WO2001090355A1 - Canal sodium humain scn12a et scn8a - Google Patents

Canal sodium humain scn12a et scn8a Download PDF

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Publication number
WO2001090355A1
WO2001090355A1 PCT/JP2000/004629 JP0004629W WO0190355A1 WO 2001090355 A1 WO2001090355 A1 WO 2001090355A1 JP 0004629 W JP0004629 W JP 0004629W WO 0190355 A1 WO0190355 A1 WO 0190355A1
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scn12a
seq
human
scn8a
cdna
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PCT/JP2000/004629
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English (en)
Japanese (ja)
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Ichiro Kanazawa
Jun Goto
Seon-Yong Jeong
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Japan Science And Technology Corporation
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Priority to CA002412306A priority Critical patent/CA2412306A1/fr
Publication of WO2001090355A1 publication Critical patent/WO2001090355A1/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • This application relates to a novel subunit SCN12A of the sodium channel of the human nervous system and its splicing variant SCN12A-s, also a human sodium channel SCN8A, human genes encoding these molecules, and cDNAs of these genes. is there. Background art
  • Membrane voltage-gated sodium channels are membrane protein molecules that are present in the cell membrane of excitable cells such as neuromuscularis. Excitable cells perform their function by generating action potentials. For example, in the case of a nerve cell, the action potential generated in the cell body propagates through the axon, thereby transmitting information as a function of the nerve cell.
  • Sodium channels are protein molecules that, along with potassium channels, are responsible for initiating and transmitting action potentials.
  • the membrane potential-dependent sodium channel is composed of a plurality of subunits, and the main functions are provided in subunits.
  • SCN1A (2q24), SCN2A (2q23-q24.3), SCN3A (2q24-q31), SCN4A (17q23.1-q25.3) .
  • SCN5A (3p21-24), SCN6A (2q21-q23), SCN8A (12q13.1), SCN9A (2q24), SCN10A (3p22-q24)
  • the number in parentheses is the human chromosome locus).
  • the corresponding disease in humans is not known, but it is also known to be due to a mutation in the mouse mutant motor endplate disease (med) Scn8a.
  • understanding the molecular biology and function of the membrane-gated sodium channel is extremely important for understanding the physiological mechanisms of humans and for identifying the direct cause of various diseases. It is.
  • substances that act on the sodium channel are drugs (eg, anesthetics, analgesics, muscle relaxants, etc.) that regulate the function of cells in which they are expressed. It is also important as a target for the development of new therapeutic agents.
  • the inventors of the present application performed a gene clone for the purpose of confirming all sodium channels expressed in the human nervous system.
  • the EST (Expressed Sequence Tags) database contained a partial sequence of SCN8A.
  • AA446997 which is not a partial sequence of SCN8A, also differs from the sequence of other known sodium channel genes.
  • This application describes a novel sodium channel SCN12A discovered by the inventors, The objective is to provide genes and cDNAs specifically. Another object of the present application is to specifically provide a sodium channel SCN8A, whose genes have been clarified by the inventors, and its gene and cDNA. Furthermore, it is an object of the present application to provide antibodies against SCN12A and SCN8A.
  • This application discloses a purified human sodium channel SCN 12A having the amino acid sequence of SEQ ID NO: 2 and a purified human sodium channel having the amino acid sequence of SEQ ID NO: 4 Provides SCN12A-S. This application also provides a purified human sodium channel SCN8A having the amino acid sequence of SEQ ID NO: 6.
  • the present application relates to a human gene encoding the SCN12A and SCN12A-S, wherein the SCN12A gene present on the third short arm of human chromosome (3p23-p21.3) and the human gene encoding the SCN8A It provides the SCN8A gene, which is located on the long arm of human chromosome 12 (12q13). Furthermore, this application provides a polynucleotide of the human SCN12A gene, which has the nucleotide sequence of SEQ ID NO: 1 and the polynucleotide having the nucleotide sequence of SEQ ID NO: 3.
