WO2002097079A2 - Gene encoding molecular motor protein and diagnosis method for the disease related to the gene - Google Patents

Gene encoding molecular motor protein and diagnosis method for the disease related to the gene Download PDF

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WO2002097079A2
WO2002097079A2 PCT/JP2002/005226 JP0205226W WO02097079A2 WO 2002097079 A2 WO2002097079 A2 WO 2002097079A2 JP 0205226 W JP0205226 W JP 0205226W WO 02097079 A2 WO02097079 A2 WO 02097079A2
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gene
polypeptide
kiflbβ
associated disease
polynucleotide
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PCT/JP2002/005226
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English (en)
French (fr)
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WO2002097079A3 (en
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Nobutaka Hirokawa
Yasuhide Hayashi
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Japan As Represented By The President Of The University Of Tokyo
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Priority to AU2002258249A priority Critical patent/AU2002258249A1/en
Priority to EP02728192A priority patent/EP1390483A2/de
Priority to JP2003500246A priority patent/JP2004534534A/ja
Publication of WO2002097079A2 publication Critical patent/WO2002097079A2/en
Publication of WO2002097079A3 publication Critical patent/WO2002097079A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to KIFlB ⁇ protein, a novel polypeptide having motor activity that transports synaptic vesicle precursor.
  • the present invention also relates to a polynucleotide encoding the polypeptide, a method for preparing the polypeptide, and use thereof, such as diagnosis method for kiflB ⁇ gene-associated disease.
  • Axonal transport supplies essential organelles and materials to nerve endings mainly by using microtubules as tracks.
  • the kinesin superfamily of molecular motor proteins (KIFs) has over 40 members potentially functioning in various aspects of axonal transport
  • Charcot-Marie-Tooth disease (CMT) , the most common inherited peripheral neuropathy in humans with a prevalence of 1/2500, is clinically characterized by weakness and atrophy of distal muscles, depressed or absent deep tendon reflexes, and mild sensory loss (Shy, M. E. et al., eds., (1999) Charcot-Marie-Tooth Disorders. Ann. New
  • CMT2A an autosomal dominant subtype of Type II CMT, was mapped to human chromosome lp35-36 (Ben Othmane, K.B. et al. (1993) Genomics 17(2),
  • An objective of the present invention is to provide a novel polypeptide having motor activity that transports synaptic vesicle precursor.
  • Another objective of the present invention is to provide a polynucleotide encoding the polypeptide, a method for preparing the polypeptide, and use thereof, such as diagnosis method for kiflB ⁇ gene-associated disease.
  • the present inventors have identified and characterized a novel isoform of the conventional mitochondrial motor KIF1B (Nangaku, M. etal. (1994) Cell 7 (7) , 1209-20; termed KIFlB ⁇ hereafter) , called
  • KIFlB ⁇ The two isoforms, splice variants of the same gene mapped on mouse chromosome 4E, share the N-terminal motor domain, but have distinct tail domains.
  • the tail domain of KIFlB ⁇ more closely resembles that of KIF1A, a motor that transports synaptic vesicle precursors (Okada, Y. et al. (1995) Cell 81(5), 769-80). Because their tail domains share no homology with each other, the cargos of KIFlB ⁇ and KIFlB ⁇ would be predicted to be different (Nakagawa, T. etal. (1997) Proc. Natl. Acad. Sci. U.S.A.
  • KIFlB ⁇ transports synaptic vesicle precursors and also characterized its functional significance in vivo , showing that knockout mice developed neurological disorders.
  • the present inventors have demonstrated that the phenotype of kiflB + ⁇ mice resembles the symptoms of Charcot-Marie-Tooth disease (CMT) . Therefore, the present inventors analyzed the KIF1B locus in CMT2A patients, and the present inventors discovered a Q98L missense mutation in the ATP-binding consensus of motor domain. Then, the present inventors have shown that the
  • the present inventors have identified a new motor protein KIFlB ⁇ that transports synaptic vesicle precursors.
  • the present inventors generated kif IB heterozygous mice that mimic human CMT2A neuropathy.
  • the evidence the present inventors present here indicates that the autosomal dominant hereditary pattern of CMT2A could be explained by haploinsufficiency of KIF1B: (1) kiflB +/ ⁇ mice reduced the KIFlB ⁇ level to half , and caused a late-onset axonopathy similar to CMT2A.
  • Human KIFlB ⁇ is closely linked to CMT2A.
  • the human Q98L mutation completely correlated with the clinical manifestation.
  • KIFlB ⁇ cDNA but not by KIFlB ⁇ , suggested that KIFlB ⁇ acts cell autonomously in neurons, which is consistent with the clinical definition of CMT2A as an axonopathy, rather than a myelinopathy .
  • kif!A ⁇ ' ⁇ mice also exhibited neurological phenotypes lethal in the newborn period, showing a reduction of the density of synaptic vesicles in the nerve terminal.
  • the phenotypes of the kiflB ⁇ / ⁇ mice were more severe than those of kiflA ⁇ ⁇ mice that could survive for about 24 hr after birth.
  • KIF1A is neuron-specific but KIFlB ⁇ has a broader spectrum of tissue distribution, so that KIFlB ⁇ could also play some roles in transporting other vesicles in non-neuronal tissues.
  • the assessment of the genetic interaction between these two related genes will be the subject of future research.
  • the conventional distal-first theory of axonal neuropathies stating that distal muscles suffer earlier and more severely than the proximal muscles, can be partially explained by the idea that longer axons need higher levels of motor molecules. Accordingly, the present inventors propose here a new disease entity of "motor proteinopathy” , representing the paradigm for motor-protein-derived pathogenesis of axonal neuropathies.
  • the present invention relates to kiflB ⁇ protein, a novel polypeptide having motor activity that transports synaptic vesicle precursor.
  • the present invention also relates to a polynucleotide encoding the polypeptide, a method for preparing the polypeptide, and use thereof, such as diagnosis method for kiflB ⁇ gene-associated disease. More specifically, the present invention relates to: [1] A substantially pure polypeptide selected from the group consisting of:
  • polypeptide that comprises the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are substituted, deleted, inserted, and/or added and that is functionally equivalent to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2;
  • [3] A vector comprising the polynucleotide of [2] .
  • [4] A host cell harboring the polynucleotide of [2] or the vector of [3].
  • a method for producing the polypeptide of [1] comprising the steps of:
  • transgenic non-human vertebrate in which expression of an endogenous polynucleotide encoding the polypeptide of [1] is suppressed.
  • the transgenic non-human vertebrate of [9] or [10] wherein the transgenic non-human vertebrate is a mouse.
