and drawings), and/ or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/ or steps are mutually exclusive.
For a better understanding of the invention and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which:- Figure 1 shows that one embodiment of a protein in accordance with the invention known as NTRP (TMCC2421-709) binds preferentially to ApoE3 compared to ApoE4. Figure 1 A shows the results of a yeast- two-hybrid screen performed using ApoE3 and ApoE4 as baits and a human brain cDNA library as a source of targets. The results are represented in a graph showing relative β-galactosidase activity of yeast co-transformed with ApoE fused to the DNA-binding domain, and NTRP 421-709 fused to the activation domain of Gal4, lamin fused to the activation domain of Gal4 or the Gal4 activation domain alone.
Figure 1 B shows the binding of ApoE and NTRP in vitro by comparing the intrinsic tryptophan fluorescence of recombinant ApoE and an NTRP-thioredoxin fusion protein when incubated alone or together. The data are represented in a graph showing the percentage change in fluorescence after co-incubation relative to the sum of fluorescence values when incubated separately. Co-incubation of ApoE3 with an NTRP -Thioredoxin fusion results in an increased fluorescence relative to the sum of individual fluorescence values. Co-incubation of ApoE4 with an NTRP -Thioredoxin fusion, and co-incubation of ApoE3 or E4 with Thioredoxin alone did not increase the total fluorescence. Data are the mean of experiments performed with four separate preparations of NTRP-thioredoxin, error bars represent the standard deviation from the mean.
Figure 2 shows the shows the structural organization and sequence similarity of embodiments of human and Drosophila NTRP protein. "CC" represents domains predicted to form coiled-coil structures, and "TM" represents regions predicted to form transmembrane domains. The percent similarity for individual conserved domains (indicated by brackets) is shown. The sequences of the region containing the conserved domains are shown in Figure 15.
Figure 3 shows the location of an enhancer trap (NTRP-Gal4) and a lethal mutation (NTRP1) in the Drosophila NTRP gene. Coding exons are in-filled boxes while non- coding exons are shown as open boxes. The structure of the Drosophila NTRP gene was obtained from flybase.org. Introns are indicated by lines, the intron indicated by a dashed line is not to scale. Figure 4 shows the expression pattern of NTRP-gal4 expression in adult flies. NTRP- gal4 was used to drive nuclear green fluorescent protein (GFP). Figure 4A shows a horizontal optical section of the optic lobe showing expression in glia (G) and in medulla neurons (mn). Figure 4B shows a vertical section of the central brain showing NTRP expression in glia associated with the mushroom body peduncle (mp), central complex (cc), and other neuropils.
Figure 5 shows the effects of region- and cell-specific depletion of NTRP. Figure 5A is a Western blot using antibody 385 that shows suppression of NTRP expression in adult Drosophila heads by RNAi driven by tubulin-Gal4 in the absence of Dcr2, each sample was prepared from 10-30 heads and the equivalent of 3 heads was loaded per lane. Figure 5B shows that global suppression of NTRP using tubulin-Gal4 in the absence of Dcr2 is mostly lethal during metamorphosis. The data is represented in a graph showing the percentage of flies that remain alive at various stages of development: H, hatching (n=263); P, pupating (n=300); E, eclosing (n=300). Figure 5C shows the result of cell- and region-specific RNAi on total NTRP levels in the head. RNAi was driven in neurons and glia (repo- and elav-gal4), glia only (repo-gal4), neurons only (elav-gal4), or using the ntrp-gal4 driver. Control flies are wi l l 8 without RNAi. NTRP expression in flies was detected by antibody 385 in an immunoblot. Each sample was prepared from 20-30 flies and the equivalent of 3.5 heads was loaded per lane. Figure 5D shows that glial-specific RNAi (repo-Gal4; n=99) for NTRP is developmental^ lethal, whereas neuronal- specific RNAi (elav-Gal4; n=108) is not developmentally lethal. The lethality of NTRP- Gal4- driven RNAi (n=95) more closely resembled that of glia-specific RNAi. The data is represented in a graph showing the percentage of larvae with RNAi for NTRP driven by the indicated Gal4 constructs that eclose as adults.
Figure 6 shows the phenotype of flies expressing mutant ntrp1. Figure 6A shows that mutant ntrp1 produces a truncated NTRP protein as determined by immunoblot using antibody 385; each lane was loaded with the equivalent of 4 heads prepared from pools of 30-35 heads of each genotype. Figure 6B shows that mutant ntrp1 is a pupal lethal allele. The results are represented by a graph showing the percentage of mutant ntrp1 flies that remain alive at various stages of development (for embryogenesis (hatching) n=300, for pupation and metamorphosis (eclosion) n=176). Figure 6C shows the survival rate of adult flies expressing wild type NTRP (circles, n=33), heterozygous for ntrp1 (squares, n=51) and homozygous mutant ntrp1 (triangles, n=35). The results show that homozygous adult mutant ntrp1 escapees die within a few days.
