DE10019119A1 - New leukotriene Bx receptor gene, LTBx, useful for diagnosis and treatment of LTBx-related diseases - Google Patents

Abstract

The human LTBx gene (I) of 5750 bp, including 5' and 3'-untranslated regions, reproduced. (I) overlaps the leukotriene B4 receptor on chromosome 14, strongly implying common transcriptional regulation of both genes. Independent claims are also included for the following: (1) transcription factors, RNA polymerases, pharmaceuticals and chemicals that modify, positively or negatively, expression of (I); (2) mRNA transcribed from (I), including splice variants and isoforms; (3) cDNA derived from the mRNA, or the intronless version of (I); (4) protein (II), and its derived peptides, derived or produced from (I), its cDNA or mRNA; (5) (monoclonal) antibodies and antisera raised against (II) or one or more of its epitopes; (6) expression systems that produce natural or recombinant (II); (7) ligand-binding studies and screening assays on native or recombinant receptors, or cells or cell membranes that contain them; (8) transgenic and knockout animals which express (II), or their splice variants, at altered levels or not at all; (9) gene therapy methods (and their development or use) based on (II) and the corresponding gene, mRNA or cDNA; and (10) (anti)sense oligonucleotides derived from (I) and their use; and (l) diagnosis and treatment of diseases that are (in)directly associated with (II). In all cases, the protein, gene, mRNA or cDNA can be modified, e.g. by amino acid or base exchanges.

Description

Invention description: LTBx

The amino acid sequence of a G protein-coupled receptor was used to Homology search in a database of a potential gene for a new G protein coupled receptor (GPCR) identified on human chromosome 14. From the Gene sequences were used to amplify the potential receptor gene and oligonucleotides its derived cDNA (mRNA) sequence prepared and for the PCR amplification of the Gene and the cDNA used. By means of these primers, the intronless gene from human genomic DNA can be cloned and sequenced.

The gene to be patented spans 5750 bases. The coding area of the gene (Items 3372 to 4538) consists of an open reading frame of 1167 bases and thus codes a protein of 389 amino acids. Hydrophobicity analysis of the amino acid sequence reveals that it is a G protein-coupled receptor with seven transmembrane domains acts. The amino acid sequence is new and previously unknown and shows the best homology (44% identity and 56% homology) to the human P2Y7 purinoceptor or to the human Leukotriene B4 receptor and it also has a relatively high homology to human Somatostatin-4 receptor and opioid receptors.

Furthermore, the patented sequence includes 3371 bases of the 5 'non-translated Region of the gene which presumably contains the promoter and 2379 bases of the 3 ' untranslated region of the gene which has a typical polyadenylation signal (AATAAA) included in headings 5018 to 5023.

We gave the receptor the name LTBx.

2. The expression of this gene was determined by means of polymerase chain reaction (PCR) and primers (sense: 5 'GAAGTAAGGAGGAGGCATG 3'; antisense: 5 ' GGTTGTAGGGTCTGCTGTC 3 ') which is the coding region of the LTBx gene flank proven. The following temperature program was used for the PCR: 94 ° C 1 min, 56 ° C 1 min, 72 ° C 3 min, 35 cycles. We were able to do the full coding cDNA (open reading frame) of the LTBx receptor from a human B cell cDNA bank reproduce. This is the expression of the mRNA of this receptor in this tissue clearly proven. PCR with genomic DNA and these primers resulted in a band of Identical size and sequencing clearly demonstrated that the gene was intronless is. Furthermore, homology searches in databases of expressed Sequence pieces of human genes (EST = expressed sequence tags) with expressed sequences high homology from human lungs, placenta, retina and testes.  