  • a method for diagnosing a kiflB ⁇ gene-associated disease comprising the step of: (a) detecting whether a subject has a mutation of a gene that is located on the allele same as the one on which a gene encoding the nucleotide sequence of SEQ ID NO: 1 is located; wherein the subject is suspected of having the kiflB ⁇ gene-associated disease if the subject has the mutation.
  • a method for diagnosing a kiflB ⁇ gene-associated disease comprising the step of:
  • a method of screening for a compound binding to the polypeptide of [1] comprising the steps of:
  • compositions for treating, alleviating, or preventing a kiflB ⁇ gene-associated disease comprising a pharmaceutically effective amount of the compound selected by the method of any one of [15] to [17] as an active ingredient, and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier comprising a pharmaceutically effective amount of the compound selected by the method of any one of [15] to [17] as an active ingredient, and a pharmaceutically acceptable carrier.
  • [20] A method for treating, alleviating, or preventing a kiflB ⁇ gene-associated disease, said method comprising the step of administering a pharmaceutically effective amount of a compound selected by the method of any one of [15] to [17] .
  • KIFlB ⁇ is a novel protein that transports synaptic vesicle precursors.
  • the nucleotide sequence of the isolated KIFlB ⁇ cDNA, which is included in this invention, is shown in SEQ ID NO: 1, and amino acid sequence of KIFlB ⁇ protein encoded by the cDNA is represented in SEQ ID NO: 2.
  • substantially pure polypeptide as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules .
  • the substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example by column chromatography, polyacrylamide gel electrophoresis , or HPLC analysis.
  • Polypeptides structurally similar to the mouse KIFlB ⁇ protein are also included in this invention as long as they have motor activity that transports synaptic vesicle precursors.
  • Such structurally analogous polypeptides also include variants of KIFlB ⁇ protein and KIFlB ⁇ proteins derived from other organisms.
  • polypeptides using, for example, standard mutagenesis methods .
  • Known methods for altering amino acids in polypeptides include site-specific mutagenesis, for example, the method of preparing deletion-mutant (Kowalski, D., et al . , 1976, J. Biochem. , 15, 4457; McCutchan, T. F. , et al., 1984, Science, 225, 626-628), Kunkel's method (Kunkel, T. A., 1985, Proc. Natl. Acad. Sci. USA, 82: 488-492; Kunkel, T. A. et al., 1987, Methods Enzymol . , 154: 367-382), Gapped-duplex method
  • a TransformerTM Site-Directed Mutagenesis Kit (CLONTECH #K1600-1) , for example, may be used.
  • the number of amino acid residues to be altered is usually 30 or less, preferably 10 or less, and more preferably 5 or less. Alteration of amino acids in polypeptides could occur spontaneously.
  • Such polypeptides having amino acid sequences different from that of the natural mouse KIFlB ⁇ protein (SEQ ID NO: 2) due to artificial or spontaneous substitution, deletion, addition, and/or insertion of amino acid residues, are also included in this invention as long as they have motor activity that transports synaptic vesicle precursors .
  • amino acid having similar properties to those of the amino acid to be substituted is preferably used for substitution. Since
  • Non-charged amino acids include Gly, Ser, Thr, Cys, Tyr, Asn, and Gin.
  • Acidic amino acids include Asp and Glu, while basic amino acids include Lys, Arg, and His.
  • Amino acids with beta-branched side chains include Thr, Val, and lie, and amino acids with aromatic side chains include Tyr, Phe, Trp, and His.
  • KIFlB ⁇ protein include a fusion protein of the mouse KIFlB ⁇ protein with other peptides.
  • Polypeptides structurally similar to the mouse KIFlB ⁇ protein having motor activity that transports synaptic vesicle precursors can be prepared using the known hybridization technique (Cell Engineering , extra issue , New Cell Engineering Experimental Protocol , 1991, Shujunsha, pp. 188-193, and Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989) , 8.46-8.52) and polymerase chain reaction (PCR) technique (Cell Engineering, extra issue, 8, New Cell Engineering Experimental Protocol , 1991, Shujunsha, pp. 171-186; Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989) , 14.1-14.35) .
  • PCR polymerase chain reaction
  • Polypeptides encoded by polynucleotides hybridizing with the mouse KIFlB ⁇ cDNA are included in this invention as long as they have motor activity that transports synaptic vesicle precursors.
  • Other organisms used for isolating such polypeptides include, for example, humans, monkeys, rats, rabbits, goats, cattle, pigs, etc., but are not limited thereto.
  • Polynucleotides encoding such polypeptides can be isolated from such sources as neurons, brains, and cultured neurons .
  • Polynucleotides encoding the KIFlB ⁇ protein derived from animals other than mice are usually highly identical with the nucleotide sequence (SEQ ID NO: 1) of mouse KIFlB ⁇ cDNA. "Being highly identical” means at least 60% or more, preferably 80% or more, and further preferably 90% or more sequence identity at the amino acid level. As used herein, "percent identity" of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990) modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993) .
  • hybridization conditions are as follows. After the pre-hybridization at 42 ° C overnight in a hybridization solution containing 25% formamide
  • the polypeptide of this invention can be prepared as either natural polypeptides or recombinant polypeptides utilizing gene recombination techniques .
  • Natural polypeptides can be prepared by subjecting tissue extracts that are supposed to contain the KIFlB ⁇ protein (for example, neurons and brains) to affinity chromatography using the antibody to the KIFlB ⁇ protein as described below.
  • recombinant polypeptides may be prepared by culturing cells transformed with polynucleotide encoding the KIFlB ⁇ protein, allowing the transformants to express the polypeptide, and recovering the polypeptide.
  • polypeptides of this invention also include partial peptides of the above-described polypeptides.
  • Partial peptides of this invention also include, for example, the C-terminal region of the polypeptide of this invention, and the peptides can be used for the preparation of antibodies.
  • Partial peptides comprising the amino acid sequence specific to the polypeptide of this invention have at least 7, preferably at least 8, more preferably at least 9 amino acid residues.
  • Partial peptides of this invention can be produced by genetic engineering techniques, known peptide synthetic methods, or by digestion of the polypeptide of this invention with appropriate peptidases.
  • This invention also relates to polynucleotides encoding the polypeptides of the invention.
  • Polynucleotides encoding the polypeptide of this invention are not particularly limited as long as they can encode the polypeptides of this invention, including cDNAs , genomic DNAs , and synthetic DNAs .
  • Polynucleotides having any desired nucleotide sequences based on the degeneracy of genetic codes are also included in this invention as long as they can encode the polypeptides of this invention.
  • an "isolated polynucleotide” is a polynucleotide the structure of which is not identical to that of any naturally occurring polynucleotide or to that of any fragment of a naturally occurring genomic polynucleotide spanning more than three separate genes.
