Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-02-08
2001-09-11
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S069100, C435S183000, C435S252300, C435S254110, C435S320100, C536S023200
Reexamination Certificate
active
06287834
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the isolation, characterization and use of a novel enzyme which belongs to a family of enzymes which catalyze the transfer of glucuronic acid from uridine diphospho-glucuronic acid to a wide variety of lipid soluble drugs, environmental chemicals and endogenous substances, and more particularly, to the characterization of, and isolation of the cDNA which encodes, a novel uridine diphospho-glucuronosyltransferase (UGT) (hereinafter UGT2B17) which has been found to conjugate androgenic compounds, particularly C
19
steroids. The use of this enzyme in an assay is also described, as are several other uses of the DNA, fragments thereof, antisense fragments thereof and antibodies thereto.
2. Description of the Related Art
The enzymes identified as UGTs are a family of enzymes which catalyze the glucuronidation process in which glucuronic acid is transferred from uridine diphospho-glucuronic acid to a wide variety of lipid soluble drugs, environmental chemicals and endogenous substances such as bilirubin, steroid hormones and thyroxine. Generally, glucuronidation occurs in the liver and kidney and is responsible for the elimination of glucuronide derivatives from the body. However, UGT activity has also been identified in numerous tissues, including prostate, testis, skin, breast, brain and ovary tissues, and in breast and prostate tumor cell lines.
The UGT family of enzymes has been classified into two subfamilies, UGT1 and UGT2. The UGT1 family are generally known to be involved in the glucuronidation of planar and bulky phenol substrates and bilirubin, however, some members of the UGT1 family can conjugate estrogens. Enzymes of the UGT2 family are divided into two subfamilies, UGT2A which includes enzymes encoded by genes expressed in the olfactory epithelium and UGT2B which includes enzymes that catalyze the glucuronidation of bile acids, C
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steroids, C
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steroids, fatty acids, carboxylic acids, phenols and carcinogens, such as benzopyrene and 2-acetylaminofluorene. Several members of the UGT2B family have been isolated from human liver and been characterized. It has been found that there is an overlap in the substrate specificity among the UGT2B enzymes.
The present invention relates to a novel UGT which is a member of the UGT2B family and which is described in detail below.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a novel uridine diphospho-glucuronosyltransferase (UGT) which is identified as UGT2B17.
It is also an object of the present invention to provide a UGT which has been shown to conjugate C
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steroids at the 3&agr;-hydroxy and 17&bgr;-hydroxy groups.
It is an additional object of his invention to provide a UGT which is involved in the conversion of androsterone to androsterone-glucuronic acid.
It is also an object of the present invention to provide a UGT which has specificity for androgenic compounds, particularly C
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steroids compounds including androsterone (ADT), testosterone, dihydrotestosterone (DHT), and androstane-3&agr;, 17&bgr;-diol (3&agr;-DIOL), and for eugenol, 4-methylumbelliferone.
It is a further object of this invention to provide nucleotide sequences for UGT2B17.
It is also an object of this invention to provide methods of using UGT2B17 in an assay to identify compounds which inhibit the activity of this enzyme or using antibodies to UGT2B17 for detecting and quantifying the enzyme.
These and other objects are discussed herein.
In particular, a novel uridine diphospho-glucuronosyltransferase, UGT2B17, has been identified and characterized. The primary protein structure UGT2B17 was found to include 530 amino acids (SEQ ID No. 2) and to have an apparent molecular weight of 53 kilodaltons (when measured by SDS-PAGE). The protein is encoded by nucleotides +52 through 1644, including the stop codon (amino acids 1 through 530), numbered in the 5′ to 3′ direction, in the following sequence (SEQ ID Nos. 1 and 2):
GGCACGAGGA AAGAAACAAC AACTGGAAAA GAAGCATTGC ATAAGACCAG G ATG TCT
57
Met Ser
1
CTG AAA TGG ATG TCA GTC TTT CTG CTG ATG CAG CTC AGT TGT TAC TTT
105
Leu Lys Trp Met Ser Val Phe Leu Leu Met Gln Leu Ser Cys Tyr Phe
5 10 15
AGC TCT GGG AGT TGT GGA AAG GTG CTG GTG TGG CCC ACA GAA TAC AGC
153
Ser Ser Gly Ser Cys Gly Lys Val Leu Val Trp Pro Thr Glu Tyr Ser
20 25 30
CAT TGG ATA AAT ATG AAG ACA ATC CTG GAA GAG CTT GTT CAG AGG GGT
201
His Trp Ile Asn Met Lys Thr Ile Leu Glu Glu Leu Val Gln Arg Gly
35 40 45 50
CAT GAG GTG ATT GTG TTG ACA TCT TCG GCT TCT ATT CTT GTC AAT GCC
249
His Glu Val Ile Val Leu Thr Ser Ser Ala Ser Ile Leu Val Asn Ala
55 60 65
AGT AAA TCA TCT GCT ATT AAA TTA GAA GTT TAT CCT ACA TCT TTA ACT
297
Ser Lys Ser Ser Ala Ile Lys Leu Glu Val Tyr Pro Thr Ser Leu Thr
70 75 80
AAA AAT GAT TTG GAA GAT TTT TTT ATG AAA ATG TTC GAT AGA TGG ACA
345
Lys Asn Asp Leu Glu Asp Phe Phe Met Lys Met Phe Asp Arg Trp Thr
85 90 95
TAT AGT ATT TCA AAA AAT ACA TTT TGG TCA TAT TTT TCA CAA CTA CAA
393
Tyr Ser Ile Ser Lys Asn Thr Phe Trp Ser Tyr Phe Ser Gln Leu Gln
100 105 110
GAA TTG TGT TGG GAA TAT TCT GAC TAT AAT ATA AAG CTC TGT GAA GAT
441
Glu Leu Cys Trp Glu Tyr Ser Asp Tyr Asn Ile Lys Leu Cys Glu Asp
115 120 125
Beaulieu Martin
Belanger Alain
Hum Dean W.
Levesque Eric
Achutamurthy Ponnathapu
Endorecherche Inc.
Rao Manjunath N.
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