Figure 7A shows the optic lobe phenotype in heterozygous and homozygous mutant NTRP1 flies where the retinal axons were detected by indirect immunofluorescence using antibody 24bl0 against chaoptin, and glia are detected using GFP expressed under the glia- specific driver repo-gal4. This optic lobe phenotype was observed in all flies homozygous for ntrp1 (n>26). Horizontal optical sections through the optic lobes of flies heterozygous for ntrp1 (images 1 and 2) show a normal appearance, however, the optic lobe of flies homozygous for mutant NTRP1 (images 3 and 4) are severely deformed. Figure 7B shows retinal axons detected by indirect immunofluorescence using antibody 24bl0 in flies with glial or neuron- specific suppression of NTRP expression by RNAi. Glia-specific RNAi (image 1) causes an optic lobe defect, whereas neuron- specific RNAi (image 2) does not cause an optic lobe defect; images are representative of 8 examples for glia-specific RNAi and 12 for neuron-specific RNAi
Figure 8 shows the results of cell- and region- specific expression of wild-type NTRP in adult flies that are homozygous for the ntrp1 allele. Figure 8A shows the expression of NTRP in head extracts of homozygous mutant NTRP1 flies which do not express wild- type NTRP (none), express wild-type NTRP in glia (repo-gal4), neurons (elav-gal4) or in both neurons and glia (elav-gal4 + repo-gal4) as detected by antibody 385 in a western blot; each lane was loaded with the equivalent of 3 heads prepared from a pool of 20-30 heads in each group. Figure 8B shows that expression of wild-type NTRP in either neurons or glia rescues the developmental lethality associated with the ntrp1 allele. Wild-type NTRP expression was driven from a UAS-NTRP construct in either the glia (repo-Gal4; n=51), neurons (elav-Gal4; n=117) or both neurons and glia (n=85). Data are presented as a proportion of ntrp1 flies eclosing compared to the expected Mendelian frequency. Figure 8C shows the lifespan of homozygous ntrp1 flies that express wild-type NTRP in glia only, neurons only, or in both neurons and glia. The lifespan of flies expressing wild-type NTRP in glia only (triangles, n=118) was shorter than that of flies expressing wild-type NTRP in neurons only (circles, n=138), or both neurons and glia (diamonds, n=54). Figure 8D shows that NTRP may influence the distribution or expression of synaptic proteins. All flies were homozygous ntrp1 flies where wild-type NTRP is expressed in glia, neurons or both. Images 1-3 are images of the optic lobe obtained from paraffin sections prepared from brains prepared on the day of eclosion and stained with antibody nc82, which recognises the component of pre-synaptic active zones known as bruchpilot. Image 1 shows the optic lobe of a homozygous ntrp1 fly expressing wild-type NTRP in both neurons and glia, image 2 shows the optic lobe of a homozygous ntrp1 fly expressing wild-type NTRP in neurons only and image 3 shows the optic lobe of a homozygous ntrp1 fly expressing wild-type NTRP in glia only. The distribution pattern of bruchpilot in flies expressing wild-type NTRP in glia only (image 3) is disrupted and patchy (indicated by an arrow in image 3) relative to that obtained in flies expressing wild-type NTRP in neurons and glia (image 1) or in neurons only (image 2). The images labelled 4 to 6 are similarly prepared sections from flies 30 days after eclosion. Flies expressing wild type NTRP in both neurons and glia (image 4) and flies expressing wild-type NTRP in neurons only (image 5), have retained specific staining for bruchpilot, whereas those expressing wild- type NTRP in glia only (image 6) have a granular appearance and have lost specific staining for bruchpilot. Images are representative of data obtained from 10-12 flies in each group. In each image the retina is marked with the letter r, the medulla neuropil, which receives the retinal axons shown in Figures 4 and 7, is marked with the letter m, and the lobula complex, which receives inputs from the medulla by the letters lc.
Figure 9 shows that homozygous NTRP1 flies (escapees) fail to expand their wings after eclosion. Figure 9A shows a representative photograph of a homozygous mutant NTRP1 fly and Figure 9B shows a heterozygous NTRP1 fly; both photographs were taken 5 hours after eclosion.
Figure 10 shows that co-expression of NTRP with human APP rescues the deleterious effect of human APP on Drosophila development and health. Figure 1 OA shows that APP-induced developmental lethality is rescued by co-expression of NTRP, but not by depletion of NTRP. For the category "developmental lethality" the bars represent the percent of young (LI) larvae of the indicated genotypes that eclose as adults. For the category M:F the bars represent the percent of total eclosing flies that are male. For the category "Wing M", the bars represent the percent of eclosing male flies that have unexpanded wings, similarly for females in the category "Wing F". Figure 10B shows the expression level of human APP when NTRP is either co-expressed with APP, or depleted in flies. The immunoblot was prepared from the heads of male flies expressing human APP (UAS-APP) that also have RNAi for NTRP (UAS-NTRP IR) or over-express NTRP (UAS-NTRP) driven by elav-Gal4. Each sample was prepared from 30 flies and the equivalent of 3 heads was loaded per lane. APP was detected with antibodies 5A3 and 1 G7 targeted against the ectodomain of APP. Similar results were obtained with antibody A5137, which is targeted at the C-terminus of APP (not shown) .
Figure 11 shows aberrant metabolism of a Drosophila APP orthologue (APPL) in mutant NTRP1 flies. Figure 11 A shows the detection of APPL by antibody dR14 in a western blot. The detected proteins correspond to (in decreasing order of molecular weight), holo-APPL, secreted APPL, and a novel APPL-reactive protein (arrow).
Figure 11B shows the detection of NTRP by antibody 385 in a western blot from the same samples as in Figure 11 A. In the protein gel, each lane is loaded with the equivalent of 4 heads from preparations made from 20-40 heads of flies collected on the day of their eclosion. All flies were isogenic.
Figure 12 shows expression patterns of various embodiments of NTRP in mouse brain as determined by in situ hybridisation. Figure 12A shows the expression pattern of NTRP (TMCC2) (image obtained from the Allen Brain Atlas). Figure 12B shows the expression pattern of TMCC1 (an NTRP paralogue) (image obtained from the Allen Brain Atlas). Figure 12C shows the expression pattern of TMCC3 (an NTRP paralogue) (image obtained from the Allen Brain Atlas). Figure 12D shows the expression of NTRP in human brain (TMCC2) (image obtained from Atlas
Antibodies). Arrows indicate examples of cells showing a positive reaction to anti- NTRP antibodies. Figure 13 shows the interaction of one embodiment of mammalian NTRP and APP. Figure 13A shows co-migration of APP (indicated by black arrows) and NTRP
(indicated by white arrows) in a 2-dimensional blue native/ SDS-PAGE of a
fractionated rat brain probed by western blot. Two blots from said gels were probed first for APP using antibody A5137 (image Al) or for NTRP using antibody 94 (image Bl); after developing these blots, the blot shown in Al was re-probed for NTRP to give the result shown in A2 (A3 is a longer exposure of A2) and the blot shown in image Bl was re-probed for presenilin 1 to give the result shown in B2. Figure 13C shows reciprocal co-immunoprecipitation of NTRP and APP from transfected cells. Cells were transfected with constructs for the expression of APP and FLAG-tagged NTRP. Immunoprecipitations were carried out as described in materials and methods.
Figure 14 shows that NTRP and apoE modulate γ-secretase activity towards APP in SHSY5Y cells. NTRP (TMCC2) and apoE interact to increase the amount of β-amyloid produced; since the production of β-amyloid is known to depend on γ-secretase, these data demonstrate modification of γ-secretase activity by NTRP and apoE. Figure 14A shows a representative example of the results of co-expressing NTRP (TMCC2), APP C99-GFP and apoE on the production of the APP intracellular domain, represented here as AICD-GFP. The levels of NTRP, APPC99-GFP, the APP intracellular domain fused to GFP (AICD-GFP), apoE and tubulin in cell lysates as assessed by SDS-PAGE and immunoblot. The arrow indicates the presence of a band representing a slower- migrating species of AICD-GFP, indicating altered γ-secretase activity towards the 99 amino acid portion of APP in the presence of NTRP and apoE. Figure 14B shows the amount of Αβΐ-40 and Αβΐ-42 produced from cells prepared in the same manner as those analyzed in Figure 14A. Levels of Αβΐ-40 and Αβΐ-42 were assessed by an ELISA assay; columns represent the amounts of each Αβ species produced relative to the amount produced when apoE3 was co-transfected with C99-GFP. There was a trend for an increase in the amount of β-amyloid produced when C99-GFP was co- transfected with either apoE isoform or NTRP; this was markedly enhanced by co- transfection of both NTRP and apoE3 or apoE41 which produced a statistically significant increase in β-amyloid production for both apoE3 and apoE4 (P<0.0001, one way ANOVA, indicated by ###); furthermore, there was a significantly higher amount of β-amyloid produced when NTRP was cotransfected with apoE4 compared to cotransfection with apoE3 (P=0.02, two-tailed Students T-test, indicated by *). Figure 14C shows another example of the effect of NTRP and apoE on APP metabolism; in this case by SHSY5Y cells stably over- expressing full-length APP, said cells were cotransfected with NTRP (TMCC2) and apoE isoforms as indicated and the amount of Αβ 1-40 and Αβ 1-42 released into the media determined by ELISA; columns represent the levels of β— amyloid produced under each condition relative to that produced when apoE3 alone was transfected. In contrast to experiments using APP-C99, these experiments showed a trend for decreased production of Αβ 1 -40 when either apoE3 or apoE4 was cotransfected with NTRP when compared to single transfections (P=0.06 to 0.1, two-tailed Students T-test, indicated by *).
