WO1997044466A1

WO1997044466A1 - Characterization and use of an isolated uridine diphospho-glucuronosyltransferase

Info

Publication number: WO1997044466A1
Application number: PCT/CA1997/000328
Authority: WO
Inventors: Alain Belanger; Dean W. Hum; Martin Beaulieu; Eric Levesque
Original assignee: Endorecherche, Inc.
Priority date: 1996-05-17
Filing date: 1997-05-16
Publication date: 1997-11-27
Also published as: US6287834B1; AU2687897A; EP0904382A1; JP2000511046A; CA2255712A1

Abstract

A uridine diphospho-glucuronosyltransferase (UGTB217) is provided. Methods of producing the enzyme and using the enzyme to identify potential compounds which inhibit or alter the activity of the enzyme are described. In addition, methods of using antibodies to localize the protein, or using the gene sequence or portions thereof for probes or using the gene sequence to produce expression-disrupting sense or antisense DNA fragments thereof, or antisense RNA, are provided.

Description

CHARACTERIZATION AND USE OF AN ISOLATED URIDINE DIPHOSPHO-GLUCURONOSYLTRANSFERASE

BACKGROUND OF THE INVENTION

Field ofthe 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 UGT2B 17) which has been found to conjugate androgenic compounds, particularly C,qSteroids. 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.

Description ofthe 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,

UGTl and UGT2. The UGTl family are generally known to be involved in the glucuronidation of planar and bulky phenol substrates and bilirubin. however, some members of the UGTl 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_|()steroids, C₁₈steroids, 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-giucuronosyltransferase (UGT) which is identified as UGT2B 17.

It is also an object of the present invention to provide a UGT which has been shown to conjugate C,₀ steroids at the 3α-hydroxy and 17β-hydroxy groups.

It is an additional object of this 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,₉ steroids compounds including androsterone (ADT), testosterone, dihydrotestosterone (DHT), and androstane-3α, 17β- diol (3α-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 UGT2B 17 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 Gin 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 He Asn Met Lys Thr He Leu Glu Glu Leu Val Gin 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 He Val Leu Thr Ser Ser Ala Ser He 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 He 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 He Ser Lys Asn Thr Phe Trp Ser Tyr Phe Ser Gin Leu Gin 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 He Lys Leu Cys Glu Asp 115 120 125 130

GCA GTT TTG AAC AAG AAA CTT ATG AGA AAA CTA CAA GAG TCA AAA TTT 489

Ala Val Leu Asn Lys Lys Leu Met Arg Lys Leu Gin Glu Ser Lys Phe 135 140 145

GAT GTC CTT CTG GCA GAT GCC GTT AAT CCC TGT GGT GAG CTG CTG GCT 537

Asp Val Leu Leu Ala Asp Ala Val Asn Pro Cys Gly Glu Leu Leu Ala

150 155 160

GAA CTA CTT AAC ATA CCC TTT CTG TAC AGT CTC CGC TTC TCT GTT GGC 585 Glu Leu Leu Asn He Pro Phe Leu Tyr Ser Leu Arg Phe Ser Val Gly 165 170 175

TAC ACA GTT GAG AAG AAT GGT GGA GGA TTT CTG TTC CCT CCT TCC TAT 633 Tyr Thr Val Glu Lys Asn Gly Gly Gly Phe Leu Phe Pro Pro Ser Tyr 180 185 190

GTA CCT GTT GTT ATG TCA GAA TTA AGT GAT CAA ATG ATT TTC ATG GAG 681 Val Pro Val Val Met Ser Glu Leu Ser Asp Gin Met He Phe Met Glu 195 200 205 210

AGG ATA AAA AAT ATG ATA TAT ATG CTT TAT TTT GAC TTT TGG TTT CAA 729 Arg He Lys Asn Met He Tyr Met Leu Tyr Phe Asp Phe Trp Phe Gin 215 220 225 GCA TAT GAT CTG AAG AAG TGG GAC CAG TTT TAT AGT GAA GTT CTA GGA 777 Ala Tyr Asp Leu Lys Lys Trp Asp Gin Phe Tyr Ser Glu Val Leu Gly 230 235 240

AGA CCC ACT ACA TTA TTT GAG ACA ATG GGG AAA GCT GAA ATG TGG CTC 825 Arg Pro Thr Thr Leu Phe Glu Thr Met Gly Lys Ala Glu Met Trp Leu 245 250 255

ATT CGA ACC TAT TGG GAT TTT GAA TTT CCT CGC CCA TTC TTA CCA AAT 873 He Arg Thr Tyr Trp Asp Phe Glu Phe Pro Arg Pro Phe Leu Pro Asn 260 265 270