  • the term therefore includes, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule in the genome of the organism in which it naturally occurs; (b) a polynucleotide incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR) , or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion polypeptide.
  • PCR polymerase chain reaction
  • the invention provides an isolated polynucleotide that encodes a polypeptide described herein or a fragment thereof.
  • the isolated polynucleotide includes a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO: 1.
  • the isolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, identical to the nucleotide sequence shown in SEQ ID NO: 1.
  • SEQ ID NO: 1 the comparison is made with the full length of the reference sequence.
  • the isolated polynucleotide is shorter than the reference sequence, e . g.
  • cDNAs encoding the polypeptides of this invention can be screened, for example, by labeling cDNA of SEQ ID NO: 1 or segments thereof, RNAs complementary to ' them, or synthetic oligonucleotides comprising partial sequences of the cDNA with 32 P, etc., and hybridizing them with a cDNA library derived from tissues (e.g., neurons, brains, etc. ) expressing the polypeptides of this invention.
  • Such cDNAs can be cloned by synthesizing oligonucleotides corresponding to nucleotide sequences of these cDNAs , and amplifying them by PCR with cDNA derived from suitable tissues (e.g. neurons, brains, etc.) as a template.
  • the genomic DNA can be screened, for example, by labeling cDNA of SEQ ID NO: 1 or segments thereof, RNAs complementary to them, or synthetic oligonucleotides comprising partial sequences of the cDNA with 32 P, etc., and hybridizing them with a genomic DNA library.
  • cDNAs encoding the polypeptides of this invention can be cloned by synthesizing oligonucleotides corresponding to nucleotide sequences of these cDNAs , and amplifying them by PCR with a genomic DNA as a template .
  • Synthetic DNAs can be prepared, for example, by chemically synthesizing oligonucleotides comprising partial sequences of cDNA of SEQ ID NO: 1, annealing them to form double strand, and ligating them with DNA ligase . These polynucleotides are useful for the production of recombinant polypeptides.
  • polypeptides of this invention can be prepared as recombinant polypeptides by inserting polynucleotides encoding the polypeptides of this invention (e.g. polynucleotide of SEQ ID NO: 1) into an appropriate expression vector, transforming suitable host cells with the vector, culturing the transformants, and purifying polypeptides expressed.
  • polypeptides of this invention is a secretory protein, it can be prepared, for example, by expressing it in mammalian cells to be secreted therefrom.
  • Expression vectors to be specifically used in E. coli include, for example, pKK223-3, pKK233-2, pJLA502 , etc.
  • the polypeptides of this invention can be expressed, for example, as fusion proteins with other polypeptides.
  • Vectors for expressing such fusion proteins include, for example, pRIT2T, pGEX-2T, pGEX-3X, etc. These fusion proteins can be easily collected using an affinity column. Only a desired polypeptide can easily excised from the fusion protein when a vector is designed to provide the thrombin- or factor Xa-cleaving site between the desired polypeptide and a partner polypeptide in the fusion protein.
  • Vectors for secreting the polypeptides extracellularly or into the periplasm include pKT280, pRIT5, etc. (Okada, M. and Miyazaki , K. ed. , Invincible Biotechnical Series, Protein Experimental Note, 1st volume, Extraction and Separation/Purification, Yodosha, 1996, pp.139-149).
  • Baculovirus vectors used in mammalian cells are, for example, pAcCAGMCSl (Muramatsu, M. , ed. , Labomanual Genetic Engineering, 3rd ed. , Maruzen, 1996, pp. 242-246).
  • Recombinant polypeptides expressed in host cells can be purified by known methods.
  • the polypeptide of this invention expressed in the form of a fusion protein, for example, with a histidine residue tag or glutathione-S-transferase (GST) attached at the N-terminus can be purified by a nickel column or a glutathione Sepharose column, etc.
  • Polynucleotides encoding the polypeptides of this invention can be applied to gene therapy for disorders caused by the mutation thereof .
  • Vectors used for gene therapy include, for example, virus vectors such as retrovirus vector, adenovirus vector, adeno-associated virus vector, vaccinia virus vector , lentivirus vector , herpesvirus vector , alphavirus vector, EB virus vector, papilloma virus vector, foamy virus vector, etc. , and non-viral vectors such as cationic liposomes, ligand-DNA complexes, gene guns, etc. (Y. Niitsu, M. Takahashi , Molecular Medicine, Vol. 35, No. 11, 1385-1395, 1998) . Gene transfer can be carried out in vivo and ex vivo .
  • the present invention also relates to polynucleotides specifically hybridizing with the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 and containing at least 15 nucleotides.
  • “Specifically hybridizing” means that polynucleotide does not significantly cross-hybridize with polynucleotides encoding other polypeptides under usual hybridization conditions as described above, preferably under stringent hybridization conditions.
  • Such polynucleotides include probes, primers, nucleotides or nucleotide derivatives (e.g.
  • cDNAs encoding the polypeptides of this invention or oligonucleotides comprising partial sequences thereof can be used for cloning genes and cDNAs encoding the polypeptides of this invention, or amplifying them by PCR.
  • the cDNAs and oligonucleotides can also be utilized for detecting polymorphism or abnormality (gene diagnosis, etc. ) of the gene or cDNAby the restriction fragment length polymorphism (RFLP) method, single strand DNA conformation polymorphism (SSCP) method, etc.
  • Antibodies of this invention include both polyclonal and monoclonal antibodies.
  • Polyclonal antibodies can be prepared, for example, according to the method described in Institute of Medical Science, Section of Carcinostasis , University of Tokyo, ed. , Cell Engineering, extra issue 8, New Cell Engineering Experimental Protocol, 1993, Shujunsha, pp. 202-217.
  • the purified polypeptides of this invention, partial peptides thereof, or peptides synthesized based on amino acid sequences of the polypeptides of this invention are injected to animals to be immunized such as rabbits, guinea pigs, mice, chickens, etc.
  • Antigenic polypeptides may be administered together with Freund's complete or incomplete adjuvants. Antigen is usually administered every several weeks. The titer can be elevated by the booster injection. Blood is periodically collected to confirm the titer elevation by ELISA, etc. After the final immunization, the blood is collected from immunized animals to obtain antisera. Antisera are purified by salting out, ion exchange chromatography, HPLC, etc. to obtain an IgG fraction. The antibody can be further purified by affinity chromatography using the immobilized antigen.