Figure 14D shows the expression NTRP (TMCC2), APP, apoE and tubulin in cell lysates of APP-expressing SHSY5Y cells as assessed by SDS-PAGE and immunoblot. In Figures 14B and 14C, columns show mean relative values obtained from 5 experiments; error bars represent the standard error of the mean. Vector refers to a pcDNA3.1 vector without an insert, included to normalize DNA levels in each transfection. The difference in effect on β-amyloid production from APP-C99-GFP and full-length APP likely results from multiple activities of NTRP towards APP; in the case of full- length APP, APP must first be trafficked to sites where BACE activity is high before β- amyloid can be generated; for APP-C99-GFP only an encounter with γ-secretase is required. These assays together may therefore measure different aspects of APP metabolism.
Figure 15 shows an alignment of various embodiments of NTRP sequences (SEQ ID No's: 1-15) and the derivation of a consensus sequence (SEQ ID No.16). Figure 16 shows a consensus sequence for NTRP and related proteins. The standard single letter code for amino acids is used. Dots indicate poor conservation of both sequence and distance between orthologues. "X" indicates relatively poor conservation of sequence, but better conservation of distance.
Figure 17 shows the results that may be obtained from a hypothetical test on a drug modifying the activity of the NTRP and apoE on γ-secretase activity and APP metabolism. In Figure 17A the potency of a test drug that suppresses β-amyloid production with or without NTRP and apoE is illustrated; the drug may be more or less potent in the presence of apoE3 or apoE4 and/ or NTRP. In Figure 17B the potency of two drugs, X and Y are compared after normalizing to their own baselines. Drug X is more potent in the presence of NTRP and/ or apoE3 or apoE4, whereas the potency of drug Y is decreased. Assays as described in Figures 17A and 17B would allow a more efficient and economical method of discovering drugs for the treatment of neurological disorders. Figure 18 shows ApoE and NTRP interact to modify β-amyloid production from
AbPPswe, i.e. the "Swedish" pathological variant of APP (APPswe, K595M/N596L). (A) NTRP and apoE significantly increase β-amyloid production from AbPPswe. Oneway ANOVA applied to the data for Abl-40 and AM -42 separately showed a significant increase in β-amyloid production (P<0.05) on co-expression of TMCC2 and apoE, indicated by *. (B) Representative western blots showing no significant effect of the expression of TMCC2 and apoE on the levels of AbPPswe. Bars indicate the relative amount of β-amyloid produced ± SEM obtained from 3 independent experiments where the indicated plasmids were transfected into SHS5Y5 cells, as assessed by ELISA. To better visualize the relative impact of apoE and TMCC2 on the production of each respective peptide, Abl-40 and A -42 levels were independently normalized to that where only apoE3 is expressed, since apoE is abundant in the brain, and apoE3 represents the most common isoform of apoE.
Examples
The inventor carried out a number of experiments, as explained in Example 1, to discover a Novel Tex-28 Related Protein (NTRP) family of proteins, and to investigate the effects of NTRP on the metabolism of APP and APP-like proteins, and, thus neurodegenerative disorders. The inventor has subsequently also named NTRP as "dementin", by virtue of its role in APP metabolism, which is known to cause dementia, i.e. Alzheimer's disease. Example 2 describes a functional assay measuring the metabolism of APP or APP-like proteins, and Example 3 describes a method for screening small molecules, tissue extracts or body fluids. Example 4 describes a method for investigating and testing therapies for conditions involving apoE, including Alzheimer's disease. Materials and Methods
Yeast-two-hybrid - A commissioned yeast two hybrid screen using apoE isoforms as baits and a human brain cDNA library as a source of targets used the MATCHMAKER system (Clontech, Palo Alto, USA). Flies - UAS-RNAi line 37338, which targets NTRP, and UAS-Dcr2 flies were obtained from the Vienna Drosophila RNAi Centre (Austria); this RNAi line is predicted to not have off-targets; unless otherwise indicated, all experiments using RNAi were performed in the presence of UAS-Dcr2. ntrp1 flies are the
PBac{RB} (CG1021)[e01970] line from the Exelixis collection at Harvard University (MA, USA); the reported insertion site was confirmed by inverse PCR. ntrp-Gal4 flies (P{GawB}NP6590) were supplied by the Drosophila Genome Resource Centre (Kyoto, Japan). UAS-APP695 (P{w[+mC]=UAS-APP695-N-myc}TW6) which may express human APP, and P{w[+mC]=UAS-GFP.nls}8 flies which may express GFP with a nuclear targeting signal were obtained from the Bloomington Stock Center (IN, USA). UAS-NTRP flies were made by amplifying the cDNA for NTRP from clone
RE27645 obtained from the Berkeley Drosophila Genome Project (CA, USA), using the primers AGCTGGTACCTTACTTCACCACCAGCGATTGCTTG (SEQ ID No.31) and AGGAGGCGGCCGCATGTCCCGGGAGCGAGCCAGCGAGGCAGC (SEQ ID No.32); the PCR product was cut with Notl and Kpnl and ligated to pUAST cut with the same enzymes; the presence of the designed insert was confirmed by sequencing, w1118 embryos were injected with DNA for UAS-NTRP at Rainbow Transgenic Flies, Inc. (CA, USA). All flies were raised on cornmeal, and when phenotypes were to be analyzed, at a density of 40-50 larvae per 10 ml of food. For the determination of lifespans, flies were kept at less than 20 flies per 10 mL vial and transferred to new food twice per week. For the analysis of wholemount brains by immunofluorescence, larval or adults brains were dissected and fixed in 4% paraformaldehyde, 1.6% L-lysine in phosphate-buffered saline for 20 minutes, washed with 1% Triton X-100 in phosphate-buffered saline (PBX) and blocked with 10% normal goat serum in PBX. For the analysis of paraffin-embedded sections, flies were fixed overnight in Carnoy fixative, processed through 3 2-h incubations in methylbenzoate at room temperature, followed by a 1 :1 mixture of methylbenzoate and paraffin at 65° C and three changes of paraffin at 65° C (45 min each). Analyses were performed on 7 μιτι sections. cDNA constructs - Human TMCC2 (Acc. No. AB007950, Kazusa, Chiba, Japan) was amplified by PCR and cloned into pcDNA3.1 (Invitrogen), functionally equivalent constructs are now available from Origene Technologies. A plasmid expressing dsRedER was obtained from Clontech, plasmids expressing apoE isoforms have been previously described (Ljungberg et a/., Neuroreport. 2002, May 7; 13(6):867-870);
constructs for the expression of APP695 and APP695 fused to the N-terminus of EGFP were gifts of C. Miller (Kings College, London), a plasmid expressing the 99 amino acid C-terminal fragment fused to EGFP with an additional Asp- Ala sequence after the signal peptide sequence as described (Lichtenthaler et a/., FEBS Lett. 1999 Jun 25;453(3):288-92.) was made by cutting APP695 fused to the N-terminus of EGFP with Bgl II and EcoRI and ligation to a cassette consisting of the oligonucleotides
GATCTATGCTGCCCGGTTTGGCACTGCTCCTGCTGGCCGCCTGGACGGCT CGGGCGGATGCAGATGCAG (SEQ ID No.33), and
AATTCTGCATCTGCATCCGCCCGAGCCGTCCAGGCGGCCAGCAGGAGCA GTGCCAAACCGGGCAGCATA (SEQ ID No.34).