GTT GAT TTT GTT GGA GGA CTT CAC TGT AAA CCA GCC AAA CCC TTG CCT 921 Val Asp Phe Val Gly Gly Leu His Cys Lys Pro Ala Lys Pro Leu Pro

275 280 285 290

AAG GAA ATG GAA GAG TTT GTG CAG AGC TCT GGA GAA AAT GGT ATT GTG 969 Lys Glu Met Glu Glu Phe Val Gin Ser Ser Gly Glu Asn Gly He Val 295 300 305

GTG TTT TCT CTG GGG TCG ATG ATC AGT AAC ATG TCA GAA GAA AGT GCC 101^' Val Phe Ser Leu Gly Ser Met He Ser Asn Met Ser Glu Glu Ser Ala 310 315 320

AAC ATG ATT GCA TCA GCC CTT GCC CAG ATC CCA CAA AAG GTT CTA TGG 1065 Asn Met He Ala Ser Ala Leu Ala Gin He Pro Gin Lys Val Leu Trp 325 330 335 AGA TTT GAT GGC AAG AAG CCA AAT ACT TTA GGT TCC AAT ACT CGA CTG 1113 Arg Phe Asp Gly Lys Lys Pro Asn Thr Leu Gly Ser Asn Thr Arg Leu 340 345 350

TAT AAG TGG TTA CCC CAG AAT GAC CTT CTT GGT CAT CCC AAA ACC AAA 1161 Tyr Lys Trp Leu Pro Gin Asn Asp Leu Leu Gly His Pro Lys Thr Lys 355 360 365 370

GCT TTT ATA ACT CAT GGT GGA ACC AAT GGC ATC TAT GAG GCG ATC TAC 1209 Ala Phe He Thr His Gly Gly Thr Asn Gly He Tyr Glu Ala He Tyr 375 380 385

CAT GGG ATC CCT ATG GTG GGC ATT CCC TTG TTT GCG GAT CAA CAT GAT 1257 His Gly He Pro Met Val Gly He Pro Leu Phe Ala Asp Gin His Asp 390 395 400

AAC ATT GCT CAC ATG AAA GCC AAG GGA GCA GCC CTC AGT GTG GAC ATC 130: Asn He Ala His Met Lys Ala Lys Gly Ala Ala Leu Ser Val Asp He 405 410 415 AGG ACC ATG TCA AGT AGA GAT TTG CTC AAT GCA TTG AAG TCA GTC ATT 1353 Arg Thr Met Ser Ser Arg Asp Leu Leu Asn Ala Leu Lys Ser Val He 420 425 430

AAT GAC CCT ATC TAT AAA GAG AAT ATC ATG AAA TTA TCA AGA ATT CAT 1401 Asn Asp Pro He Tyr Lys Glu Asn He Met Lys Leu Ser Arg He His 435 440 445 450

CAT GAT CAA CCG GTG AAG CCC CTG GAT CGA GCA GTC TTC TGG ATT GAG 1449 His Asp Gin Pro Val Lys Pro Leu Asp Arg Ala Val Phe Trp He Glu 455 460 465

TTT GTC ATG CGC CAT AAA GGA GCC AAG CAC CTT CGG GTC GCA GCC CAC 149" Phe Val Met Arg His Lys Gly Ala Lys His Leu Arg Val Ala Ala His 470 475 480

AAC CTC ACC TGG ATC CAG TAC CAC TCT TTG GAT GTG ATA GCA TTC CTG 1545 Asn Leu Thr Trp He Gin Tyr His Ser Leu Asp Val He Ala Phe Leu 485 490 495 CTG GCC TGC GTG GCA ACT ATG ATA TTT ATG ATC ACA AAA TGT TGC CTG 1593 Leu Ala Cys Val Ala Thr Met He Phe Met He Thr Lys Cys Cys Leu 500 505 510

TTT TGT TTC CGA AAG CTT GCC AAA ACA GGA AAG AAG AAG AAA AGG GAT 1641 Phe Cys Phe Arg Lys Leu Ala Lys Thr Gly Lys Lys Lys Lys Arg Asp 515 520 525 530