  • a monoclonal antibody can be prepared, for example, according to the method described in Koike, T. & Taniguchi, M. 1991, Jikken
  • Antibodies thus prepared are used for the affinity purification of the polypeptides of this invention. They can also be used for the test and diagnosis of disorders caused by abnormal expression and structural abnormality of the polypeptides of this invention and for detection of the expression level of the polypeptide. Abnormality in expression and structure of the polypeptides of this invention can be examined and diagnosed by extracting polypeptides from, for example, tissues, blood or cells, and detecting the polypeptides of this invention using Western blotting, immunoprecipitation, ELISA, etc. Antibodies of this invention can be applied to the antibody treatment. For antibody treatment, they are preferably humanized or human antibodies.
  • Humanized monoclonal antibodies can be prepared according to, for example, a method described in H. Isogai, 1988, Jikken Igaku, Vol.6, No. 10, 55-60. A method using molecular biological techniques as described in T. Tsunenari et al., 1996, Anticancer Res., 16, 2537-2544 can also be used. Human antibodies can be prepared by immunizing mice, in which the immune system is replaced by the human system, with the polypeptide of this invention. This invention also relates to a transgenic non-human vertebrate harboring in an expressible manner an exogenous polynucleotide encoding the polypeptide of this invention.
  • Such animals include animals expressing the exogenous polynucleotide, and animals that can induce the expression of the exogenous polynucleotide.
  • Expression may be a systemic expression or may be cell, tissue, or organ specific. It may also be period specific. Furthermore, it may be those where expression is induced by external stimuli, or those where expression is. altered (induced or suppressed) in the later generation due to crossing.
  • the transgenic non-human vertebrate of this invention can be produced, for example, by transfecting a vector expressing the polynucleotide of this invention into a fertilized egg. Transfection of polynucleotide can be performed by treating mixture of the vector and the egg with calcium phosphate, or by microinjection under an inverted microscope .
  • the vector of this invention can be transfected into an embryonic stem cell (ES cell) and the selected ES cell can be introduced into a fertilized egg (blastocyst) by microinjection (Gordon, J. W. et al., 1980, Proc. Natl. Acad. Sci. USA 77: 7380-7384) .
  • the obtained fertilized egg can be transplanted into the oviduct of a recipient that has undergone pseudopregnancy by mating a vas deferens ligated male individual, and an offspring can be obtained.
  • an offspring can be obtained.
  • polynucleotide from the tail of the offspring retention of the transfected polynucleotide can be confirmed by PCR (Katsuki, M. ed.
  • promoters used to express the polypeptide of this invention in vivo include systemically expressing promoters, tissue specific promoters, and period specific promoters (Sauer, B., 1998, Methods 14: 381-92).
  • Cre-loxP system and yeast FLP system may be used .
  • a transgenic animal having a Cre recombinase gene downstream of a site specific or period specific promoter is produced, and separately a transgenic animal retaining a vector in which polynucleotide encoding the polypeptide of this invention is linked downstream of a general purpose promoter is prepared.
  • a stop codon, a transcription termination signal, or the like is inserted between loxP sequences and, then, is placed between a promoter and the polynucleotide encoding the polypeptide of this invention.
  • the polypeptide of this invention can be expressed along with expression of Cre (Sauer, B., 1998, Methods 14:381-92) .
  • the transgenic non-human vertebrate of this invention includes transgenic non-human vertebrate in which expression of an endogenous polynucleotide encoding the polypeptide of this invention is suppressed by substitution, deletion, addition, and/or insertion at least a portion of the polynucleotide.
  • Such transgenic animals may be produced, for example, by gene targeting.
  • Such transgenic non-human vertebrates can be produced, for example, by a method comprising the steps of:
  • step (b) transfecting the vector into a mammalian embryonic stem cell; (c) selecting, from cells obtained in step (b) , cells in which the vector has been integrated into their genome by homologous recombination ;
  • step (d) preparing embryos of the mammals; (e) injecting the cell obtained in step (c) to the embryo obtained in step (d) ;
  • step (f) developing the embryo obtained in step (e) ;
  • a specific method for gene targeting can be carried out, for example, by the method indicated in the literature (Capecchi, M. R. (1989) Science 244, 1288-1292).
  • transgenic non-human vertebrates in which expression of an endogenous polynucleotide encoding the polypeptide of this invention is suppressed can be prepared by utilizing an antisense method.
  • an antisense method (1) a vector containing
  • DNA encoding RNA complementary to the transcription product of polynucleotide encoding the polypeptide of this invention can be transfected into a mammalian embryonic stem cell as mentioned above,
  • the vector can be administered by methods such as intracerebroventricular administration (Kagiyama,
  • the transgenic non-human vertebrate of this invention is especially useful as a model for kiflB ⁇ gene-associated diseases , such as Charcot-Marie-Tooth disease type 2A.
  • kiflB ⁇ gene-associated diseases such as Charcot-Marie-Tooth disease type 2A.
  • These transgenic non-human vertebrates may be utilized for molecular analyses of kiflB ⁇ gene-associated diseases and for development of therapeutic or preventive drugs for kiflB ⁇ gene-associated diseases.
  • mammals especially rodents such as mice and rats, are preferably used.
  • the testing and diagnosis of this invention can be carried out by detecting structural variations or abnormal expression of the kiflB ⁇ gene.
  • detection of structural variations of the kiflB ⁇ gene can be performed by amplifying the kiflB ⁇ gene from genomic DNA or mRNA from a sub ect by PCR and such, and then by determining the nucleotide sequence. The presence or absence, and the type of mutation are determined by comparison to the nucleotide sequence of the normal gene.
  • mutation includes polymorphisms.
  • kiflB ⁇ gene mutation that reduces motor activity, such as ATPase activity of KIFlB ⁇ protein and cell motility in vivo , is judged to be risk factors of kiflB ⁇ gene-associated disease onset and progress .
  • abnormal expression of the kiflB ⁇ gene for example, expression of the kiflB ⁇ gene in patient-derived neurons is analyzed by microarray analysis , quantitative or semi-quantitative RT-PCR, immunohistochemical staining, Western blotting, and such.
  • Abnormal expression is determined by comparison to expression in normal tissues. Abnormal expression includes rise, decline, and loss of expression. Decline in kiflB ⁇ gene expression is judged to be risk factors for kiflB ⁇ gene-associated disease onset and progress .
  • the testing and diagnosis of this invention includes risk testing performed before kiflB ⁇ gene-associated disease onset, and classification performed after kiflB ⁇ gene-associated disease onset, testing for preparing a therapeutic plan, and so on.
  • kiflB ⁇ gene-associated disease onset structural variation or abnormal expression of the kiflB ⁇ gene is detected by testing the neuronal tissue, and the presence or absence and the type of variation and abnormality is determined.
  • the probability of detecting kiflB ⁇ gene-associated disease in early stage can be elevated by setting a frequent preventive medical examination schedule.
  • a reasonable prediction of prognosis or selection of treatment is possible through classification or distinction of kiflB ⁇ gene-associated disease.