  • FIG. 1 is a schematic diagram showing a set of primers used for PCR amplification of SCN12A, and an EST sequence and a known sequence on which these primers were synthesized.
  • FIG. 2 is a schematic diagram of the SCN12A full-length cDNA clone.
  • FIG. 3 is a schematic diagram showing the relationship between the structure of each cDNA of SCN12A and SCN12A-S and the clones obtained by RT-PCR and RACE.
  • FIG. 4 shows the results of chromosomal locus identification of SCN12A by FISH.
  • the metaphase chromosome plate shows a hybridization signal of SCN 12A on human chromosome 3p23-p21.3 (arrow).
  • the vertical bar indicates the position of SCN 12A.
  • FIG. 5 shows the results of Northern blot analysis in which the expression of SCN12A in each organ was examined.
  • the upper row shows the results of various adult tissues, and the lower row shows the results of examining each central nervous system tissue.
  • FIG. 6 shows the results of in situ hybridization of SCN12A using rat organs.
  • a DIG-labeled RNA probe was used specifically for SCN12A.
  • Panel F shows the results of SCN12A RT-PCR using human cell lines.
  • the cell lines are glioblastoma U215, neuroblastoma LAN-5 and SHSY-5 ⁇ .
  • FIG. 8 is a schematic diagram showing a set of primers used for PCR amplification of SCN8A and a known sequence from which these primers were synthesized.
  • FIG. 9 is a schematic diagram showing the relationship between the structure of SCN8A cDNA and clones obtained by RT-PCR and RACE.
  • FIG. 10 is a schematic diagram of the SCN8A full-length cDNA clone. BEST MODE FOR CARRYING OUT THE INVENTION
  • the sodium channels SCN12A and SCN12A-S of the present invention are different transcripts expressed from the same gene, respectively.
  • SCN12A is composed of 179 "! Amino acids shown in SEQ ID NO: 2, and comprises SCN12A-S S consists of 1444 amino acids shown in SEQ ID NO: 4 (hereinafter, these two may be simply referred to as “SCN12A”).
  • the sodium channel SCN8A of the present invention is a transcript expressed from the cDNA having the nucleotide sequence shown in SEQ ID NO: 1 and consists of 1980 amino acids shown in SEQ ID NO: 1.
  • Each of SCN12A and SCN8A of the present invention can be isolated from a known method, that is, a method of isolating from human various organs (eg, brain, spleen, small intestine, placenta, spinal cord, etc., as confirmed in the Examples below). It can be obtained by a method of preparing a peptide by chemical synthesis based on the amino acid sequence provided by the application, or by a method of producing by recombinant DNA technology using the polynucleotide provided by this application.
  • a known method that is, a method of isolating from human various organs (eg, brain, spleen, small intestine, placenta, spinal cord, etc., as confirmed in the Examples below). It can be obtained by a method of preparing a peptide by chemical synthesis based on the amino acid sequence provided by the application, or by a method of producing by recombinant DNA technology using the polynucleotide provided by this application.
  • RNA is prepared by in vitro transcription from a vector having the polynucleotide (SEQ ID NO: 1, 3 or 4) of the present invention, and this is used as a type III protein.
  • the SCN12A or SCN8A protein can be obtained by performing in vitro translation as described above.
  • this recombinant vector is used to transform Escherichia coli, Bacillus subtilis, yeast, animal and plant cells, etc., these transformants can be used. Protein can be expressed in large quantities.
  • the translation region of the polynucleotide of the present invention is recombined into a vector having an RNA polymerase promoter, and the RNA polymerase corresponding to the promoter is contained.
  • RNA polymerase promoter examples include T7, T3, and SP6.
  • vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T718, ⁇ 7 / 319, pBluescript II, and the like.
  • an expression vector having an origin, a promoter, a ribosome binding site, a DNA-cloning site, a terminator, and the like that can be replicated in the microorganism is used.
  • the polynucle of the present invention An expression vector may be prepared by recombination of the nucleotide translation region, a host cell may be transformed with the expression vector, and the transformant may be cultured. At this time, by adding a start codon and a stop codon before and after the arbitrary translation region, a protein fragment containing the arbitrary region can be obtained.