TAG TTATATCAAA AGCCTGAAGT GGAATGACCA AAAGATGGGA CTCCTCCTTT 1694

ATTCCAGCAT GGAGGGTTTT AAATGGAGGA TTTCCTTTTT CCTGCGACAA AACGTCTTTT 1754

CACAACTTAC CCTGTTAAGT CAAAATTTAT TTTCCAGGAA TTTAATATGT ACTTTAGTTG 1814

GAATTATTCT ATGTCAATGA TTTTTAAGCT ATGAAAAATA ATAATATAAA ACCTTATGGG 1874

CTTATATTGA AATTTATTAT TCTAATCCAA AAGTTACCCC ACACAAAAGT TACTGAGCTT 1934 CCTTATGTTT CACACATTGT ATTTGAACAC AAAACATTAA CAACTCCACT CATAGTATCA 1994

ACATTGTTTT GCAAATACTC AGAATATTTT GGCTTCATTT TGAGCAGAAT TTTTGTTTTT 2054

AATTTTGCCA ATGAAATCTT CAATAATTAA AAAAAAAAAA AAAAAAAAAA AAA 2107

The open reading frame of 1590 bases is flanked by a 5'- untranslated region of 51 base pairs and a 3'- untranslated region of 463 base pairs. A more detailed description ofthe

sequences will be provided infra. The present invention includes methods for the synthetic production of

UGT2B17, as well as peptides that are biologically functionally equivalent, antibodies to UGT2B17, and uses ofthe antibodies to detect and quantify the enzyme.

The nucleotide sequence which encodes UGT2B17 and recombinant expression vectors which include the sequence may be modified so long as they continue to encode a functionally equivalent enzyme. Moreover, it is contemplated, within the invention, that codons within the coding region may be altered, inter alia, in a manner which, given the degeneracy of the genetic code, continues to encode the same protein. It is believed that nucleotide sequences analogous to SEQ ID No. 1. or those that hybridize under stringent conditions to the coding region of SEQ ID No. 1 (or its complement), are likely to encode a UGT2B17 functionally equivalent to that encoded by the coding region of SEQ ID No. 1, especially if such analogous nucleotide sequence is at least 1500 nucleotides. and most preferably at least 1590 nucleotides in length. As

used herein, except where otherwise specified, "stringent conditions" means O.lx SSC

(0.3 M sodium chloride and 0.03M sodium citrate) and 0.1% sodium dodecyl sulphate (SDS) and 60° C.

It is also likely that tissues or cells from human or non-human sources.

particularly tissues or cells which include steroids, include a UGT2B17 sufficiently analogous to human UGT2B17 to be used in accordance with the present invention. In particular, cDNA libraries prepared from cells, as described above, may be screened

with probes in accordance with well known techniques prepared by reference to the nucleotides disclosed herein, and under varying degrees of stringency, in order to identify analogous cDNAs in other species. These analogous cDNAs are preferably at least 92% homologous to SEQ ID No. 1, and most preferably at least 95% homologous.

They preferably include stretches of perfect identity at least 10 nucleotides long, more preferably stretches of 15, 20 or even 30 nucleotides of perfect identity. Appropriate probes may be prepared from SEQ ID No. 1 or fragments thereof of suitable length. preferably at least 15 nucleotides in length. Confirmation with at least two distinct probes is preferred. Alternative isolation strategies, such as polymerase chain reaction

(PCR) amplification, may also be used. Homologous UGT2B17s so obtained, as well as the genes encoding them, can be used in accordance with the invention in all ofthe ways for using SEQ ID

No. 2 and SEQ ID No. 1, respectively.

Recombinant expression vectors can include the entire coding region for UGT2B17 as shown in SEQ ID No. 1. the coding region for UGT2B17 which has been modified as discussed herein, portions of the coding region for human UGT2B17 or analogous coding regions from other animals as described above, an antisense construct

to UGT2B17. or portions of antisense constructs to UGT2B17. In the context of the invention, "isolated" means having a higher purity than exists in nature, but does not require purification from a natural source. Isolated nucleotides encoding UGT2B 17 may be produced synthetically, or by isolating cDNA obtained from a cDNA library prepared from mRNA encoding UGT2B17. or by any other method known in the art. In one embodiment, the invention provides an isolated nucleotide sequence encoding uridine diphospho-giucuronosyltransferase 2B17. said sequence being, sufficiently homologous to SEQ ID No. 1 or a complement thereof, to hybridize under stringent conditions to the coding region of SEQ ID No. 1 or a complement thereof and said sequence encoding an enzyme which catalyzes the conversion of androsterone to androsterone-glucuronic acid.

In a further embodiment, the invention provides an isolated nucleotide sequence comprising at least thirty consecutive nucleotides identical to thirty consecutive nucleotides in the coding region of SEQ ID No. 1. or the complement thereof. In an additional embodiment, the invention provides a nucleotide sequence comprising nucleotides 52 to 927 of SEQ ID No. 1.