Antibodies - Antibody 94: recombinant TMCC2 residues 252 to 648, corresponding to the conserved region (see Figure 15) prior to the predicted transmembrane domain, was produced as described below and injected into rabbits at Harlan Teklad (UK). Antibody 94 was used at a concentration of 1 :1000 in 5% milk, 150 mM NaCl, 50 mM Tris.Cl pH 7.4. Antibody 385 raised against the peptide QSANADVLGSERLQ (SEQ ID No.35) (from Orosophila NTRP) was raised in rabbits at Eurogentec (Belgium) and used at a concentration of 1 : 1000 in 5% milk, 150 mM NaCl, 50 nM Tris.Cl pH 7.4; anti-APPL antibodies dR-14 and dC-12 targeted to the C-terminal region of the APPL ectodomain (communication from Santa Cruz Biotechnology) were obtained from Santa Cruz Biotechnology and used at a concentration of 1:100 in PBS 0.2% fish gelatin, 0.1% Triton X-100; APPL antigen recognition was best preserved when samples were stored at -80°C. Mouse monoclonal antibodies 5 A3 and 1G7 against the APP ectodomain were gifts of S. Soriano. Rabbit polyclonal antibody A5137 directed against the final 20 amino acids of human APP was a gift of C. Miller (Kings College, London). Goat polyclonal anti-apoE was obtained from Calbiochem, and goat anti-presenilin-1 C- terminal fragment (C20) was obtained from Santa Cruz Biotechnologies. Mouse anti- Golgi 58K and rabbit anti-BiP were purchased from Sigma Aldrich. Mouse monoclonal antibodies 24bl0 (anti-chaoptin), 8D12 (anti-repo), and nc82 (anti-bruchpilot) were obtained from the Developmental Studies Hybridoma Bank (The University of Iowa, USA). Western blots - Western blots were routinely developed using the ECL system
(Amersham).
Analysis of TMCC2 '-related sequences - Collected TMCC2-related sequences were clustered based on homology using the ClustalW program (Larkin et al, Bioinformatics. 2007 Nov l;23(21):2947-8; Higgins et al, Gene. 1988 Dec 15;73(l):237-44.237) followed by manual refinement, and a phylogenetic analysis was performed using the service at phylogeny.fr (Dereeper et al, Nucleic Acids Res. 2008 Jul l;36(Web Server issue):W465- 9465). Coiled-coiled domains were predicted by the TMHMM service (Krogh et al, Journal of Molecular Biology, Jan 2001, 305(3):567-580).
Production of Recombinant NTRP - A cDNA encoding TMCC2 (Acc. No. AB007950, Kazusa, Chiba, Japan) from amino acid Met79 to Arg640 (adjacent to the first predicted t ansmembrane region) was amplified by PCR and cloned into the pBADThio vector (Invitrogen); the 5' primer contained an in-frame coding sequence for the FLAG epitope (DYKDDDDK) (SEQ ID No.36). This resulted in a construct encoding a TMCC279 640 -thioredoxin protein (TMCC2-TrxA) in which the codons for the first exon were replaced by codons for thioredoxin, followed by the FLAG sequence, truncated TMCC2, and at the C-terminus, a six-His tag. Expression was induced by adding arabinose (final concentration, 0.2%) to a log-phase culture of transformed Escherichia coli at 37 °C. Cell pellets were lysed in 6 M guanidine-HCl, 150 mM NaCl, and 20 mM Tris-Cl, pH 8.0, and cleared by centrifugation at 15,000 ^ for 1 h. The supernatant was loaded onto a Ni2+-chelating sepharose column, and proteins were eluted with a gradient of 0-150 mM imidazole in 6 M guanidine-HCl, 150 mM NaCl, and 20 mM Tris-Cl, pH 8.0. After dialysis against 1.0 mM HCl, fractions TMCC2-TrxA were identified by SDS-PAGE and staining with Coomassie-blue, readjusted to 6 M guanidine-HCl, 150 mM NaCl, and 20 mM Tris-Cl, pH 8.0, and passed over a Ni2+- chelating column as described above. Fractions containing TMCC2-TrxA were dialyzed against 1 mM HCl and lyophilized. TMCC2-TrxA was dissolved in 6.0 M guanidine HCl, 1.0 M L-arginine, 150 mM NaCl, 200 mM trimethylamine n-oxide, 40 mM ammonium bicarbonate, and 10 mM dithiothreitol. Proteins were refolded by rapid dilution with 10 volumes of 1.0 M L-arginine, 150 mM NaCl, 200 mM trimethylamine n-oxide, 100 mM ammonium bicarbonate, and 10 mM dithiothreitol and dialyzed against 150 mM NaCl, 100 mM ammonium bicarbonate, and 100 mM trimethylamine n-oxide.