  • SCN12A can be expressed as a fusion protein with another protein. By cutting this fusion protein with an appropriate protease, only SCN12A can be obtained.
  • the expression vector for Escherichia coli include a pUC system, a pBluescript pET expression system, and a pGEM expression system.
  • the expression region for a eukaryotic cell having a promoter, a splicing region, a poly (A) addition site, and the like is used as the translation region of the polynucleotide of the present invention. First, it is recombined and introduced into eukaryotic cells.
  • Eukaryotic cells include, but are not limited to, monkey kidney cells COS7, mammalian cells such as Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, African Xenopus egg cells, etc. Not something.
  • known methods such as an electroporation method, a calcium phosphate method, a ribosome method, and a DEAE dextran method can be used.
  • a known separation operation is combined to isolate and purify the target protein from the culture. For example, treatment with denaturing agents such as urea or surfactants, ultrasonic treatment, enzyme digestion, salting out, solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing , Ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.
  • the SCN12A of the present invention includes a peptide fragment (5 or more amino acid residues) containing any partial sequence in the amino acid sequence of SEQ ID NO: 2 or 4.
  • SCN8A also includes a peptide fragment of 5 amino acids or more in the amino acid sequence of SEQ ID NO: 6. These peptide fragments can be used as antigens for producing antibodies.
  • the SCN12A or SCN8A of the present invention also includes a fusion protein with any other protein.
  • the gene of the present invention is a human gene encoding the above-mentioned SCN12A, and is a gene present in the third short arm of human chromosome (3p23-p21.3).
  • Another gene of the present invention is a gene encoding the above-mentioned SCN8A, which is present on the long arm of human chromosome 12 (12q133).
  • genes can be isolated from an existing genomic library using, for example, each polynucleotide of the present invention or its partial sequence as a probe.
  • the polynucleotide (cDNA) of the present invention can be cloned, for example, from cDNA derived from human cells.
  • cDNA is synthesized using poly A + RNA extracted from human cells as type II. Examples of the synthesis method include the Okayama-Ichi Berg method (Mol. Cell. Biol. 2: 161-170, 1982), the Gubler-Hoffman method ( ⁇ Gene 25: 263-269, 1983), and the cabbing method (Gene 150: 243-250). , 1994) can be used.
  • a commercially available human cDNA library can also be used.
  • an oligonucleotide is synthesized based on the nucleotide sequence of an arbitrary portion of the cDNA of the present invention, and this is used as a probe. Screening by one or plaque hybridization may be performed. In addition, oligonucleotides that hybridize to both ends of the cDNA fragment of interest are synthesized, and using this as a primer, the cDNA of the present invention is purified from mRNA isolated from human cells by RT-PCR. It can also be prepared.
  • polynucleotide of SEQ ID NO: 1, 3 or 5 in which one or more nucleotides are added, deleted and / or substituted by other nucleotides are also included in the polynucleotide of the present invention.
  • the polynucleotide of the present invention includes a DNA fragment (10 bp or more) containing any partial sequence of the nucleotide sequence of SEQ ID NO: 1, 3 or 5. It also includes a DNA fragment consisting of a sense strand and an antisense strand. These DNA fragments can be used as probes for gene diagnosis.
  • the antibody of the present invention can be obtained as a polyclonal antibody or a monoclonal antibody by a known method using SCN12A or SCN8A itself or a partial peptide thereof as an antigen.
  • SCN12A or SCN8A itself or a partial peptide thereof as an antigen.
  • the (mRNA) fraction was purified.
  • the mRNA purified in (1) was converted into type ⁇ , and single-stranded DNA was synthesized using a random primer.
  • This one Using the strand DNA as type III, a partial sequence of SCN12A of 1832 bp was obtained by nested PCR.
  • the primer used in this PCR has high homology between the EST sequence AA446997 and the known sodium channel ⁇ subunit (human ⁇ and rat R), and the sequence is well conserved. It was designed based on the part that was.