In another embodiment, the invention provides a nucleotide sequence comprising nucleotides 204 to 723 of SEQ ID No. 1. In a further embodiment, the invention provides an isolated nucleotide sequence comprising SEQ ID No. 1.

In a further embodiment, a protein encoded by SEQ ID No. 1 is provided.

In another embodiment, isolated nucleic acid which encodes SEQ ID No.

2 is provided.

In a further embodiment, an isolated uridine diphospho- glucoronosyltransferase 2B17 enzyme having the same amino acid sequence as the

amino acid sequence shown in SEQ ID NO. 2 is provided. In another embodiment, the invention provides a recombinant expression vector comprising a promoter sequence operably linked to a coding sequence encoding uridine diphospho-giucuronosyltransferase 2B17, said coding sequence being

sufficiently homologous to SEQ ID No. 1 or a complement thereof, to hybridize under stringent conditions to the coding region of SEQ ID No. 1 or a complement thereof and said coding sequence encoding an enzyme which catalyzes the conversion of androsterone to androsterone-glucuronic acid. SEQ ID NO. 1 and other DNA sequences encoding SEQ ID NO. 2 may be used as the coding region of a vector in accordance with the invention.

In a further embodiment, the invention provides host cells transformed or transfected with such vectors, and a method for producing uridine diphospho- glucuronosyltransferase 2B17 comprising the steps of preparing a recombinant host transformed or transfected with the vector of claim 3 and culturing said host under conditions which are conducive to the production of uridine diphospho- glucuronosyltransferase 2B 17 by said host.

In an additional embodiment, the invention provides a kit for detecting

antibodies to uridine diphospho-glucuronosyltransferase 2B17 comprising an immobilized antigenic composition comprising uridine diphospho- glucuronosyltransferase 2B 17 or an antigenic fragment thereof and means for detecting a complex of said immobilized antigen and an antibody to said antigen.

In a further embodiment, a kit for detecting uridine diphospho- glucuronosyltransferase 2B17 comprising an immobilized antibody composition comprising antibodies to uridine diphospho-glucuronosyltransferase 2B17 or an antigenic fragment thereof and means for detecting a complex of said immobilized

antibodies and uridine diphospho-glucuronosyltransferase 2B17 is provided.

In another embodiment, the invention provides antisera to purified or recombinant diphospho-glucuronosyltransferase 2B17.

In an additional embodiment, the invention provides a method of altering the concentration of an androgenic compound in a tissue comprising the step of administering uridine diphospho-glucuronosyltransferase 2B17.

In a further embodiment, the invention provides a method for detecting a localized concentration of uridine diphospho-glucuronosyltransferase 2B 17 comprising administering labelled antibodies to said uridine diphospho-glucuronosyltransferase 2B 17 and thereafter detecting said label.

In another embodiment, the invention provides a method of blocking the synthesis of uridine diphospho-glucuronosyltransferase 2B17, comprising the step of introducing a nucleotide sequence of at least 30 consecutive nucleotides in the coding region of SEQ ID No. 1 , or the complement thereof.

In a further embodiment, a method of altering androgenic activity in a tissue comprising the step of administering uridine diphospho-glucuronosyltransferase

2B17 is provided. In an additional embodiment, the invention provides a method for

detecting an alteration in the level of androgenic activity in a sample comprising the step of determining a concentration of uridine diphospho-glucuronosyltransferase 2B17.

In another embodiment, the invention provides a method of detecting uridine diphospho-glucuronosyltransferase 2B17 in a test sample comprising contacting

said test sample with antibodies to uridine diphospho-glucuronosyltransferase 2B17 and measuring formation of an immunocomplex between said antibodies and said uridine diphospho-giucuronosyltransferase 2B 17.

In another embodiment, the invention provides a method of detecting antibodies to uridine diphospho-glucuronosyltransferase 2B17 in a test sample comprising contacting said test sample with uridine diphospho-glucuronosyltransferase

2B17 and measuring formation of an immunocomplex between said antibodies and said uridine diphospho-glucuronosyltransferase 2B17.

Other features and advantages of the present invention will become apparent from the following description of the invention which refers to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a general diagram of the steroidogenesis scheme which illustrates the role of uridine glucuronosyltransferases;

Figure 2 is a map of a pCMV vector which is exemplary of one that can be used to transfect host cells in accordance with the invention;

Figure 3 provides the results of an assay to determine the specificity of UGT2B 17 for androgenic compounds:

Figures 4A and 4B are Lineweaver-Burk plots of ADT and DHT. and

3α-DIOL and testosterone (TESTO), respectively; and

Figure 5 provides a Southern blot indicating the presence or absence of UGT2B17 in various tissues.