In Vitro Association ofTMCC279-640-TrxA andApoE - The intrinsic fluorescence of TMCC2-TrxA, apoE3, apoE4 and thioredoxin was measured with a Hitachi F2000 fluorimeter. Proteins were excited at 295 nm, and fluorescence emission was measured from 300 to 450 nm. All protein concentrations were 1.0 μΜ, and all measurements were performed at 25°C in 150 mM NaCl, 100 mM ammonium bicarbonate, and 100 mM trimethylamine n-oxide after co-incubation for 1 h. The change in total fluorescence at 342 nm on co-incubation is expressed as a percent of the sum of the fluorescence intensity measured for each protein alone. Four independent assays using separately purified and prepared samples of TMCC2-TrxA were performed. Recombinant apoE3, apoE4 and thioredoxin were gifts of Yvonne Newhouse and Karl Weisgraber (Gladstone Institute of Neurological Disease, San Francisco, USA) .
Sucrose-gradient fractionation of mouse and rat brain - One whole adult mouse or rat brain was homogenized by 50 strokes of a dounce homogenizer in 50 mM imidazole.HCl, pH 7.0, 0.25 M sucrose, 1 mM EDTA and protease inhibitors (Complete, Boehringer
Mannheim); 10 ml was used for a mouse brain and 50 ml for a rat brain.. The homogenate was centrifuged at 600 g for 10 min. and the supernatant loaded onto a 1.9 M to 0.25 M sucrose gradient in the above buffer and centrifuged for 95,000 g for 18 h. 1 mL fractions were collected from the bottom of the tube and analyzed by Western blot or stored at -80°C.
Two-dimensional Blue-native I SOS PAGE of rat brain extracts - A TMCC2- enriched rat brain fraction was prepared as follows. The TMCC2- containing sucrose-gradient fraction prepared as above was diluted 10-fold with 50 mM imidazole.HCl, pH 7.0, 0.25 M sucrose, 1 mM EDTA and protease inhibitors (Complete, Boehringer Mannheim) and centrifuged at 1,500 g for 5 min., TMCC2 was enriched in the pellet. The pellet was resuspended in 1% Triton X-100, 1% NP-40 and 1% Coomassie blue R-250 in 50 mM imidazole.HCl pH 7.0 and centrifuged at 100,000 g for 20 min. to remove insoluble material. The supernatant was fractionated by electrophoresis on a 4-13% acrylamide gradient gel as described (Wittig et a/. Nat. Protoc. 2006;l (l):418-28.). Gel strips were excised and incubated in 100 mM Tris.Cl pH 6.8, 1% SDS, 10 mM DTT for 30 min. and inserted between the plates of a previously poured SDS-PAGE gel without a stacking gel. A stacking gel was then poured around and under the strip and allowed to polymerize. The gel was run and the proteins transferred to polyvinyl difluoride membranes (Millipore) and analyzed by western blot in a conventional manner.
Co-immunoprecipitation - Cells were co-transfected with TMCC2 (NTRP) tagged at the C- terminus with the FLAG sequence (DYKDDDDK) and APP695 using
Lipofectamine2000 (Invitrogen); 24 hr after transfection cells were lysed in phosphate- buffered saline, 1% Triton X-100, protease inhibitors (Complete, Boehringer
Mannheim) and phosphatase inhibitors (10 mM NaF, 4 mM β-glycerophosphate). Lysates were centrifuged at 100,000 g to remove insoluble material and incubated overnight with protein A/ G beads (Santa Cruz Biotechnology) previously coupled to M2 anti-FLAG antibody (Sigma Aldrich), antibody A5137 directed against the C- terminus of APP, or no antibody. The beads were washed 6 times in the above buffer over a period of 2 h. Proteins bound to the A5137 anti-APP antibody were eluted with SDS-containing gel loading buffer, and proteins bound to the M2 anti-FLAG antibody were eluted with the FLAG peptide as recommended by the supplier (Sigma Aldrich) . Eluted proteins were separated by SDS-PAGE and transferred to nitrocellulose for analysis by western blot. TMCC2 was detected using the rabbit antibody 94 directed raised against recombinant TMCC2, APP was detected using antibody A5137.
Assessment of the impact ofTMCC2 on β— amyloid production - β— amyloid production from SHS5Y5 cells was assayed using ELISA kits purchased from Millipore and used as directed by the manufacturer. SHSY5Y cells were routinely cultured in 42% F12 media (Gibco), 42% EMEM media (Sigma), 15% bovine calf serum, supplemented with glutamate, non-essential amino acids and sodium pyruvate. SHSY5Y cells were seeded into 6-well dishes and cultured for 48 h before being transfected with the constructs indicated. Transfections were achieved using Lipfectamine2000 (Invitrogen), as directed by the manufacturer, using 2 μΐ Lipofectamine2000 per 1 μg of plasmid DNA per well for 4 h., when the transfection mixture was replaced with normal culture media. A total of 1 g of each plasmid DNA was used, and empty pcDNA3.1 used to normalize the amount of DNA in each transfection. Cells were incubated in the transfection mixture for 4 hr when it was removed and replaced with fresh Optimem (Invitrogen), media and cells were harvested after 36 hr.
Example 1 - Discovery of the NTRP family of proteins
To isolate novel proteins that differentially bind to apoE3 and apoE4, which may lead to the identification of new models for the role of apoE in AD, a yeast-two-hybrid screen was performed using apoE3 and apoE4 as baits and a human brain cDNA library as a source of targets. The results of the yeast-two-hybrid screen are shown in Figure 1 A. This screen yielded a clone (i.e. SEQ ID NO.l) that has been called NTRP that corresponds to residues 421 to 709 of a protein (i.e. SEQ ID NO. 3). It can be seen that in this assay, this clone, NTRP, binds to apoE3 more strongly than it does to apoE4. The inventor confirmed direct binding of NTRP and apoE in vitro by comparing the intrinsic tryptophan fluorescence of recombinant apoE and an NTRP-Thioredoxin (NTRP-TrxA) fusion protein when incubated alone or together. The combined fluorescence of apoE3 and NTRP-TrxA, but not apoE4 and NTRP-TrxA, increased when co-incubated. As shown in Figure IB, TrxA alone had no effect on the net fluorescence of either apoE3 or apoE4. As the NTRP portion of NTRP-TrxA contains no tryptophans, the increase in fluorescence may be attributed to a change in the local environment of one or more of the tryptophans in apoE3 on binding to NTRP.
Definition of the NTRP protein family
Using BLAST (Altschul et a/., ] Mol Biol. 1990 Oct 5; 215(3):403-10) and publicly- available resources, the inventor collected sequences which related to NTRP (SEQ ID No.l), and found that it was a member of a novel family of highly conserved proteins found in a wide variety of animal species. Human Tex28 has been reported (Hanna et al., Genomics. 1997 Aug l;43(3):384-6). However, its function has not yet been investigated. The inventor found that the human, rat and mouse genomes contain four NTRP orthologues each and the Drosophila genome contains one orthologue.
Examples of the NTRP family members are provided in Table 1.
Table 1 - NTRP protein identities in human, rat, mouse. Drosophila and C. elegans
NTRP protein Unigene Chromosomal SEQ ID
Code location NO.