  • Est-f1 oligonucleotide of SEQ ID NO: 7
  • Est-f2 oligonucleotide of SEQ ID NO: 8
  • Na-r1 oligonucleotide of SEQ ID NO: 9
  • Na-r2 oligonucleotide of SEQ ID NO: 10
  • the single-stranded DNA is highly homologous between known sodium channel ⁇ - subunits (human ⁇ and rat R) as shown in FIG. 8, and the sequence is well conserved.
  • a partial sequence of a sodium channel containing a known sodium channel ⁇ -subunit was obtained by nested PCR using an oligonucleotide designed based on the portion as a primer. One of them, 1806 bp, has a very high homology of 91% in base sequence and 98% in amino acid sequence. It was confirmed that this was a partial sequence of human SCN8A cDNA.
  • a primer was synthesized based on the nucleotide sequence of the SCN12A RT-PCR product obtained in (2) above, and 5′-RACE and 3′-RACE were performed using Marathon-ready cDNA (Clontech) as a type II. CDNA, cDNAs with 5'-side and 3'-side adapters were obtained.
  • the primer for 5'-RACE was Race-5 (oligonucleotide of SEQ ID NO: 11)
  • the primer for 3'-RACE was Race-3a (oligonucleotide of SEQ ID NO: 12) and Race-3b (sequence of SEQ ID NO: 12). No. 13 (oligonucleotide).
  • AP1 oligonucleotide of SEQ ID NO: 14
  • AP2 oligonucleotide of SEQ ID NO: 15
  • 5′-RACE and 3′-RACE were similarly performed on SCN8A to obtain 5′- and 3′-side cDNAs.
  • the 5 'end was obtained by PCR using a primer designed based on the 5' end cDNA sequence.
  • the relationship between the obtained cDNA fragments is as shown in FIG. (4) Subcloning
  • the cDNA fragment of SCN12A obtained by RT-PCR and RACE was subcloned into the plasmid vector pBleuscript IISK (+).
  • the plasmid of the above (4) was propagated and purified, subjected to a re-quenching reaction by a dye terminator and a die primer method, and the reaction product was analyzed by a fluorescent sequencer-377A (Perkin Elmer / ABI).
  • Northern blot was performed using an SCN12A cDNA fragment as a probe, using an RNA plot membrane (manufactured by Clontech) prepared from human various organ poly A + RNA.
  • the probe - were labeled with [32 P] dCTP.
  • the results of the hybridization were analyzed with a Storm 830 image analyzer (Molecukar Dynamics).
  • the chromosomal locus was identified by fluorescence in situ hybridization (FISH).
  • SCN8A cDNA containing full-length ⁇ RF was obtained by RT-PCR, and these were subcloned into the plasmid vector pSP64TR (FIG. 10). .
  • SCN12AcDNA is 6528 bp in length, has an ORF of 5373 bp, and encodes a 1791 amino acid residue protein.
  • SCN 12A-SCDNA is 5728 bp in length, has an ORF of 4332 bp, and encodes a protein with 1444 amino acid residues. All proteins identify four transmembrane domains common to the sodium channel subunit.
  • Table 1 also shows homology with known sodium channel subunits, chromosomal locus, expression site, and relationship with disease.
  • the homology with the known ⁇ -subunit is about 37-73%, which is quite distant from the conventionally known ones.Therefore, SCN12A has a different function from the known sub-unit. It is suggested that
  • SCN12A has a serine amino acid residue in the SS2 segment of the transmembrane domain, as does Scn10a PN3 / SN $ and NaNZSNS2, which are known tetrodotoxin resistant (TTX-R) sodium channels. It was considered to be a TTX-R sodium channel. '
  • the results of Northern blot analysis are as shown in FIG. 5, and the mRNA size of SCN 12A was about 7.0 Kb, and expression in brain, spleen, small intestine, placenta and spinal cord was observed. Expression in other tissues was below the detection sensitivity in Northern blots.
