WO1998029545A1 - NOUVELLES SEQUENCES DES PROTEINES p56 AFFECTANT LES CANAUX K-ATP - Google Patents

NOUVELLES SEQUENCES DES PROTEINES p56 AFFECTANT LES CANAUX K-ATP Download PDF

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Publication number
WO1998029545A1
WO1998029545A1 PCT/US1997/022522 US9722522W WO9829545A1 WO 1998029545 A1 WO1998029545 A1 WO 1998029545A1 US 9722522 W US9722522 W US 9722522W WO 9829545 A1 WO9829545 A1 WO 9829545A1
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WIPO (PCT)
Prior art keywords
sequence
sequences
chart
nucleic acids
composition
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PCT/US1997/022522
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English (en)
Inventor
Michael J. Bienkowski
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Pharmacia & Upjohn Company
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Priority to JP53003198A priority Critical patent/JP2002514064A/ja
Priority to CA002274310A priority patent/CA2274310A1/fr
Priority to EP97954679A priority patent/EP0951547A1/fr
Priority to AU59570/98A priority patent/AU5957098A/en
Publication of WO1998029545A1 publication Critical patent/WO1998029545A1/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 invention relates to potassium channels and the p56 protein.
  • This invention describes the full length sequence of human p56 protein (p56- 1), a related homolog (p56-2) and the nucleic acids that code for these proteins.
  • the sequences are provided in Charts 1, 2, 3, and 4 and the sequence listings of the application.
  • the protein's entire amino acid sequence of p56 is provided in Chart 1 and the DNA coding for p56 is provided in Chart 2.
  • the DNA and amino acids are disclosed Charts 3 and 4.
  • Equivalents and obvious homologues are disclosed. Also disclosed by reference to PCT/US95/14124, published 30 May 1996 as WO 96/16088, hereby incorporated by reference, are cloning and other useful vectors for the sequence.
  • Type I ATP-sensitive K channels (IK-ATP-* were fi rst described in cardiac muscle (1) and have subsequently been characterized in skeletal muscle (2) vascular smooth muscle (3) and the ⁇ -cell of the pancreas (4-6). These type I channels are inhibited by micomolar concentrations of intracellular ATP and are insensitive to voltage and Ca (7).
  • the ⁇ -cell IR-ATP channel is the most well understood in functional terms and it serves as a metabolic sensor that controls the release of insulin. Glucose stimulation of the ⁇ -cell results in an increase in the intracellular ATP/ADP ratio via glycolysis and the increase in ATP inhibits channel conductance.
  • IR-ATP channels dominate the resting membrane potential of the ⁇ cell, inhibition of channel conductance results in a depolarization that activates voltage- n i , dependent Ca channels and the resultant increase in intracellular Ca triggers insulin release.
  • Pharmacological agents like sulfonylureas [IK_ATP blocker] or diazoxide [IR-ATP opener] stimulate or inhibit insulin release, respectively (8).
  • the IR.ATP channels serve a similar function, coupling cell metabolism and electrical activity. These channels have a low open probability under resting conditions and are activated by a decline in the intracellular ATP/ADP ratio in response to either ischemic injury and/or exercise (2,9).
  • ⁇ K-ATP channels are unique among potassium channels because their activity can be regulated by a wide variety of structurally diverse pharmacological agents (10-15). These include both channel blockers ⁇ eg. sulfonylureas, guanidines and cyanoguanidines) and channel openers (eg. diazoxide, pinacidil, chromakalim, and minoxidil sulfate).
  • channel blockers eg. sulfonylureas, guanidines and cyanoguanidines
  • channel openers eg. diazoxide, pinacidil, chromakalim, and minoxidil sulfate.
  • I- p56 Purification of I- p56 from A10 cells and protein microsequence analysis identified a unique twelve amino acid residue sequence tag derived from the NH2 terminus of I-p56 and this identification was substantiated by the preparation and characterization of an antipeptide antibody that recognizes this sequence tag.
  • This amino acid sequence tag was used to query expressed sequence tag (EST) databases to identify EST clones that potentially encode the human ortholog of the p56 sequence.
  • EST expressed sequence tag
  • Complete sequence analysis of multiple EST clones revealed a full-length cDNA that encoded the human ortholog of rat p56.
  • Human p56-l and p56-2 were characterized by determining their tissue distribution of expression, their glycosylation in vitro and their ability to reconstitute the I-probe