Human TMCC1 Hs.477547 3q21.3 2
Human NTRP Hs.6360 lq32.1 3
(TMCC2)
Human TMCC3 Hs.370410 12q22 4
Human Tex 28 Hs.672606 Xq28 5 Rat TMCC1 Rn.198698 4q42 6
Rat NTRP (TMCC2) Rn.106120 13ql 3 7
Rat TMCC3 Rn.19106 7ql3 8
Rat Tex28 none Xq37 9
assigned
Mouse TMCC1 Mm.425352 6qE3 10
Mouse NTRP Mm.273785 lqE4 11
(TMCC2)
Mouse TMCC3 Mm.23047 10qC2 12
Mouse Tex28 Mm.475616 X 13
Drosophila (CGI 021) Dm.4403 III 14
C. ekgans (C15H9A) Cel.23608 X 15
NTRP Consensus NA NA 16
sequence
As illustrated in Figure 2, (and shown in Figure 15), the NTRP proteins in human and Drosophila are highly similar, with conserved regions showing a percent similarity ranging from 41 % to 84% in amino acid sequence and are thus likely to function in a similar manner.
An alignment of NTRP-related sequences showed that they are highly conserved in the C-terminal portion of the molecule (shown in Figure 15), which in the embodiment of NTRP referred to as TMCC2 spans residues 247-709. The N-terminal region varies considerably among orthologues, though it is conserved in homologues. A minimal consensus sequence that defines the NTRP family was derived using the "seaview" software (Galtier et a/., Comput Appl Biosci. 1996 Dec; 12(6):543-8), followed by manual refinement, and is shown in Figure 16.
All NTRP sequences analyzed are predicted to possess two closely located
transmembrane (TM) domains near the C-terminus and two coiled-coil (CC) domains, as illustrated in Figure 2, but no leader peptide, suggesting that they are intracellular multipass transmembrane proteins. Otherwise, NTRP proteins do not have sufficient similarity to known and studied proteins for sequence analysis to reliably predict their function, or to determine if a functional relationship exists between apoE, NTRP and molecular mechanisms in neurodegeneration.
Drosophila NTRP functions in brain development
The expression pattern of Drosophila NTRP was analysed by an enhancer trap in the NTRP locus. Figure 3 illustrates schematically where the enhancer trap (NTRP-gal4) is inserted in the gene for NTRP. As shown in Figure 4, NTRP-gal4 was expressed in both neurons and glia. Moreover, as illustrated in Figure 5, experiments in which NTRP level in Drosophila was depleted by either neuron- or glia- specific RNAi also confirmed that NTRP was expressed in both neurons and glia.
NTRP expression was efficiently suppressed by RNAi using tubulin-Gal4, which is expressed in all tissues (Figure 5A); tubulin-gal4-driven suppression of NTRP expression was almost completely lethal during development, showing that flies deficient in NTRP would not develop into adults. As illustrated in Figure 5C and Figure 5D, a more restricted suppression of NTRP using glia- or neuron-specific methods of suppressing NTRP production showed that glia-specific suppression of NTRP recapitulated the developmental effect of global suppression of NTRP.
Suppression of NTRP expression in a glia-specific or global manner was partly lethal during development, while flies with neuronal suppression of NTRP expression were viable.
The inventor identified a recessive pupal-lethal hypomorphic allele of the Drosophila NTRP gene (NTRP1) a piggyBac transposon insertion (see Figure 3) that leads to the production of a truncated peptide, as is shown in Figure 6A, and as indicated by an arrow. As shown in Figure 6B, similar to the effect of global RNAi for NTRP (Figure 5B), homozygous NTRP1 flies died mainly during metamorphosis, and as shown in Figure 6C, homozygous NTRP1 escapees died within a few days. The DNA encoding this embodiment of NTRP thus contains a pathological mutation. Referring to Figure 7, examination of the brains of newly eclosed NTRP1 flies showed a severe fully penetrant (26 out of 26) neuroanatomical phenotype indicative of arrested brain growth that was readily observed in the optic lobe. As shown in Figure 7B, this phenotype was partly recapitulated by glia-specific RNAi, but not neuron-specific RNAi.
While neuronal RNAi for NTRP did not prevent development, evidence for a neuronal function of NTRP was shown, since the developmental lethality associated with the NTRP1 allele was rescued by expression of wild-type NTRP in either neurons or glia. In addition, as shown in Figure 8C, mutant NTRP1 flies rescued by expression of wild- type NTRP in neurons or in both neurons and glia had a normal lifespan, whereas those rescued by expression in glia alone had a shorter lifespan. The expression of wild-type NTRP in the rescue experiment is shown in Figure 8A. Taken together, the above data indicate a role for both glial and neuronal NTRP in Drosophila both brain development and in longevity.
Neurodegeneration in ntrp1 flies.
As shown in Figure 8C, NTRP1 flies with glia-only expression of wild-type NTRP had a significantly reduced lifespan compared with those expressing wild-type NTRP in both neurons and glia, suggesting that the shorter lifespan was caused by a deficiency of NTRP in neurons. Referring to Figure 8D, the inventor determined that ntrp1 flies without wild-type NTRP in neurons had neurodegeneration. Figure 8D, images 1 to 3 show paraffin sections of the optic lobe of ntrp1 flies fixed on the day of eclosion and stained by indirect immunofluorescence for bruchpilot, a pre-synaptic protein. Images 1, 2 and 3 show the optic lobe of ntrp1 flies expressing wild-type NTRP in neurons and glia, neurons only, or glia only, respectively. Images 4 to 6 show the optic lobe of flies of the same genotype fixed 30 days after eclosion. These data show that at both times points, flies expressing wild-type ntrp in neurons had specific staining for a presynaptic proteintein (bruchpilot), however ntrpl flies expressing wild-type NTRP only in glia had a disrupted staining pattern on the day of eclosion (image 3), and no specific staining 30 days later (image 6). Although not wishing to be bound by any particular hypothesis, the inventor believes that these data show that neuronal expression of pathogenic variants of NTRP leads to disruption of neuronal function and eventually to neurodegeneration. NTRP interacts genetically with APP-like proteins in Drosophila
Referring to Figure 9, in addition to the neuroanatomies phenotype, 85% of mutant NTRP1 flies (30 out of 35) showed a failure of wings to expand after eclosion. This phenocopies over- expression of human APP in Drosophila, which in addition to affecting wing expansion, is partly lethal during development, and affects males more severely than females. The inventor therefore examined if NTRP would modify the effect of APP over-expression in Drosophila.
As shown in Figure 10A, co-expression of NTRP and APP protected against APP- induced developmental lethality and against wing-expansion defects. Neuronal RNAi for NTRP did not rescue the wing-expansion defect. As shown in Figure 10B, these effects were not correlated with changes in the level of human APP expression.