  • the testing of this invention is also useful for determining whether the drug obtained by the screening of this invention is effective. These testing enable realization of tailor-made treatment according to each individual kiflB ⁇ gene-associated disease.
  • Polynucleotides containing the kiflB ⁇ gene nucleotide sequence or a portion of their complementary strand may be a polynucleotide containing the nucleotide sequence or a portion of its complementary strand of the gene in mRNA, cDNA, or genomic DNA, and may be, for example, a sequence in the protein coding region, 5' and 3' UTS, intron, exon, and a sequence in the transcription regulatory region.
  • the above-mentioned polynucleotide is preferably a polynucleotide containing continuous nucleotides within the kiflB ⁇ gene nucleotide sequence or their complementary strand of at least 15 or more nucleotides, preferably 16 or more nucleotides, and more preferably 17, 18, or 20 or more nucleotides.
  • These polynucleotides may be DNA or RNA. It may also be those that contain modified nucleotides.
  • Polynucleotides may be used in testing as probes, as immobilized polynucleotides in DNA microarray, or as primers for RT-PCR and such.
  • the antibodies are useful for detecting expression of the kiflB ⁇ gene at the protein level, and for performing tests through protein structure analyses.
  • the present invention provides test or diagnostic reagents for kiflB ⁇ gene-associated disease including these polynucleotides or antibodies.
  • the polynucleotides and- antibodies may be bound to a carrier or to a support. For example, it may be in the form of a DNA microarray and such.
  • polynucleotides and antibodies may be combined appropriately with solvents and solutes.
  • the test and diagnostic reagents of this invention are provided, for example, as aqueous solutions.
  • the reagent of this invention may be a testing and diagnosing kit that is placed into an appropriate container, including casing and/or instructions.
  • test and diagnostic reagent of this invention preferably mentions on its container, casing or instructions that it can be used for testing and diagnosis of kiflB ⁇ gene-associated disease including Charcot-Marie-Tooth disease type 2A, otherwise, such information indicated in other printed matter, URL, and such are related to the reagents.
  • the present invention also relates to a method of screening for a compound binding to the polypeptide of this invention.
  • the screening method of this invention comprises: (a) contacting a test compound with the polypeptide of this invention, (b) detecting the binding activity between the polypeptide and the test compound, and (c) selecting a compound that binds to the polypeptide of this invention.
  • Polypeptides binding to the polypeptide of this invention can be screened, for example, by applying culture supematants or extracts of cells, which are expectedly express polypeptides binding to the polypeptide of this invention, to an affinity column to which the polypeptide of this invention is attached (immobilized) , and purifying polypeptides specifically binding to the column.
  • polypeptides binding to the polypeptide of this invention can be screened by the West Western blotting method or two hybrid system.
  • a cDNA library using a phage vector is prepared from tissues or cells (for example, brains, neurons, etc.) which expectedly express polypeptides binding to the polypeptide of this invention, and polypeptides are expressed from cloned cDNA on agarose, transferred to filter, fixed, and reacted with the labeled polypeptide of this invention to detect plaques expressing binding polypeptides.
  • the polypeptide of this invention is expressed as a fusion protein with a test protein such as GAL4 DNA-binding region and GAL4 transcription activating region, and the binding of the polypeptide of this invention to the test protein is detected by the expression of a reporter gene linked downstream of a promoter having the binding sequence of the GAL4 DNA-binding protein.
  • screening methods include (1) the method comprising contacting synthetic compounds, natural proteins or random phage peptide display library with the immobilized polypeptide of this invention and detecting binding molecules and (2) the method comprising isolating compounds binding to the polypeptide of this invention using a high-through put based on combinatorial chemical technique.
  • Test samples used for screening include, for example, cell extracts, expression products of a gene library, synthetic low molecular weight compounds, synthetic peptides, natural compounds, etc., but are not limited thereto. Those test samples used for screening may be labeled prior to use as the occasion demands . Labels include, for example, radioactive and fluorescent ones, etc., but are not limited to them.
  • this invention also relates to a method of screening for a candidate compound for treating, alleviating, or preventing a kiflB ⁇ gene-associated disease using the transgenic non-human vertebrate of this invention.
  • a transgenic non-human vertebrate in which expression of an endogenous polynucleotide encoding the polypeptide of this invention is suppressed by substitution, deletion, addition, and/or insertion at least a portion of the polynucleotide can be used for the screening method of the present invention because it exhibits symptoms of kiflB ⁇ gene-associated diseases, such as Charcot-Marie-Tooth disease type 2A.
  • the screening method of this invention comprises the following steps of:
  • a candidate compound used in the screening of this invention may include natural or synthetic compounds, various organic compounds, natural or synthetic saccharides, proteins, peptides, products of gene libraries, cell extracts, and bacterial or fungal components. These candidate compounds may be orally or parenterally administered to the transgenic non-human vertebrate for kiflB ⁇ gene-associated disease.
  • polypeptides of this invention or compounds isolated by the screening methods of this invention are used as drugs, they may be administered to patients as they are or as pharmaceutical preparations produced by known methods. They can be formulated together with, for example, pharmaceutically acceptable carriers or media such as sterilized water, physiological saline, vegetable oil, emulsifiers, suspending agents , surfactants, stabilizers, etc. They may be administered to patients by methods well known in the art, for example, by intra-arterial, intravenous, subcutaneous injection. They can also be administered intranasally , intrabronchially, intramuscularly, or orally. Doses may vary depending on the body weight and age of patients as well as administration method, and can be suitably selected by those skilled in the art.
  • gene therapy may be performed by inserting the DNA into a vector for gene therapy.
  • Doses of DNA and method for its administration may vary depending on the body weight, age and symptoms of patients, but can be suitably selected by those skilled in the art.
  • Figure 1 shows a diagram and photographs representing molecular cloning and antibody characterization of mouse KIFlB ⁇ .
  • A Schematic representation of kif IB cDNAs .
  • Figure 2 shows photographs representing association of mouse KIFlB ⁇ with synaptic vesicle precursors.
  • A Nycodenz density-gradient floatation assay indicating that KIFlB ⁇ was present in the vesicle fraction containing synaptotagmin. Arrows indicate the floated peak.
  • B GST pull-down assay of KIFlB ⁇ -associated vesicles. Synaptic vesicle proteins were specifically detected in the vesicles associated with KIFlB ⁇ tail region (aa 885-1770) , but not with the truncated construct ( ⁇ PH; aa885-1528) lacking the pleckstrin homology (PH) domain.
  • C Immunoprecipitation of the vesicle fraction of mouse brain using an anti-KIFlB ⁇ and several anti-synaptic vesicle protein antibodies .
  • KIF17 was used for control .