  • known sites of expression of the sodium channel sigma subunit are nervous tissues such as brain, spinal cord and dorsal root ganglion, and muscular tissues such as skeletal muscle, cardiac muscle and uterus.
  • SCN12A is expressed in the brain, spinal cord, spleen, small intestine, and placenta, but hardly expressed in skeletal muscle, extensor muscle, and uterus, and has an expression pattern that is not known. It was confirmed that they were different.
  • SCN12A showed olfactory bulb, granular layer of cerebellum, dentate gyrus of hippocampus, central ependymal cells of spinal cord, spleen Germinal centers and placental vegetative germ layers showed strong expression (Fig. 6).
  • SCN12A was expressed in both nervous cells and glial cells in the nervous system, and human material in the dorsal root ganglion (DRG). Although no RT-PCR band was observed due to the above problem, nested RT-PCR showed that SCN12A was also expressed in the dorsal root ganglion (FIG. 7).
  • the SCN 8A cDNA has a total length of 7053 bp, has an ORF of 5940, and encodes a protein of 1980 amino acid residues.
  • SCN8A four transmembrane domains common to sodium channel ⁇ -subunit are identified.
  • Table 2 shows the homology with the known sodium channel ⁇ -subunit gene cDNA.
  • the homology with the mouse and rat homologous genes was 86% and 94%, respectively.
  • relatively high homology was observed with SCN1A, SCN2A, and SCN3A.
  • this application provides a novel sodium channel subunit and its cDNA. These inventions will contribute greatly to elucidation of the physiological mechanisms involved in excitable cells, identification of the causes of various human diseases, and development of new therapeutic agents.

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Abstract

Cette invention concerne une sous-unité α SCN12A du canal sodium du système nerveux humain ; sa variante d'épissage SNC12A-s ; une autre sous-unité SNC8A du canal sodium humain ; des gènes humains codant pour ces molécules ; et des ADNc des ces gènes. SNC12A, dont la séquence d'acides aminés est SEQ ID NO :1, est codé par un polynucléotide (ADNc) dont la séquence de base est SEQ ID NO :1. SNC12A-s, dont la séquence d'acides aminés est SEQ ID NO :4, est codé par un polynucléotide (ADNc) dont la séquence de base est∫i⊃ ∫/i⊃SEQ ID NO :3. SNC8A, dont la séquence d'acides aminés est SEQ ID NO:6, est codé par un polynucléotide (ADNc) dont la séquence de base est∫i⊃ ∫/i⊃SEQ ID NO : 3∫i⊃.∫/i$m(g)
PCT/JP2000/004629 2000-05-23 2000-07-11 Canal sodium humain scn12a et scn8a WO2001090355A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998038302A2 (fr) * 1997-02-26 1998-09-03 F. Hoffmann-La Roche Ag Sous-unite alpha de canal sodique sensible a la tetrodotoxine
JP2000201684A (ja) * 1999-01-11 2000-07-25 Japan Science & Technology Corp ナトリウムチャンネルｓｃｎ８ａ

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998038302A2 (fr) * 1997-02-26 1998-09-03 F. Hoffmann-La Roche Ag Sous-unite alpha de canal sodique sensible a la tetrodotoxine
JP2000201684A (ja) * 1999-01-11 2000-07-25 Japan Science & Technology Corp ナトリウムチャンネルｓｃｎ８ａ

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Nicholas W. Plummer, et al., "Exon Organization, Coding Sequence, Physical Mapping, and Plymorphic Intragenic Markers for the Human Neuronal Sodium Channel Gene SCN8A", GENOMICS (1998), Vol. 54, No. 2, pages 287-296. *
S. D. DIB-HAJI, et al., "NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy", Proc. Natl. Acad. Sci. USA (1998), Vol. 95, pages 8963-8968. *
Seon-Yong Jeong, et al., "Identification of a Novel Human Voltage-Gated Sodium Channel alpha Subunit Gene, SCN12A", Biochemical and Biophysical Research Communications, (2000, Jan.), Vol. 267, No. 1, pages 262-270. *

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CA2412306A1 (fr) 2001-11-29

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