Character: MBHB-RECIPE-10 (continued description)

3. The amino acid sequence of the LTBx receptor derived from the cDNA sequence is 389 Amino acids long. Hydrophobicity analysis of the amino acid sequence gives a putative Secondary structure of the protein as an integral membrane protein with probably seven transmembrane domains. The protein contains a potential N-glycosylation site in the N- terminal area (amino acid positions 41). Furthermore, there is a in the amino acid sequence contain potential protein kinase C phosphorylation sites (amino acid positions 246), their optional phosphorylation may be involved in the modulation of the receptor function can. A potential casein kinase II is located in position 322 of the amino acid sequence Phosphorylation site. A typical heme is located in amino acid positions 199 to 204. Binding motif of the cytochrome C family, which binds a heme iron complex can

4. Classification and potential functions of the patented LTBx receptor and of his gene (or cDNA; mRNA)

The receptor belongs to the large gene amile of G protein-coupled receptors (GPCRs). Within this large family he belongs to the sub-family of the "Class A rhodopsin-like" Receptors. It has the highest homology to the leukotriene B4 receptor and homology Purinoreceptors (especially P2Y type 7, which is apparently identical to the leukotriene B4 receptor is in the literature and in gene databases) to somatostatin receptors (especially type 4) and low homology to opioid receptors. It is most likely that the receptor belongs to the group called "chemoattractant belongs to a family of receptors (e.g. leukotriene B4, N-formyl peptide and Platelet activating factor receptors), which, after activation by the appropriate Agonists, chemotactic and cytotoxic functions such as B. Neutrophil migration mediate against intruders / inflammation of the body (immune response). The gene for that The patentable LTBx receptor is located on chromosome 14 in the immediate vicinity of the gene for the leukotriene B4 receptor, with which it even partially overlaps. One is likely of the two genes were created by gene duplication from the other during evolution. The 3 'end of the LTBx receptor gene to be patented can simultaneously be 5' untranslated region of the gene for the leukotriene B4 receptor act. Since the Sequence of the leukotriene B4 receptor identical to the sequence of the purinoreceptor P2Y7 which is also located on chromosome 14 and has nanomolar affinity for ATP (Adenosine triphosphate), it may be that the receptor to be patented also by ATP can be activated and, like the P2Y7 receptor, regulates cardiac Muscle contraction is involved.

Finding ESTs of the gene / receptor to be patented in human lung, placenta, However, retina and testes as well as in B lymphocytes speak for a function analogous to that Leukotriene B4 receptor.  

MBHB-RECIPE-10: amino acid sequence LTBx

MBHB Recipe-10: LTBx cDNA sequence

Claims (16)

1. The gene shown including the 5 'and 3' untranslated region and the finding, that the LTBx receptor gene matches the gene for the leukotriene B4 receptor Chromosome 14 overlaps, which is a likely common regulation of the Transcription of the two genes implies. 2. Transcription factors, RNA polymerases and pharmaceuticals as well as chemicals that make up the Affect gene expression in a positive or negative manner. 3. The messenger RNA transcribed from the gene, including that derived from it Splice variants and isoforms. 4. The cDNA derived from the mRNA or from the intronless gene. 5. The protein derived or produced from the mRNA (or the gene or the cDNA), and peptides derived therefrom. 6. Antibodies or antisera which are directed against one or more epitopes of the protein or against the whole protein. 7. Monoclonal antibodies or antisera against one or more epitopes of the Protein or against the whole protein. 8. Expression systems (eukaryotic cell lines, yeast cells, Xenopus oocytes, insect cell Systems, baculovirus systems, bacterial expression systems) which contain the protein mentioned express (native or recombinant). 9. Ligand binding studies and screening assays on native or recombinant receptors or cells or cell membranes that contain this receptor. 10. Transgenic animals and knock-out animals, which these (or the corresponding Species variant) Express receptor in changed density or not at all. 11. Methods of gene therapy which relate to this receptor or its gene or its extend mRNA (cDNA) and their development and application. 12. Sense and antisense oligonucleotides derived from this gene as well their application. 13. The diagnosis and treatment of diseases with this receptor in direct or communicating indirectly. 14. Methods for diagnosing diseases associated with this receptor (or its gene, mRNA) are connected in a direct or indirect manner such. B. Hybridization techniques, sequencing, SSCP, RFLP, Northern blot, Southern blot, Western blot, expression arrays, antibodies, mutation analyzes. 15. The use of the information or the expressed receptor to develop new ones Pharmaceuticals, compounds and chemicals and the evaluation of existing ones Pharmaceuticals, compounds and chemicals as well as for the development of new technologies or evaluation of existing technologies. 16. The patent should also extend to points 1 to 15 for modified proteins, and genes, cDNA and mRNA sequences (amino acid changes, base changes).