  • Ashcroft, S. J.H. and Ashcroft F.M. "Properties and function of ATP-sensitive K + channels" Cell Signal 2:197-214 (1990).
  • Clone 56-1 contained a 1515 bp open-reading frame that encoded a 505 amino acid polypeptide with a predicted Mr for the mature protein of 52 kDa, close to the expected p56.
  • the predicted sequence contained a signal peptide followed by a mature NH2-terminus that showed 11/14 exact matches with the rat p56 NH2-terminal sequence disclosed in PCT/US95/14124, published 30 May 1996 as WO 96/16088.
  • the predicted polypeptide for clone 56-1 also contained 3 canonical acceptor sites for Asn-linked glycosylation.
  • the predicted amino acid sequence has no significant homology to known sequences.
  • the predicted amino acid from clone 56-1 was analyzed using a variety of secondary structure prediction algorithms.
  • a Rossman fold was detected near the NH2-terminus, indicating that this protein was likely to bind nucleotides like ATP.
  • no predicted transmembrane segments were detected but an endoplasmic reticulum retention signal (KTEL-versus-the canonical KDEL) near the COOH-terminus was scored.
  • KTEL-versus-the canonical KDEL endoplasmic reticulum retention signal
  • a ⁇ -turn- ⁇ membrane association motif similar to the P (pore)-region of voltage-gated-K + -channels, was also detected. It appears that this polypeptide may associate with other K-channel pore-forming polypeptides (e.g.
  • Kir 6.1 or 6.2 and regulate K channel activity. See, Inagaki, N., Tsuura, Y., Namba, N., Masuda, K, Gonoi, T., Horie, M., Seino, Y., Mizuta, M. and Seino, S. "Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle and heart" J. Biol. Chem. 270: 5691-5694 (1995). AND.
  • sequences can also be used in screens and assays for the detection of biologically active compounds. Additional descriptions of, procedures for and examples of these types of vectors, plasmids, cells, screens and assays can be found in case 6001. NCP, serial number 08/709,923 filed 9 September 1996, hereby incorporated by reference. In particular page 16 relating to procedures for preparing and using clones in assays especially relevant and incorporated by reference. In a similar manner additional descriptions of, procedures for and examples of these types of vectors, plasmids, cells, screens and assays can be found in WO 94/19464, PCT/US94/01210, published 1 September 1994, hereby incorporated by reference. In particular, the pages relating to procedures for preparing and using clones in assays is especially relevant and incorporated by reference.
  • the second p56 sequence was discovered according to the following procedures. Various data bases were queried with the amino acid sequence of human p56 using the FASTA search tool. In addition to identifying the known EST matches to p56, three additional ESTs that shared approximately 38-48% identity with p56 were also scored [447210, 2607571 and 2663551]. Alignment of the 5' EST sequence reads for these 3 clones with the p56 sequence showed that it was unlikely that the EST sequences overlapped and that clone 2607571 was most likely to be full-length. These clones were obtained and clone 2607571 was completely sequenced.
  • This clone contained a 2.6 kb insert complete with a 1482 bp open reading frame that showed 41% shared identity with human p56. Motifs that were common to both predicted amino acid sequences include (1) a signal sequence, (2) Rossman fold, (3) canonical acceptor sites for Asn-linked glycosylation [3 in p56-l and 6 in p56-2] and (4) an ER retention signal at the COOH-terminus [KTEL].
  • Each expression plasmid was linearized by digestion with Notl and capped cRNA synthesized using T7-RNA polymerase. These cRNAs were then used to direct the synthesis of S-methionine labeled protein using rabbit reticulocyte lysates ⁇ canine pancreatic microsomes. The radiolabeled proteins were fractionated by SDS-PAGE and visualized by fluorography of the dried gel. P56-1 and p56-2 have unglycosylated M_ values of 52 kDa and 50 kDa, respectively. In the presence of canine pancreatic microsomes, multiple bands with higher M_ values, including 56kDa, were also detected, indicating the addition of Asn-linked oligosaccharides to p56-l. In contrast to p56-l, p56-2 did not appear to be glycosylated under these conditions.
  • the blots were then visualized by hybridization to coding sequence DNA probes prepared from either human p56-l or human p56-2.
  • the p56-l probe visualized a 6.0 kb transcript that was expressed at the highest level in skeletal muscle and lower levels detected in heart and pancreas. Minor signal was also detected in brain, placenta and liver while no transcript was detected in either lung or kidney under these conditions.