  • synaptic vesicle proteins synaptotagmin, synaptophysin, and SV2 were specifically detected in the vesicles co-immunoprecipitated with KIFlB ⁇ .
  • COX I cytochrome oxidase I, a marker for mitochondria.
  • D Immuno EM indicating the colocalization of KIFlB ⁇ (10-nm gold) and synaptic vesicle proteins (5-nm gold) on axonal vesicles. Bar, 100 nm. The percentages of the number of vesicles carrying both signals over the number of all the vesicles in randomly chosen areas were indicated. **,p ⁇ 0.001 (chi-square for independence test) .
  • Figure 3 shows diagrams and photographs representing targeted disruption of mouse kif IB gene.
  • A The targeting strategy for kif IB knockout. Strategy for genomic Southern blotting by Hindi is also indicated. A+T/pau, AT-rich pausing signal; pBS, pBluescriptII-SK(+) ; He, Hindi .
  • B Genomic Southern blotting in which the recombinant allele gave an additional band of 2.1-kb.
  • C Immunoblot analysis of crude extracts from 18.5 dpc mouse brains with anti-KIFlB ⁇ and anti-KIFlBO antibodies , respectively. In both cases, no bands were detected from kiflB ⁇ / ⁇ samples.
  • D Appearance of a newborn litter.
  • MdD dorsal medullary reticular nucleus
  • MdV ventral medullary reticular nucleus
  • VLRt ventrolateral reticular nucleus. Bar, 500 ⁇ m.
  • H-I Frontal sections of cerebral hemispheres of kiflB +/+ (H) and kiflB ⁇ / ⁇ (I) embryos on 18.5 dpc, with defective formation of commissural fibers. Arrowheads, anterior commisure; arrows, internal capsule. Bar, 600 ⁇ .
  • J Reduced density of synaptic vesicles in the nerve terminals at the anterior horn region of the spinal cord of kiflB ⁇ / ⁇ mice on 18.5 dpc. #l-#4 represents four different regions counted.
  • Figure 4 shows diagrams and photographs representing neuronal cell death in primary culture of kiflB ⁇ / ⁇ hippocampus .
  • A Time course of the primary culture of each genotype. Note severe neuronal death of the kiflB ⁇ /_ neurons with a significant delay in differentiation. Arrows, dead cells. Bar, 20 ⁇ m.
  • B Survival and differentiation curves of plated neurons of each genotype. Relative index of the neuronal cell number was calculated taking the initial cell number per unit area as 100% .
  • C-E Rescue experiments .
  • C Rescued culture of kiflB ⁇ / ⁇ hippocampal neurons on the fifth day of infection.
  • Green fluorescence indicates the neurons expressing respective adenoviral vectors expressing the kif IB isoforms tagged by EGFP. Note that the neurons reached highly differentiated stages only with the kiflB ⁇ construct. Arrows, dead cells. Bar, 10 ⁇ .
  • D Quantification of the rescue efficiency of kiflB ⁇ / ⁇ neurons. * p ⁇ 0.001. ⁇ , kiflB ⁇ ; a , kiflBa; N, non-infected control.
  • E Immunoblot of infected fibroblast lysates using an anti-GFP antibody. Each EGFP-tagged protein gave a band of the expected molecular weight.
  • Figure 5 shows photographs representing decreased levels of the synaptic vesicle proteins in kiflB +/ ⁇ sciatic nerve axons.
  • A Immunoblotting of the sciatic nerve extracts. 10 ⁇ g of protein was loaded in each lane. The protein levels in kiflB +/ ⁇ nerves are indicated as a percentage of those in kiflB +/+ nerves.
  • B Immunoblotting of pooled organelles in sciatic nerves 2 hr after the ligation. The protein levels from 1-cm regions proximal (Prox) and distal (Dist) to the ligated sites were compared.
  • C Immunoblotting of the spinal cord extracts prepared as in (A) .
  • D Protein staining by Coomassie Brilliant Blue of the extracts from sciatic nerves and spinal cords. 10 ⁇ g of total protein was loaded in each lane.
  • FIG. 6 shows diagrams and a photograph representing chronic peripheral neuropathy in kiflB +/ ⁇ mice.
  • A Typical posture of 1.5-year-old mice. Note that the kiflB +/ ⁇ mouse could not support its own body weight.
  • C-D Electrophysiological analysis of the sciatic nerves of 12-month-old mice.
  • C Typical traces of the evoked action potentials at the planter muscles.
  • D Quantification of the motor nerve conduction velocities (MNCV) and the amplitudes of evoked muscle potentials. Although the amplitude was reduced, the MNCV did not significantly change, suggesting an axonal origin of the disease
  • Figure 7 shows diagrams and photographs representing a loss-of-function mutation in human KIFIB gene from CMT2A patients.
  • A Pedigree of family #694. Circles, females; squares, males; cross-hatched, deceased individuals; open figures, unaffected; closed figures, affected.
  • B Mobility shifts in the PCR-SSCP analyses for the P-loop exon 3. Arrows, shifted bands specifically observed in the affected individuals .
  • C Sequence electropherogram in the P-loop region. In the sample from affected individual, A and T simultaneously read in the mutated codon, resulting in a heterozygous Q98L point mutation.
  • FIG. 8 shows photographs representing immunoblotting for the spatiotemporal expression pattern of KIFlB ⁇ .
  • A Wide distribution of KIFlB ⁇ in 4-week-old mouse nervous system.
  • B Continuous developmental expression of KIFlB ⁇ in the brain. 20 ⁇ g of protein was loaded on each lane.
  • Figure 9 shows photographs representing central, but not peripheral origin of the newborn apnea phenotype in kiflB ⁇ / ⁇ mice.
  • A Electron micrographs of the type II alveolar epithelial cells in the newborn lungs of the respective genotypes. Note that secreted lamellar bodies (arrows) were found in both samples, suggesting the function of the lung epithelium was preserved in kiflB ⁇ / ⁇ mice. Bar, 2 ⁇ m.
  • B Electron micrographs of the newborn intercostal muscles of the respective genotypes. The normal development of the muscle fibers in kiflB ⁇ / ⁇ mice excludes a muscular origin of the phenotype. Bar, 2 ⁇ m.
  • C Electron micrographs of the anterior horn regions of the spinal cord, showing the density of synaptic vesicles in the nerve terminals was significantly reduced in kiflB ⁇ / ⁇ mice. See text. Bar, 400 nm.
  • Figure 10 shows photographs representing defective development of the facial nuclei in kiflB ' ' mice.
  • A HE-stained 7 ⁇ m-thick paraffin-embedded cross-sections of the medulla oblongata at the respective stages of respective genotypes.