DETAILED DESCRIPTION OF THE INVENTION

Glucuronidation is an irreversible enzymatic reaction in the pathway of steroid metabolism which is catalyzed by a uridine diphospho-glucuronosyltransferase (UGT). The UGTs catalyze the conjugation of the sugar acid moiety of uridine diphospho-glucuronic acid to steroids. It is shown that the formation of steroid

glucuronides (-G) completely inactivates the androgens. Therefore. UGT enzymes can be used to regulate the concentration of substrates in tissues. A cDNA encoding the enzyme, UGT2B17, has been isolated and

encodes a protein having 530 amino acids with a measured molecular weight of 53 kilodaltons. The coding portion includes nucleotides +52 through 1644, including the stop codon (and encodes amino acids +1 through 530), numbered in the 5' to 3' direction. The protein structure contains a hydrophobic signal peptide at amino acids 5

through 12 which is responsible for directing the protein into the endoplasmic reticulum.

In addition, the leader sequence contains a positively charged lysine at position 4 and terminates with a possible cleavage site at the cysteine residue at position 23. Further,

UGT2B 17 has a hydrophobic transmembrane region between amino acids 494 and 510

followed by a positively charged lysine residue. The UGT2B17 also includes three potential asparagine-1 inked glycosylation sites (NXS/T) at amino acid residues 65, 316, and 483.

A general diagram of steroidogenesis and the role of UGTs is provided is

Figure 1. In this figure, ER is an estrogen receptor; AR is an androgen receptor: El is estrone; E2 is estradiol; HSD is hydroxysteroid dehydrogenase: and DHEA is dehydroepiandrosterone.

It has been shown that the carboxy terminal region of the enzyme, which includes amino acids 290 through 530, contains a domain which is critical for the binding of uridine diphospho-glucuronic acid (UDPGA). Further, it has been shown that a domain for substrate specificity is present in the amino terminal region, particularly between amino acid residues 54 and 227.

The UGT2B 17 enzyme can be produced by incoφorating the nucleotide sequence for the coding portion of the gene into a vector which is then transformed or transfected into a host system which is capable of expressing the enzyme. The DNA can be maintained transiently in the host or can be stably integrated into the genome of the host cell.

In particular, for the cloning and expression of UGT2B17. any common expression vectors, such as plasmids, can be used. These vectors can be prokaryotic expression vectors including those derived from bacteriophage λ such as λgtl 1 and λEMBL3. E. coli strains such as pBR322 and Bluescript (Stratagene); or eukaryotic vectors, such as those in the pCMV family. A vector incoφorating an isolated human cDNA (nucleotides 36 to 1870 of Sequence ID No. 1, ATCC Deposit No. 98228) for UGT2B17 was placed on deposit at the American Type Culture Collection (ATCC,

Rockville, MD), in accordance with the terms of the Budapest Treaty, and will be made

available to the public upon issuance of a patent based on the present patent application.

The gene may also be obtained by screening human prostate and LNCaP (a human prostatic adenocarcinoma cell line) cell cDNA libraries with probes derived from all or part of SEQ ID No. 1.

Vectors which can be used in the practice of the invention generally include appropriate replication and control sequences which are compatible with the host system into which the vectors are transfected. A promoter sequence is generally included. For prokaryotes, some representative promoters include β-lactamase. lactose, and tryptophan. In mammalian cells, commonly used promoters include, but are not limited to. adenovirus, cytomegalovirus (CMV) and simian virus 40 (SV40). The vector can also optionally include, as appropriate, an origin of replication, ribosome binding sites, RNA splice sites, polyadenylation sites, transcriptional termination sequences and/or a selectable marker. It is well understood that there are a variety of vector systems with various characteristics which can be used in the practice of the invention.

A map of the pCMV vector, which is an example of a vector which can be used in the practice ofthe invention, is provided in Figure 2.

Commonly known host systems which are known for expressing an enzyme, and which may be transfected with an appropriate vector which includes the gene for UGT2B17 can be used in the practice of the invention. These host systems include prokaryotic hosts, such as E. coli, bacilli, such as Bacillus subtilus. and other enterobacteria. such as Salmonella, Serratia, and Pseudomonas species. Eukaryotic microbes, including yeast cultures, can also be used. The most common of these is

Saccharomyces cerevisiae, although other species are commercially available and can be used. Furthermore, cell cultures can be grown which are derived from mammalian cells. Some examples of suitable host cell lines include embryonal kidney (293), SW-

13, Chinese hamster ovary (CHO), HeLa. myeloma. Jurkat. COS-1. BHK. W138 and madin-darby canine kidney (MDCK). In the practice of the invention, the 293 cells are preferred.