  • BLAST searching revealed 20 ESTs derived from 11 different tissues matching the query with the complete p56-l DNA sequence.
  • ESTs 5 from breast (4 normal/1 tumor), 3 from prostate (1 normal/2 tumor), 2 from brain, 2 from colon tumors, 2 from kidney and single ESTs from stomach, uterus, pituitary, nasal polyp, thyroid and mononuclear cells.
  • the human p56-2 probe visualized a 2.8 kb transcript that was expressed at the highest level in heart, brain, pancreas and placenta. No signal for p56-2 was observed in lung, liver, skeletal muscle or kidney under these conditions.
  • p56-2 transcripts were widely expressed in brain regions, with the highest levels in the amygdala and lower levels in the hippocampus, the caudate nucleus, the corpus callosum, the substantia nigra and the subthalamic nucleus. Twenty ESTs matched the p56-2 query sequence and these were derived from 13 different tissues including 3 from pancreas, 3 from lung tumors, 2 from synovial membrane, 2 from leukocytes, 2 from hippocampus and single EST matches from heart, kidney, bladder, small intestine, adrenal, breast, prostate and nasal polyp.
  • Chart 1 provides the sequences for the full length of the p56-l protein.
  • the sequences in Chart 1 include the signal or leader sequence.
  • the sequences in Chart 1 are also in sequence listing no. 1.
  • Chart 2 provides the cDNA residues that code for the p56-l protein and it includes untranslated sequences. All of the sequences in Chart 2 are provided for in sequence listing no. 2.
  • the p56-l DNA sequence shown in Chart 2 contains one of many possible lengths of poly A tail, included as part of the cDNA sequence.
  • This full-length cDNA contains: 1) a 5' untranslated sequence (alignment positions 1-39), 2) a coding sequence (alignment positions 40-1554), 3) a stop codon "TGA” (alignment position 1555-1557) and 4) a 3' untranslated sequence (alignment positions 1555-1724 with poly A).
  • the coding sequence beginning with ATG at position 40 starts with a signal sequence that ends at position 123 while the mature sequence begins at position 124. These positions are noted on Charts 1 and 2 below.
  • the signal sequence or leader sequence is a hydrophobic region, usually about 20-25 amino acids, here 28 aa, at the N-terminus that 'signals' attachment of the ribosome to the endoplasmic reticulum and aids in the extrusion of the nacent polypeptide chain into the lumen of the ER.
  • This signal sequence is cleaved off in the lumen by the signal peptidase.
  • the untranslated sequences may have important regulatory functions such as governing mRNA stability and the like.
  • the poly A track is added after transcription, the length is variable, often from 10-200 A's, here we show a 27 track poly A.
  • Charts 3 and 4 disclose the p56-2 proteins and the DNA that code for those proteins.
  • Chart 3 provides the sequences for the full length of the p56-2 protein.
  • the sequences in Chart 3 include the signal or leader sequence.
  • the sequences in Chart 3 are also in sequence listing no. 3.
  • Chart 4 provides the cDNA residues that code for the p56-2 protein and it includes untranslated sequences. All of the sequences in Chart 2 are provided for in sequence listing no. 4.
  • the p56-2 DNA sequence shown in Chart 4 contains one of many possible lengths of poly A tail, included as part of the cDNA sequence.
  • This full-length cDNA contains: 1) a 5' untranslated sequence (alignment positions 1-35), 2) a coding sequence (alignment positions 36-1517), 3) a stop codon "TGA” (alignment position 1518-1520) and 4) a 3' untranslated sequence (alignment positions 1518-2567 with poly A).
  • the coding sequence beginning with ATG at position 36 starts with a signal sequence. These positions are noted on Charts 3 and 4 below.
  • Chart 3 Amino acid sequence of the p56-2 protein.
  • Chart 4 Nucleotide sequence of the coding region for the p56-2 protein .
  • ADDRESSEE Pharmacia and Upjohn, Co., Intel. Prop. Law (1920-32-LAW)
  • Ala Ala Ala Ala Ala Ala Gly Gly Asp Ala Pro Pro Gly Lys lie Ala Val Val 20 25 30
  • Gly Ala Gly lie Gly Gly Ser Ala Val Ala His Phe Leu Gin Gin H s 35 40 45 Phe Gly Pro Arg Val Gin lie Asp Val Tyr Glu Lys Gly Thr Val Gly 50 55 60
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • AGGAGGTCAT GGAGAAGTTC ATGAGGATCT ATAAGTACCA GGCCCACGGC TATGCCTTCT 540
  • GCAACAGCTC TGACTTCTAT GACATCGTGG TCATCGCCAC CCCCCTGCAC CTGGACAACA 960 GCAGCAGCAA CTTAACCTTT GCAGGCTTCC ACCCGCCCAT TGATGACGTG CAGGGCTCTT 1020
  • GCTCCCGCCC CACGCTCCCG AGGTTTGCAC TCCATGACCA GCTCTTCTAC CTCAATGCCC 1380
  • CTTCAGACTT GGTTTCTTAG CTAGAAACCA GAAGACTACG GGAGGGAATA TAAGGCAGAG 1860
  • CTCCAAGCTT CCTGGCAACC AGTGGGAAAA GAAACATGCG AGGCTGTAGG AAGAGGGAAG 2220

