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
1999-10-04
2002-12-24
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S004000, C435S011000, C436S013000, C530S300000, C530S350000
Reexamination Certificate
active
06498019
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel protein having a lecithin-cholesterol acyltransferase-like activity etc. and a DNA coding for the protein.
BACKGROUND ART
Cholesterol is an important lipid constituting the animal cell membrane and defining its character. Moreover, it is a precursor of steroid hormones, thus being a substance essential to animal life. However, due to the recent changes in dietary habit and ecology, arteriosclerosis and other adult diseases arising from pathological intracellular accumulation of cholesterol are now presenting a serious problem so that elucidation of the mechanisms of cholesterol metabolism in the body is being awaited.
In the efflux of cholesterol from the peripheral cells, high density lipoprotein (hereinafter sometimes referred to briefly as HDL) is suspected to play a cardinal role and this assumption has been supported by the epidemiologic finding of an inverse correlation between risk for coronary artery disease and plasma HDL levels and the experimental finding that HDL in culture medium stimulates cholesterol efflux from cells and decreases the intracellular concentration of cholesterol (Journal of Lipid Research, 37, 2473, 1996). In the reverse cholesterol transport system, lecithin-cholesterol acyltransferase (hereinafter sometimes referred to briefly as LCAT) is involved to a significant extent.
LCAT transfers the &bgr;-acyl group (fatty acid) of lecithin (phosphatidylcholine) to the 3&bgr;-OH group of cholesterol, so that it consumes the equivalent moles of lecithin and unesterified cholesterol and produces the equivalent moles of cholesteryl ester and lysolecithin (Journal of Lipid Research, 9, 155, 1968). In the circulation, most of LCAT exists in HDL to show activity and a portion of the cholesteryl ester produced in the HDL is taken up and metabolized in the liver, while another portion of the ester migrates into the nonpolar core of the HDL particle to give rise to a mature HDL rich in cholesteryl esters. Owing to the concentration gradient resulting from the consumption of unesterified cholesterol in HDL, the HDL continuously absorbs cholesterol from the other cell membranes. In this manner, LCAT together with HDL is in charge of the reverse cholesterol transport from peripheral tissues to the liver, thus contributing to the anti-atherosclerotic action of HDL (Biochimica et Biophysica Acta, 1084, 205, 1991).
In familial LCAT deficiency which is an inheritable disease, the reverse cholesterol transport system is lacking so that characteristic tissue damages occur from deposits of cholesterol, leading to coroneal opacity, hemolytic anemia associated with a morphological abnormality of erythrocytes, and proteinuria and renal failure due to kidney impairment (Lancet, 388, 778, 1991). In addition to gene abnormalities, various illnesses involving plasma lipid abnormalities cause changes in LCAT activity. For example, LCAT activity is reportedly elevated by hypercalorism or in obesity and hypertriglyceridemia (Clinical Science, 38, 593, 1970) and decreased in malnutrition, abetalipoproteinemia, and Tangier disease.
LCAT is a 416-residue polypeptide synthesized in the liver, and exists as a glycoprotein with a molecular mass of 59-68 KDa (Journal of Biological Chemistry, 254, 7456, 1979). In the blood, most of LCAT exists in HDL and in the expression of its activity, Apo AI, the principal apoprotein of HDL, acts as the cofactor to stimulate LCAT activity (FEBS Letters, 15, 355, 1971). There exist a variety of mutant LCAT genes corresponding to the variation in enzyme defect and clinical picture in various cases of familial LCAT activity deficiency, and they are discharging significant functions in the metabolism of plasma lipoproteins.
So far, only one kind of LCAT has been reported and the existence of any analogous protein having similar activity has not been predicted.
Meanwhile, in arteriosclerosis, thrombus formation, and post-PTCA restenosis, abnormality of vascular tonus, enhancement of inflammatory reactions, and abnormality of the coagulation-fibrinolysis system, which stem from endothelial cell impairment, occur to cause a remodeling of the blood vessel with the proliferation and transformation of vascular smooth muscle cells as a cardinal pathological picture. The changes on the molecular level which occur in the course of formation of vascular lesions are now being understood in terms of a group of transcription factors controlling the expression of individual genes (Kurabayashi et al., Modern Medicine, 52, 2340, 1997). In such a specifically expressed gene, there is a promoter (enhancer-repressor) sequence which functions only ad hoc and this promoter domain controls the transcription levels of the mRNA encoding the protein.
Some of those promoters are known to be hormone-dependent or growth factor-dependent, and by utilizing them, several drug screening systems and transgenic animals have already been created and actually the systems used in the screening for drugs and the animals used in the analysis of vital functions.
The present invention has for its object to provide a novel protein having LCAT and other activities, a precursor protein thereof, a partial peptide, a salt of either of them, a signal peptide, a DNA coding for said protein, precursor protein, partial peptide or signal peptide, a recombinant vector, a transformant, a method of producing said protein, a pharmaceutical composition comprising said protein or DNA, an antibody against said protein, a screening method/screening kit for a compound promoting or inhibiting the LCAT activity of said protein, a compound obtained by using said screening method, a pharmaceutical composition comprising said compound, a promoter for a novel protein having LCAT and other activities, a screening method/screening kit for a compound promoting or inhibiting the promoter activity, a compound obtained by using the screening method mentioned just above, and a pharmaceutical composition comprising the compound.
The inventors of the present invention did intensive research to accomplish the above-mentioned objects and succeeded in cloning a cDNA having a novel nucleotide sequence from each of human heart-, human kidney-, and mouse kidney-derived cDNA libraries, and discovered that the protein encoded by those cDNA clones is a lecithin-cholesterol acyltransferase-like protein (hereinafter sometimes referred to as the LCAT-like protein). The inventors further cloned the genomic DNA of said LCAT-like protein and by a promoter activity assay, found a promoter for said LCAT-like protein.
DISCLOSURE OF INVENTION
The present invention, provides:
(1) a protein comprising an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or a substantial equivalent thereto, a precursor protein thereof or a salt thereof,
(2) a protein or precursor protein according to the above item (1), which comprises an amino acid sequence represented by any one of SEQ ID NO:4 to SEQ ID NO:8,
(3) a protein or precursor protein according to the above item (1), which possesses lecithin-cholesterol acyltransferase-like activity,
(4) a partial peptide of the protein according to the above item (1), or a salt thereof,
(5) a signal peptide comprising an amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, or a substantial equivalent thereto,
(6) a DNA which comprises a DNA having a nucleotide sequence coding for the protein or precursor protein according to the above item (1),
(7) a DNA according to the above item (6), which comprises a nucleotide sequence represented by any one of SEQ ID NO:12 to SEQ ID NO:19,
(8) a DNA which comprises a DNA having a nucleotide sequence coding for the signal peptide according to the above item (5),
(9) a DNA according to the above item (8), which comprises a nucleotide sequence represented by SEQ ID NO:20 to SEQ ID NO:22,
(10) a recombinant vector comprising the DNA according to the above item (6),
(11) a transformant which is transformed by the recombinant vector according to the above item (10),
(12) a method for
Chao Mark
Foley Shannon A.
Housel James
Ramesh Elaine M.
Takeda Chemical Industries Ltd.
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