UGT2B17, whether recombinantly produced as described herein, purified from nature, or otherwise produced, can be used in assays to identify compounds which inhibit or alter the activity of the enzyme. In particular, since UGT2B17 is shown to catalyze the glucuronidation process, this enzyme can be used to identify compounds which interfere with this process. It is preferred that the enzyme be obtained directly from the recombinant host, wherein following expression, a crude homogenate is prepared which includes the enzyme. A substrate ofthe enzyme, such as androsterone and a compound to be tested are then mixed with the homogenate. The

activity of the enzyme with and without the test compound is compared. Numerous

methods are known which can be used to indicate the effects of the test compound on the activity of the substrate for easy detection of the relative amounts of substrate and product over time. For example, it is possible to label the substrate so that the label also stays on any product that is formed. Radioactive labels, such as C'⁴ or H\ which can be quantitatively analyzed are particularly useful.

It is preferred that the mixture of the enzyme, test compound and substrate be allowed to incubate for a predetermined amount of time. In addition, it is preferred that the product is separated from the substrate for easier analysis. A number of separation techniques are known, for example, thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), spectrophotometry, gas chromatography, mass spectrophotometry and nuclear magnetic resonance (NMR). However, any known method which can differentiate between a substrate and a product can be used. In addition, compositions which include the protein, or a fragment thereof or antibodies to the protein, or to antigenic fragments thereof can be produced.

These compositions are contacted with samples, such as body fluids or tissues which are suspected of containing UGT2B17 antibodies or UGT2B17. After contact, known methods are used to determine the extent to which antigen/antibody complexes are formed.

The preferred techniques for detecting the formation of antigen/antibody complexes include, but are not limited to. enzyme-linked immunosorbent assay

(ELISA). indirect fluorescence assay, latex agglutination, radioimmunoassay (RIA). and liposome-based assay. Alternatively, a Western blot technique may be used, in which case the bands are detected by visual inspection, and substantial appearance of dark bands may be taken as a positive indication. In addition, in vivo assays may be used, wherein labelled antibodies against the antigen are used to detect the presence of

UGT2B17 /Λ vivo.

In preferred embodiments of the invention, the antigen or antibody compositions of the invention are immobilized and contacted with the sample to be tested. After washing away the sample and any antibodies or antigens which did not bind, standard methods are used to determine the extent to which the antigens and antibodies are bound.

Before contacting a test sample with antigenic or antibody compounds in accordance with the invention, it is preferred (but not necessary) that the antigenic or antibody composition be immobilized using conventional techniques (e.g. ELISA). In an embodiment, liposome-based assays may be used, as described in more detail below. For conventional immobilization, polystyrene plates, for example, may be incubated with antigenic or antibody suspensions made in accordance with the invention.

Alternatively, for example, antigens isolated as protein bands on electrophoretic gel may be transferred to a nitrocellulose sheet by known methods. See Towbin et al., Proc.

Nat'l. Acad. Sci.. 76: 4350-54 (1979); Bumette et al., Biochem.. 1 12: 195-203 (1981). Numerous other techniques are known in the art for binding antigens or antibodies to substantially inert substrates.

Bound antigens in accordance with the invention are preferably contacted with a dilute fluid which includes the sample to be tested for presence of antibody to UGT2B17. The antigen and sample are preferably incubated for at least 5 to

15 minutes. Less time is needed when incubation proceeds at or near human body

temperature, about 37°C. Incubation at other temperatures, for instances 4°C, is also proper, but generally requires additional incubation time. Preferred incubation time at 37° is from about 5 minutes to about 90 minutes. The bound antigens should then be rinsed to remove any unbound antibodies, i.e., those which are not specific for the antigens. Preferably, rinsing proceeds with a buffer solution such as PBS T, PBS TT or Tris/T ween/Sodium chloride/azide. Multiple rinsings are preferred.

During incubation, UGT2B17 specific antibodies bind to the immobilized antigens to create antigen/antibody complexes. All unbound antibodies are substantially removed during the rinsing procedure. Due to the high specificity of the antigens of the invenuon, antibodies which are not specific for UGT2B17 are substantially removed by the rinsing. Naturally, if the tested sample did not contain UGT2B17 specific antibodies, the immobilized antigens would be substantially free of human antibody, and subsequent testing for antigen/antibody complexes should not indicate a substantial presence of such complexes. On the other hand, if the tested sample were rich in UGT2B17-specifιc antibodies, these antibodies should have bound to the immobilized antigens to form a large quantity of antigen/antibody complex for subsequent detection. Detection of antigen/antibody complex may be achieved by a wide variety of known methods. Preferred methods include but are not limited to enzyme- linked immunosorbent assay, latex agglutination. Western blot technique or indirect immunofluorescence assay. Typically, the UGT2B17-specifιc antibodies complexed with immobilized antigen are detected by contact with labelled or otherwise detectable second antibodies specific for the immunoglobulin being tested for (the anti-UGT2B17).