However, APP may interact directly with NTRP (see Figure 13; explained more below) and suppress the effect of APP by sequestering it. The inventor next examined preparations from the heads of newly eclosed NTRP1 flies for the Drosophila homologue of APP, APPL, which is expressed in both neurons and glia. With reference to Figure 11 , this showed that heterozygous and homozygous mutant NTRP1 flies, but not isogenic control flies, accumulated a ~50 kDa protein that reacted with antibodies targeted to APPL (indicated by an arrow), indicating aberrant metabolism of APPL. Thus, Drosophila NTRP interacts genetically both with human APP, and it's Drosophila orthologue, APPL.
Expression of NTRP in human and rodent brain
The inventor also investigated mammalian APP and NTRP for evidence of interaction. Data available from the Allen Brain Atlas (www.brain-map.org) and from Atlas Antibodies (www.atlasantibodies.com) show expression of NTRP and related genes in mouse and human brains (see Figure 12).
Interaction of mammalian NTRP and APP
As shown in Figure 13A the existence of a complex containing both NTRP and APP in rat brains was suggested by two-dimensional blue-native/ SDS-PAGE, which showed co-migration of these two proteins in front of the migration position for the presenilin 1 complex (13B2). Referring to Figure 13C, an interaction between human NTRP and APP was also indicated by co-immunoprecipitation assays, where immune complexes isolated from cells co-transfected with APP and NTRP contained both proteins.
Alteration of APP metabolism, β-amyloid production, and γ -secretase activity by NTRP
The inventor next determined that NTRP modulated APP metabolism and the activity of γ-secretase and also modulated the production of β-amyloid. With reference to Figure 14A the inventor showed that production of the APP intracellular domain (AICD) was modified by NTRP. Figure 14A is a western blot showing a novel band that the inventor believes represents a γ- secretase product that is generated in the presence of NTRP and apoE (indicated by an arrow). Alteration of γ-secretase activity, and secretion of β-amyloid was also shown when NTRP and apoE caused an increase in β-amyloid production from APP-C99-GFP, as shown in Figures 14B and 14C. Since the cleavage of the C99 fragment of APP is known to depend strictly on γ- secretase, this shows that NTRP may modulate γ-secretase activity.
Conclusion
Taken together, these data indicate that NTRP -like proteins interact with APP-like proteins in Drosophila and mammals, and that disruption of NTRP metabolism leads to altered metabolism of both APP-like proteins and proteins involved in
neurotransmission, and to neurodegeneration. Since (i) the Drosophila and mammalian sequences of NTRP are so similar, (ii) Drosophila NTRP interacts with both human APP and Drosophila APPL, (iii) rat NTRP and APP co-migrate on native gels, and (iv) human NTRP and APP co-immunoprecipitate, (v) NTRP interacts with apoE in an isoform-specific manner to alter β-amyloid production, (vi) NTRP interacts with apoE to alter γ-secretase activity, the inventor believes that embodiments of NTRP and related proteins play an important role in the metabolism of APP and APP-like proteins in humans. Given that APP and APP-like proteins contribute to neurodegenerative disorders, the inventor believes that embodiments of NTRP and related proteins play a role in such diseases, including Alzheimer's disease.
Animals with general, cell- or region-specific regulated expression of NTRP, or similar proteins, or which express modified forms of NTRP, or similar proteins, or which have RNAi-mediated regulation of NTRP protein production may therefore be used to model neurodegeneration, to investigate mechanisms in neurodegeneration, or to test drugs for impacts on neuronal function specifically, or brain function generally, or for impacts on diseases such as neurodegeneration. Such animals may also be used to investigate the metabolism of APP and APP-like proteins, the activity of γ-secretase, or to test compounds that may alter the metabolism of such proteins.
Furthermore, since NTRP can bind ApoE in an isoform-specific manner, animals with modified expression of NTRP or related proteins, or with the expression of modified forms of NTRP may be used to investigate and test therapies for conditions which involve apoE, including Alzheimer's disease, perse.
Example 2
The metabolism of APP or similar proteins, such as APPL, APLP1 or APLP2, and other γ-secretase substrates may be assessed in an assay using NTRP. In this assay, NTRP is increased or depleted relative to its physiological levels and the cellular distribution of APP assessed by, for example sucrose gradient centrifugation of by measurement of it's normal processing, such as β-amyloid. Such assays may also use modified forms of NTRP that are partially active, such as NTRP1 described above. Example 3
A screen for drugs intended for the treatment of conditions such as Alzheimer's disease may also be assessed by this invention as illustrated in Figure 17. For example, a test compound may be included in a cell-based or in vitro assay that measures the production of APP metabolites, such as β-amyloid as described above. This compound may already be suspected to inhibit or increase β -amyloid production or to interact with apoE. By incorporating or removing NTRP and apoE, or either alone, in or from the assay an improved assessment of the merits of the compound can be made. The compound may, for example, be either less effective or more effective in the presence of NTRP and/ or apoE. Such compounds could include those aimed at targeting lipid metabolism, such as those known as statins. Such compounds may also be targeted at insulin metabolism, such as rosiglitazone, pioglitazone or drugs with a similar mechanism of action. Such compounds may include those targeted at γ-secretase, such as LY450139 (hydroxylvaleryl monobenzocaprolactam). Such compounds may be those targeted at apoE, such as those described in US patent number US 2006/0073104 Al . Such compounds may also consist of fragments of apoE or closely related peptides.
Example 4
A screen for modifiers of γ-secretase activity is shown in Figure 14A and 14B. Example 5
Co-expression of apoE and NTRP with the "Swedish" pathological variant of APP (APPswe, K595M/N596L) showed a significant effect of both apoE isoforms and NTRP on β-amyloid secretion (see Figure 18). Thus, NTRP interacts differentially with the neurodegeneration-risk versus normal versions of two separate classes of protein, apoE and APP.

Claims

Claims
1. An isolated polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's: 1—16, or a functional variant or functional fragment thereof, wherein the polypeptide, variant or fragment thereof is capable of: (i) influencing amyloid precursor protein (APP) metabolism; (ii) influencing the activity of γ-secretase; (iii) modulating the progression of neurodegeneration; and/or (iv) binding to apolipoprotein E.
2. An isolated polypeptide according to claim 1 , wherein the polypeptide, variant or fragment alters the rate of cleavage of APP by one of the secretases (e.g. γ-secretase,
OC-secretase or BACE), or alters the site at which a secretase preferentially cleaves APP, or regulates the cellular trafficking of APP, or alters the rate of generation or cellular activity of the APP intracellular domain, or alters the rate at which APP reaches the cell surface.
3. An isolated polypeptide according to either claim 1 or claim 2, wherein the polypeptide, variant or fragment is capable of increasing the activity of γ-secretase in a manner that increases the production of β-amyloid by at least 20%, 50%, 100%, 150%, 200%, 250% or at least 300% compared to the amount of β-amyloid which is produced in the absence of the polypeptide, variant or fragment thereof.