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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne la séquence complète de la protéine humaine p56 (p56-1), un homologue apparenté (p56-2) et les acides nucléiques codant pour ces protéines. Lesdites séquences sont décrites dans les diagrammes 1, 2, 3 et 4, ainsi que dans les listes des séquences de la présente demande.
PCT/US1997/022522 1996-12-30 1997-12-18 NOUVELLES SEQUENCES DES PROTEINES p56 AFFECTANT LES CANAUX K-ATP WO1998029545A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP53003198A JP2002514064A (ja) 1996-12-30 1997-12-18 Ｋ−ＡＴＰチャンネルに作用する蛋白質ｐ５６の新規配列
CA002274310A CA2274310A1 (fr) 1996-12-30 1997-12-18 Nouvelles sequences des proteines p56 affectant les canaux k-atp
EP97954679A EP0951547A1 (fr) 1996-12-30 1997-12-18 NOUVELLES SEQUENCES DES PROTEINES p56 AFFECTANT LES CANAUX K-ATP
AU59570/98A AU5957098A (en) 1996-12-30 1997-12-18 Novel sequences of p56 proteins which affect k-atp channels

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3556096P 1996-12-30 1996-12-30
US60/035,560 1996-12-30

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WO1998029545A1 true WO1998029545A1 (fr) 1998-07-09

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072811A2 (fr) * 2001-03-13 2002-09-19 Bayer Aktiengesellschaft Regulation de la proteine de type lyase prenylcysteine humaine

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994019464A2 (fr) * 1993-02-19 1994-09-01 The Upjohn Company Sequence d'adn humain codant une vanne a potassium dependant de l'atp du rein
WO1996016088A1 (fr) * 1994-11-17 1996-05-30 Pharmacia & Upjohn Company Proteine agissant sur les canaux katp

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994019464A2 (fr) * 1993-02-19 1994-09-01 The Upjohn Company Sequence d'adn humain codant une vanne a potassium dependant de l'atp du rein
WO1996016088A1 (fr) * 1994-11-17 1996-05-30 Pharmacia & Upjohn Company Proteine agissant sur les canaux katp

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DUBRAY C ET AL: "Pharmacological characterisation of endogenous and recombinant K-ATP channels using voltage-sensitive dyes and novel instrumentation.", FASEB JOURNAL, vol. 10, no. 3, 8 March 1996 (1996-03-08), pages A137, XP002064581 *
ISOMOTO S ET AL.: "A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K-channel", J. BIOL. CHEM., vol. 271, no. 40, 4 October 1996 (1996-10-04), pages 24321 - 24324, XP002064580 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072811A2 (fr) * 2001-03-13 2002-09-19 Bayer Aktiengesellschaft Regulation de la proteine de type lyase prenylcysteine humaine
WO2002072811A3 (fr) * 2001-03-13 2004-07-15 Bayer Ag Regulation de la proteine de type lyase prenylcysteine humaine

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NZ513844A (en) 2001-09-28
JP2002514064A (ja) 2002-05-14
EP0951547A1 (fr) 1999-10-27
CA2274310A1 (fr) 1998-07-09
AU5957098A (en) 1998-07-31

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