If the test sample is human sera, for example, the detectable second antibody is specific

for human immunoglobulin. The labelled second antibodies may be specific for any human antibody, such as IgG or IgA. When acute sero-conversion is suspected, an IgM test using a labelled second antibody specific for IgM may be appropriate. The second antibodies are preferably incubated with the immobilized antigens for about 5 minutes

to about 2 hours, preferably 30 minutes to 60 minutes at a temperature of about 20°C to about 37°C. Then, the antigens are washed with a buffer solution (preferably multiple times) in order to remove all unbound labelled antibody. The washings will remove substantially all labelled antibody except that which has bound to immunoglobulin

present on the antigens. Of course, substantially the only human immunoglobulin present at this point should be UGT2B 17-specifιc antibody. Hence, the presence of UGT2B 17-specific antibody may be indirectly measured by determining the presence or absence ofthe labelled second antibody.

There are many known techniques for detecting the label, which vary with the type of label used. For instance, fluorescein-labelled antibody may be detected by scanning for emitted light at the characteristic wavelength for fluorescein. Alternatively, an enzyme label is detected by incubation with appropriate substrate and detection of any enzymatic activity, preferably activity resulting in a color change. Such activity can be determined by visual inspection or can be read automatically by a spectrophotometer set at the appropriate wavelength. For example, the enzyme label may be horseradish peroxidase and the substrate may be H₂O₂ and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) which

produces in the presence of the enzyme, a compound detectable by a spectrophotometer set at 414 nm.

In Western blotting, the positive signal may be detected when an enzyme is conjugated to the second antibody. Incubation with appropriate substrate enzymatically produces a color product in the immediate vicinity of the antigenic band resolved by this process. The presence of a reactive band may be detected by visual inspection. In an indirect immunofluorescence assay, fluorescein-labelled second antibodies may be detected by fluorescence-activated detectors, or by visual inspection.

A liposome-based assay may involve the presence of fluorescein, an enzyme or a substrate inside a Hposome onto whose surface UGT2B17 antigens are expressed. These liposomes are incubated with a diluted body fluid sample to be tested, and are thoroughly washed. Any liposomes with immunoglobulins on their surface forming an antigen/antibody complex may be recognized by attaching a second antibody, specific to the immunoglobulin being tested for, onto the inside walls of a polystyrene tube containing the liposomes. Liposomes having antibody bound to their surfaces will become immobilized on the tube walls, and non-immobilized will be

washed away. The liposomes can be lysed with, for instance, detergent, or complement, and the enzyme or substrate that was in the interior is now free to react with the complementary substrate (or enzyme) in the solution in the tube. Enzymatic activity, preferably a color change reaction could be detected by visual inspection or spectrophotometric color determination. It is also possible to use antibodies to UGT2B17 or to an antigenic portion thereof to detect antigens in a test sample. The techniques and methodology used are similar to those described above, except that antibodies are immobilized,

samples suspected of containing UGT2B17 are tested and labelled anti-UGT2B17 is used to detect formation of complexes between UGT2B17 and the immobilized antibodies.

UGT2B17-specifιc test kits can be constructed for detecting antibodies or antigens using several different techniques for detection. A test kit for antibody or antigen detection may include a compartmented enclosure containing a plurality of wells, plates which were coated prior to use with UGT2B 17 antigen or antibody, and

ELISA materials for enzyme detection, and a color change indicator. Naturally, a variety of enzymes and developers can be used.

A second test kit for detecting antibodies using the Western blot technique may be comprised of a container, cover, nitrocellulose sheet, and a

polyacrylamide slab gel in the presence of sodium dodecyl sulfate. surfactants. pH modifiers, dried non-fat milk and materials for enzyme detection including a color change indicator, such as DAG in Tris with hydrogen peroxide. This Western blot analysis kit also contains peroxidase-labelled goat or rabbit anti-human immunoglobulin and a source of UGT2B 17 antigens.