4. An isolated polypeptide according to any preceding claim, wherein the polypeptide, variant or fragment increases neurodegeneration if it comprises a pathological mutation or if the gene encoding it comprises a pathological mutation.
5. A conjugate comprising the polypeptide according to any one of claims 1-4, and an apolipoprotein.
6. A conjugate according to claim 5, wherein the apolipoprotein comprises apolipoprotein E.
7. A conjugate according to either claim 5 or claim 6, wherein the apolipoprotein is apolipoprotein E3 (apoE3) or apolipoprotein E4 (apoE4).
8. Use of a polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's:l-16, or a functional variant or functional fragment thereof, for: (i) influencing amyloid precursor protein (APP) metabolism; (ii) influencing the activity of γ-secretase; (iii) modulating the progression of neurodegeneration; and/ or (iv) binding to apolipoprotein E.
9. A method of altering the activity of a polypeptide comprising an amino acid sequence substantially as set out in any one of SEQ ID No's: 1-16, or a functional variant or functional fragment thereof, the method comprising contacting the polypeptide, functional variant or functional fragment thereof, with an apolipoprotein.
10. A method according to claim 9, wherein the apolipoprotein comprises
apolipoprotein E, for example apoE3 or apoE4.
11. An isolated nucleotide sequence encoding the polypeptide, variant or fragment thereof according to any one of claims 1 -4.
12. An isolated nucleotide sequence according to claim 11, wherein the nucleotide sequence comprises a nucleic acid sequence substantially as set out in any one of SEQ ID No's: 17 to 30, or a functional variant or a fragment thereof.
13. A genetic construct comprising the nucleotide sequence according to either claim 11 or claim 12.
14. A recombinant vector comprising the genetic construct according to claim 13.
15. A host cell comprising the genetic construct according to claim 13, or the recombinant vector according to claim 14.
16. A transgenic host organism comprising at least one host cell according to claim 15.
17. A host organism according to claim 16, wherein the genome of the organism comprises a nucleic acid sequence encoding a polypeptide, variant or fragment according to any one of claims 1 -4, optionally wherein the polypeptide, variant or fragment, or its encoding nucleic acid thereof comprises a pathological mutation.
18. A host organism according to claim 17, wherein a pathological mutation is an insertion into an intron that alters the production of normal NTRP, for example the ntrp1 allele described herein.
19. An assay for screening for a compound that is a modulator of a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof, the assay comprising the steps of:
(a) providing an assay system in which a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof is either over-expressed or under-expressed compared to the corresponding physiological level of expression;
(b) introducing a test compound into the assay system; and
(c) determining either γ-secretase activity or APP metabolism,
wherein an alteration in either γ-secretase activity or APP metabolism in the presence of the test compound compared to γ-secretase activity or APP metabolism in the absence of the test compound indicates that the test compound is a modulator of the polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof.
20. A method of screening for a therapeutic agent useful in the prophylaxis or treatment of a neurodegenerative disorder, the method comprising the steps of:
(a) providing an assay system in which a polypeptide selected from the group
consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof is either over-expressed or under-expressed compared to the
corresponding physiological level of expression;
(b) introducing a test compound into the assay system; and
(c) determining either γ-secretase activity or APP metabolism,
wherein an alteration in either γ-secretase activity or APP metabolism in the presence of the test compound compared to γ-secretase activity or APP metabolism in the absence of the test compound is an indication of the ability of the test compound to modulate the neurodegenerative disorder.
21. An assay for screening for a compound that is a modulator of apolipoprotein E (apoE), the assay comprising the steps of:
(a) providing an assay system comprising apoE or a fragment thereof, and in which a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof is either over-expressed or under-expressed compared to the corresponding physiological level of expression;
(b) introducing a test compound into the assay system; and
(c) determining either γ-secretase activity or APP metabolism,
wherein an alteration in either γ-secretase activity or APP metabolism in the presence of the test compound compared to γ-secretase activity or APP metabolism in the absence of the test compound indicates that the test compound is a modulator of apoE.
22. An assay for screening for a compound that is a modulator of the binding between apolipoprotein E (apoE) and a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof, the assay comprising the steps of:
(a) contacting apoE and a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof;
(b) introducing a test compound; and
(c) determining the impact of the test compound on the binding between apoE and the polypeptide, variant or fragment thereof, wherein an alteration in the structure of apoE and/ or the polypeptide, variant or fragment thereof, or in the association therebetween, indicates that the test compound is a modulator of the interaction between apoE and the polypeptide, variant or fragment.
23. An assay or method according to any one of claims 19-22, wherein the test compound is a therapeutic agent useful in the prophylaxis or treatment of a neurodegenerative disorder.
24. An assay or method according to any one of claims 19-23, wherein the assay system is a non-human animal model, for example, a transgenic ape, monkey, mouse, rat, fish, ferret, sheep, dog, cat, worm or Orosophila.
25. A modulator of a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof, for use in treating, ameliorating, or preventing a neurodegenerative disorder.
26. A modulator according to claim 25, wherein the neurodegenerative disorder is selected from a group consisting of Alzheimer's disease, Parkinson's disease,
Huntington's disease, Spinocerebellar type 1, type 2, and type 3, and Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia or Schizophrenia.
27. A modulator according to claim 26, wherein the neurodegenerative disorder is Alzheimer's disease.
28. A modulator according to any one of claims 25-27, wherein the modulator comprises a modifier of γ-secretase, or a drug that is involved in lipid metabolism, such as a member of the group of drugs known as statins; for example, but not limited to, simvastatin, atorvastatin or lovastatin, or a drug from the thiazolidinedione class of drugs, for example, but not limited to pioglitazone or rosiglitazone.
29. A modulator according to any one of claims 25-27, wherein the modulator is a negative modulator of the function of a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof.
30. A modulator according to claim 29, wherein the negative modulator comprises an RNAi or an siRNA molecule, which is capable of inhibiting or slowing expression of the polypeptide.
31. A modulator according to claim 30, wherein the RNAi or siRNA molecule comprises a nucleotide sequence which is specific for, or complementary to, at least a region of one or more of the nucleic acid sequences substantially as set out in any one of SEQ ID No's: 17 to 30.
32. An anti-neurodegenerative disease composition comprising a therapeutically effective amount of a modulator of a polypeptide selected from the group consisting of SEQ ID No: 1 to 16 or a functional variant or functional fragment thereof, and optionally a pharmaceutically acceptable vehicle.
33. A process for making the composition according to claim 32, the process comprising combining a therapeutically effective amount of a modulator of a polypeptide selected from the group consisting of SEQ ID No:l to 16 or a functional variant or functional fragment thereof, with a pharmaceutically acceptable vehicle.
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