  • the size of the facial nuclei gradually increased in kiflB +/+ embryos, while that in kiflB ⁇ / ⁇ embryos significantly decreased, indicating the functional significance of the kiflB gene in neuronal development and survival in vivo . Bar, 400 ⁇ m.
  • FIG 11 shows photographs representing mitochondrial staining in cultured hippocampal neurons of the respective genotypes probed by Mitotracker (Molecular Probes) . Note that the mitochondria were distributed similarly in the neuronal axons of both genotypes.
  • Anti-synaptophysin and anti-SV2 monoclonal antibodies were kindly provided by Drs. A. Davies and I. Jones (Inst Viol & Enviol Microbiol) .
  • Anti-SNAP-25 and anti-Mintl (mLin-10) ; anti-syntaxin; anti-COX I; anti-GFP antibodies were respectively purchased from BD Transduction Laboratories; Sigma; Molecular Probes; and Clontech.
  • Mouse kiflB ⁇ cDNA was cloned from a 4-week-old ICR mouse brain cDNA library probed with a cDNA fragment of the kiflB motor domain (Nangaku, M. et al. (1994) Cell 79(7) , 1209-20) . Sequencing of this cDNA has revealed that this new and longer isoform consisted of 1770 amino acids, with a predicted molecular weight of 199 kD (GenBank accession no. AB023656; Nakagawa, T. etal. (1997) Proc. Natl. Acad. Sci. U.S.A. 94(18), 9654-9).
  • N-terminal motor domain (1-660 aa) was 100% identical to that of KIFlB ⁇ at the nucleotide level. Other domains of the two, however, shared no significant homology, suggesting that an alternative splicing mechanism generated two different motors from a single gene (Fig. 1A) .
  • a rabbit polyclonal antibody specific to the KIFlB ⁇ tail domain was raised against a synthetic peptide TTTFESAITPSESSGYDSADVESLVDREKELAC (SEQ ID NO: 3) corresponding to the 1505-1536 aa, which was not homologous to KIFIA tail.
  • the present inventors characterized the cargo organelles of KIFlB ⁇ and detected a single band of the predicted size in brain and bands of other sizes (presumably splicing variants or post-translationally modified forms) in other tissues (Fig. IB; Conforti, L. et al. (1999) Mamm. Genome 10(6), 617-22; Gong, T. W. et al. (1999) Gene 239 (1) , 117-27) .
  • This antibody did not crossreact with KIFIA because the present inventors had chosen a KIFlB ⁇ -specific sequence as an antigen.
  • KIFlB ⁇ was widely distributed both in cell bodies and axons of neurons (Figs. IC and D, also see Fig.
  • the present inventors generated knockout mice by gene targeting.
  • the present inventors replaced a 2.2-kb portion containing the coding region of the ATP-binding consensus P-loop of the motor domain with a positive selection cassette (Fig. 3) .
  • mouse kiflB genomic clones were obtained from a ⁇ EMBL3 genomic library of ES cell line Jl (Tanaka, Y. et al . (1998) Cell 93 (7) , 1147-58).
  • a 2.2-kb region spanning between the P loop exon and a part of the upstream exon (P-l) was replaced by a poly (A) -less neo-selection cassette, and the knockout procedures were performed as described previously
  • mice were backcrossed with C57BL/6J females in a specific pathogen free environment, and two independent lines were used simultaneously.
  • Wild-type kif IB alleles were detected by PCR amplification using the following primer set: 5 '-CGCTAGGGTTAAAGCACACGCTAC-3 ' (SEQ ID NO: 4) and 5'-TGAACTTAGAAATCCACCTGCCTC-3' (SEQ ID NO: 5).
  • neo transgene was amplified as described previously (Tanaka, Y. etal. (1998) Cell 93 (7), 1147-58) .
  • kiflB ⁇ / ⁇ pups were delivered in normal Mendelian ratios , but died within 30 minutes of birth. Their pulmonary alveoli did not expand and thus apnea could well explain the cause of newborn lethality (Fig. 3E) .
  • This apnea probably did not have a peripheral origin, because the histology and ultrastructure of the lung and the intercostal muscles involved in respiration did not reveal any significant defects (see Fig. 9) .
  • Newborn kiflB '/ ⁇ pups displayed multiple neurological abnormalities. They were insensitive to pinching stimuli (Table 1) , and their body posture was severely lordotic with dropping forelimbs
  • Fig. 3D representing an overall reduction of motosensory neural function.
  • kiflB ⁇ / ⁇ brains were reduced approximately 10% in size compared to the control.
  • the cellularity, organization, and development of the brain stem nuclei and commissural fibers were significantly affected (Figs. 3F-I, also see Fig. 10).
  • the number of neuronal cell bodies was less than 25% of that in the control.
  • Neuronal loss in the kiflB '/ ⁇ respiratory center, or dorsal and ventral medullary reticular nuclei (MdD and MdV) may be the primary cause of the newborn apnea.
  • the cellularity and organization of the hippocampus was also affected (Figs. 3H and I).
  • the present inventors quantified synaptic vesicle density.
  • Anterior horn of the spinal cord at the C4 level was processed for conventional electron microscopy.
  • Sixty areas of synapses were randomly chosen from the electronmicrographs of 100 nm-thick sections, and quantified for four pairs of littermates as described previously (Yonekawa, Y. et al. (1998) J. Cell Biol. 141(2), 431-41).
  • the present inventors found significant reduction in the density of synaptic vesicles of the nerve terminals (Fig. 3J, also see Fig. 9C) .
  • KIFlB ⁇ acts cell autonomously in neurons
  • the present inventors then cultured kiflB +/+ or kiflB ⁇ / ⁇ hippocampal neurons to assess the neuronal loss phenotype in vi tro .
  • the neurons were observed for five successive days after plating, classified into differentiation stages 0-4 with established criteria for neuronal differentiation (Goslin, K. and Banker, G. (1991) Rat hippocampal neurons in low-density culture. In Culturing Nerve Cells, G. Banker and K. Goslin, ed. (Cambridge, MA; MIT Press) , pp. 251-82) , and quantified.
  • the culture procedure and quantitative observation were performed as described previously (Goslin, K. and Banker, G.
  • Adenoviral vectors encoding EGFP-tagged isoforms were first used to infect fibroblasts to verify expression by immunoblotting (Fig. 4E) .
  • Fig. 4E immunoblotting
  • the full-length cDNA of KIFlB ⁇ and ⁇ was tagged with EGFP (Clontech) at the C-termini , and ligated into pAdex-Wtl vector with a CAG promoter to produce the recombinant adenovirus, as described previously
  • KIFlB ⁇ or KIFlB ⁇ adenovirus on the day of plating, and the survival rate was observed after 5 days.
