Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-05-05
2002-08-13
Stucker, Jeffrey (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06432674
ABSTRACT:
TECHNICAL FIELD
The present invention relates to steroid hormone binding proteins, genes thereof, and production and use of the proteins and the genes.
BACKGROUND ART
It is generally thought that steroid hormones exert their physiological influence by regulating transcriptional activities. Very recently, however, steroids that exhibit their activities rapidly without acting on genes have become widely known, but this cannot be explained by the above theory. Evidence of this rapid action of steroids has been shown for every steroid in many species and tissues. Examples include the rapid action of aldosterone on lymphocytes and vascular smooth muscle (Wehling, M. (1995) Cardiovasc. Res. 29(2), 167-171), vitamin D3 on epithelial cells, progesterone on sperm (Revelli, A.; Modotti, M.; Piffaretti-Yanez, A.; Massobrio, M.; and Balerna, M. (1994) Hum Reprod 9 (5), 760-766), neurosteroids on neurons, and estrogen on blood vessels. The signal recognition and transduction mechanisms of these activities are currently being studied. As a result, it is now becoming clear that the signal recognition and transduction system resembles cascade systems of membrane receptors and the second messengers, such as those of catecholamines and peptide hormones, since many of the activities depend on phospholipase C, phosphoinositide turnover, intracellular pH, intracellular calcium, protein kinase C, tyrosine kinases, etc., (Baran, D. T. (1994) J Cell Biochem 56 (3), 303-306; de Boland, A. R. and Nemere, I. (1992) J Cell Biochem 49 (1), 32-36). Although the physiological or pathological relevance is not clear, it has been presumed that the rapid action of steroids can also be observed in vivo in the cardiovascular system, the central nervous system, and the reproductive system. It was expected that these receptors would be cloned soon and that the relationship between the rapid action of steroids and their clinical effects would be clarified (Wehling, M. (1997) Annu Rev Physiol 59, 365-393; and Wehling, M. (1995) J Mol Med 73 (9), 439-447).
In recent years, the progesterone membrane binding protein (PMBP), a membrane binding type that differs from usual steroid hormone receptors of the intranuclear transcription regulation type, was finally cloned for the first time from a pig (Falkenstein, E.; Meyer, C.; Eisen, C.; Scriba, P. C.; and Wehling, M. (1996) Biochem Biophys Res Commun 229 (1), 86-89). This protein was purified from the microsome fraction, has a hydrophobic region near its N terminus, and shows no homology to existing steroid receptors. To date, a putative human homologue of PMBP, the “putative progesterone binding protein gene” (LOCUS, HSPROGBIN; accession number, Acc.Y12711), and a putative rat homologue, “25Dx” (LOCUS, RNU63315; accession number, Acc.U63315), have been isolated. The pig PMBP has been well characterized, and it has been reported to bind not only to progesterone but also to corticosterone, cortisol, promegestone, and testosterone (Meyer C. (1996) Eur. J. Biochem. 239, 726-731).
The discovery of these membrane-bound steroid hormone binding proteins suggests the existence of a mechanism in the organism for regulating hormone action that differs from the one for the receptors involved in the intranuclear transcription regulation. Therefore, it should be possible to develop novel drugs that distinguish the affinities or biological activities of the membrane-bound steroid hormone binding proteins from those of the intranuclear transcription regulation type receptors using the membrane-bound steroid hormone binding proteins.
DISCLOSURE OF THE INVENTION
An objective of the present invention is to provide a novel steroid hormone binding protein having homology to PMBP and its gene, and also methods for producing it and uses thereof.
In order to achieve the above objective, the present inventors discovered ESTs that are inferred to be parts of a cDNA encoding a protein having homology to PMBP (Falkenstein, E.; Meyer, C.; Eisen, C.; Scriba, P. C.; and Wehling, M. (1996) Biochem Biophys Res Commun 229 (1), 86-89), which is a membrane-bound steroid hormone binding protein. The present inventors then extracted a consensus sequence from the sequence information of the ESTs and performed a polymerase chain reaction on human genes using primers designed according to the consensus sequence. As a result, they have succeeded for the first time in isolating the gene encoding a novel steroid hormone binding protein having homology to PMBP from a human.
The present invention relates to a novel steroid hormone binding protein having homology to PMBP and its gene, and also methods for producing it and uses thereof. More specifically, it relates to:
(1) a protein comprising the amino acid sequence of SEQ ID NO:4,
(2) a protein having a steroid hormone-binding activity, comprising the amino acid sequence of SEQ ID NO:4 wherein one or more amino acids are substituted, deleted, and/or added,
(3) a DNA encoding the protein of (1) or (2),
(4) a vector carrying the DNA of (3),
(5) a transformant expressibly retaining the DNA of (4),
(6) a method for producing the protein of (1) or (2), the method comprising culturing the transformant of (5),
(7) an antibody that binds to the protein of (1),
(8) a method for screening a compound that binds to the protein of (1) or (2), the method comprising selecting a compound that binds to the protein of (1) or (2) by contacting a test sample with the protein of (1) or (2),
(9) a compound that binds to the protein of (1),
(10) the compound of (9) isolable by the method of (8),
(11) a method for screening a compound that specifically binds to the protein of (1) or (2) or a steroid hormone receptor of the intranuclear transcription regulation type, the method comprising selecting a compound that specifically binds to the protein of (1) or (2) or the steroid hormone receptor of the intranuclear transcription regulation type by contacting a test sample with the protein of (1) or (2) and the steroid hormone receptor of the intranuclear transcription regulation type,
(12) a compound that specifically binds to either the protein of (1) or (2) or the steroid hormone receptor of the intranuclear transcription regulation type,
(13) the compound of (12) isolable by the method of (11), and
(14) a DNA comprising at least 15 nucleotides, which specifically hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO:3.
The present invention also relates to a protein of human origin, “hSMBP2,” which has homology to the pig membrane-bound progesterone binding protein (progesterone membrane binding protein (PMBP)) (Falkenstein, E.; Meyer, C.; Eisen, C.; Scriba, P. C.; and Wehling, M. (1996) Biochem Biophys Res Commun 229 (1), 86-89). The nucleotide sequence of the hSMBP2 cDNA isolated by the present inventors is shown in SEQ ID NO:3, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO:4.
FIG. 1
compares the amino acid sequence of hSMBP2 and those of proteins having homology to it, pig PMBP, rat 25Dx, and hSMBP1. hSMBP1, like hSMBP2, is a human gene isolated by polymerase chain reaction (PCR) using primers designed according to consensus sequences extracted from multiple ESTs having homology to pig PMBP. A gene identical to hSMBP1 has been registered in a database (Acc. Y12711). As apparent from
FIG. 1
, although hSMBP2 has significant homology to pig PMBP, it has a somewhat lower degree of homology except near its C terminus, unlike hSMBP1, which has a high degree of homology as a whole. hSMBP2 is thus presumed to be a protein belonging to the same family but having a different spectrum of steroids to bind.
The steroid hormone binding protein of the present invention is thought to be a membrane-bound type, which differs from the usual intranuclear transcription regulation receptors. This suggests that it may be involved in regulating hormone action in the organism by a different mechanism than the intranuclear transcription regulation receptors. It therefore may be possible to develop drugs that have few side effects using the steroid hormone
Chugai Seiyaku Kabushiki Kaisha
Fish & Richardson P.C.
Seharaseyon Jegatheesan
Stucker Jeffrey
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