  • the infection efficiency was over 90%, and 50-80% of infected kiflB +/+ neurons had survived by day 5.
  • infection with KIFlB ⁇ , but not KIFlB ⁇ expressing virus significantly rescued the viability of kiflB _/ ⁇ neurons (Figs. 4C-D) .
  • Any potential deficiency in mitochondrial transport caused by lack of KIFlB ⁇ was apparently compensated for with redundant mitochondrial motors (such as conventional kinesins KIF5A, B, and C: Tanaka, Y. etal. (1998) Cell 93 (7) , 1147-58; Kanai, Y.
  • the present inventors then assessed the protein content of the peripheral nerves of heterozygotes, to test if specific transport of synaptic vesicle precursors was impaired by haploinsufficiency of the motor protein.
  • the sciatic nerves of 2-month-old kiflB +/ ⁇ mice were chosen for the study, because they did not show apparent phenotypes that could develop secondary defects .
  • the present inventors compared levels of several proteins in the peripheral nerves of kiflB +/+ and kiflB +/ ⁇ mice, in the steady state (Fig. 5A) and in a cumulative manner by nerve ligation for 2 hr (Fig. 5B) .
  • the nerve ligation experiment further indicated that the transport of synaptic vesicle proteins was specifically decreased (Fig. 5B) .
  • the amount of synaptic vesicle proteins being accumulated at the proximal portion in heterozygote axons was decreased compared to that in wild type axons, while the amount of synaptic plasma membrane proteins being accumulated at the proximal portion was not changed between the heterozygote and wild type axons.
  • Staining for total protein also showed no obvious difference in the levels of major cytoskeletal proteins (Fig. 5D) .
  • the reduction in synaptic vesicle protein transport was unlikely to be due to a nonspecific overall impairment of axonal transport.
  • mice After being trained three times, the retention time of the mice was recorded for a maximum of 60 sec as described previously (Kadotani, H. etal. (1996) J. Neurosci. 16(24) , 7859-67) .
  • the mice were placed on a rotating rod accelerating from 4 to 40 rpm in 5 minutes, and the retention time was measured essentially as described previously (Jones, B. J. and Roberts , D. J. (1968) J. Pharm. Pharmacol. 20(4) , 302-4) . Three trials were given per day for five consecutive days. As a result, the present inventors detected significantly shorter retention times for 12-month-old kiflB +/ ⁇ mice than for controls (Table 2) .
  • the present inventors analyzed the evoked compound action potentials of 12-month-old sciatic nerves recorded at the plantar muscles following direct stimulation at the midthigh level (Fig. 6C) .
  • Peripheral nerve conduction velocity was determined for the sciatic nerve as described previously (Robertson, A. etal. (1993) Acta Neuropathol . (Berl) 86(2), 163-71) with slight modifications. All of the recordings were performed being unaware of the genotypes.
  • CMT2A an autosomal dominant subtype of Type II Charcot-Marie-Tooth disease
  • KIFlB-based neuropathy in humans.
  • the present inventors suspected this because of the similarity of the mouse phenotype to the human disease and because KIFIB is linked to the mapped CMT2A interval on chromosome 1.
  • the present inventors analyzed a previously well-characterized human pedigree of CMT2A, with an autosomal dominant hereditary pattern (Fig. 7A; Saito, M. et al. (1997) Neurology 49 (6) , 1630-5) .
  • High-molecular-weight DNA was prepared from peripheral blood leukocytes from 12 Japanese individuals in CMT2A family #694 (Saito, M. etal. (1997) Neurology 49 (6) , 1630-5). Based upon a complete exon-intron structure of human KIFIB gene (Yang, H.W. et al. (2001) Oncogene, 20(36), 5075-83), each of the 47 exons was amplified by genomic PCR as described (Yang, H.W. et al. (2001) Oncogene, 20(36), 5075-83) and subjected to SSCP and/or direct sequence analysis.
  • SSCP analysis was performed using low-pH buffer system to separate the longer fragments (Kukita, Y. et al. (1997) Hum. Mutat. 10(5), 400-7).
  • PCR products were purified and sequenced directly with ABI sequencers (Perkin Elmer) .
  • ABI sequencers Perkin Elmer
  • SNPs single nucleotide polymorphisms
  • the present inventors detected that an abnormal SSCP mobility shift in exon 3 completely correlated with the clinical manifestation. It was detected in all four affected people in this pedigree, but not in eight unaffected siblings or 95 healthy unrelated Japanese controls (Fig. 7B) .
  • Exon 3 of human kiflB ⁇ orthologue was amplified using the following primer set, 5'-TGGAAGCAATCAAGTAAGTATAGA-3' (SEQ ID NO: 6) and 5'-CCCCGCATAATGTTCAAGCC-3' (SEQ ID NO: 7) under a condition of 30 sec at 95°C, 30 sec at 60°C, and 30 sec at 72°C for 35 cycles, followed by a 10-min extension at 72°C. Then the present inventors directly sequenced the PCR product and revealed a heterozygous A->T point mutation, which transformed Q 98 to L in the affected individuals (Fig. 7C) .
  • the present inventors measured microtubule-activated ATP turnover rates of the wild type and Q98L recombinant KIFlB ⁇ full-length proteins, which were 3.6 ⁇ 0.5 and 0.0 ⁇ 0.4 nmol/mg/min , respectively .
  • the present inventors further expressed Q98L or wild type KIFlB ⁇ in Vero cells to test their motility in vivo .
  • Vero cells were transfected with cationic lipids, cultured for 24 hr, and immunostained with the anti-KIFlB ⁇ polyclonal antibody and an anti-OC-tubulin monoclonal antibody DM1A (Sigma) . Due to its motility toward the plus ends of the microtubules, overexpressed wild type KIFlB ⁇ protein accumulated at the cell periphery (Fig. 7D, left) . In contrast, the Q98L mutant protein remained and aggregated in the perinuclear region (Fig. 7D, right) . These data suggested that the Q98L point mutation resulted in a functional loss of motor activity.
  • the present invention provides KIFlB ⁇ protein, a novel polypeptide having motor activity that transports synaptic vesicle precursor.
  • the present invention also provides a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, a host cell harboring the vector, a method for preparing the polypeptide, and a method for diagnosing kiflB ⁇ gene-associated disease.
  • the present invention provides a method of screening for a compound that ameliorates the symptom of the kiflB ⁇ gene-associated disease.
  • the polypeptide of the present invention a gene encoding the polypeptide, and a compound that ameliorates the symptom of the kiflB ⁇ gene-associated disease are useful for developing a therapeutic or preventive agent for kiflB ⁇ gene-associated disease, for example, Charcot-Marie-Tooth disease type 2A.

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Cited By (1)

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