Another UGT2B 17-specific test kit for detecting antibodies or antigens using the indirect immunofluorescence assay may include a compartmentai container with UGT2B17 antigens or antibodies, phosphate buffered saline and a fluorescent labelled conjugate. Finally, a different UGT2B17-specific test kit for detecting antibodies or antigens uses liposomes and comprises a container, fluorescent marker- (or enzyme- or substrate-) filled Hposome with UGT2B17 antigens or antibodies on their surface, and a surface-active agent. In this assay, the container might be a precoated tube or well with the appropriate conjugate.

The extent of detection ofthe antigen/antibody complex which should be considered a positive signal depends upon the detection mean chosen, but may be defined generically as a value greater than the mean plus one ( 1 ) interval of standard

deviation from the results observed from a negative control group, all other parameters (e.g. dilution of sample, time of incubation) being held constant. In some embodiments, where a higher specificity is desired, mean plus two or mean plus three standard deviations may be utilized.

It is also contemplated that the gene for UGT2B17 or a portion thereof can be used to produce antisense nucleic acid sequences for inhibiting expression of UGT2B17 in vivo. Thus, the activity of the enzyme and the levels of its substrates (e.g. androgens) may be increased where desirable. In general, antisense nucleic acid sequences can interfere with transcription, splicing or translation processes. Antisense sequences can prevent transcription by forming a triple helix or hybridizing to an opened loop which is created by RNA polymerase or hybridizing to nascent RNA. On the other hand, splicing can advantageously be interfered with if the antisense sequences bind at the intersection of an exon and an intron. Finally, translation can be affected by blocking the binding of initiation factors or by preventing the assembly of ribosomal subunits at the start codon or by blocking the ribosome from the coding portion of the mRNA. preferably by using RNA that is antisense to the message. For further general information, see Helene et al., Biochimica et Biophysica Acta. 1049:99-125 (1990), which is herein incoφorated by reference in its entirety.

An antisense nucleic acid sequence is an RNA or single stranded DNA sequence which is complementary to the target portion of the target gene. These antisense sequences are introduced into cells where the complementary strand base pairs with the target portion of the target gene, thereby blocking the transcription, splicing or translation of the gene and eliminating or reducing the production of UGT2B17. The length of the antisense nucleic acid sequence need be no more than is sufficient to interfere with the transcription, splicing or translation of functional UGT2B17.

Antisense strands can range in size from 10 nucleotides to the complete gene, however. about 10 to 50 nucleotides are preferred, and 15 to 25 nucleotides are most preferred.

Although it is contemplated that any portion ofthe gene could be used to produce antisense sequences, it is preferred that the antisense is directed to the coding

portion of the gene or to the sequence around the translation initiation site of the mRN A or to a portion of the promoter. It is preferred to use a portion including amino acids 1 through 295 and, most preferable to use a portion which includes amino acids 54 and

227.

As is well understood in the art, the sequences can be modified in various manners in order to increase the effectiveness ofthe treatment. In particular, the sequences can be modified to include additional RNA on the 3' end of the RNA which can form a haiφin-loop structure and thereby prevent degradation by nucleases. In addition, the chemical linkages in the backbone of the oligonucleotides can be modified to prevent cleavage by nucleases.

There are numerous methods which are known in the art for introducing the antisense strands into cells. One strategy is to incoφorate the gene which encodes

UGT2B17 in the opposite orientation in a vector so that the RNA which is transcribed from the plasmid is complementary to the mRNA transcribed from the cellular gene. A strong promoter, such as pCMV. is generally included in the vector, upstream of the gene sequence, so that a large amount of the antisense RNA is produced and is available for binding sense mRNA. The vectors are then transfected into cells which are then

administered. It is also possible to produce single stranded DNA oligonucleotides or antisense RNA and incoφorate these into cells or liposomes which are then administered. The use of liposomes, such as those described in WO95/03788. which is herein incoφorated by reference, is preferred. However, other methods which are well understood in the art can also be used to introduce the antisense strands into cells and to administer to these patients in need of such treatment. The following is a description of the materials and methods used in the isolation of a UGT2B17 cDNA clone. In addition, the materials and methods used in the expression of UGT2B17 and the determination of substrate specificity are provided below. These examples are intended to be illustrative of the invention and it is well understood by those of skill in the art that modifications and alterations in the procedure and source and/or different techniques can be used within the scope of the invention. In addition, it is well understood that the gene can be produced using recombinant techniques.

Materials. UDP-glucuronic acid and all aglycons were obtained from Sigma Chemical Co. (St. Louis, MO) and ICN Pharmaceutical. Inc. (Montreal. Canada).

Radioinert steroids were purchased from Steraloids Inc. (Wilton, NH). [9,11-TTJ androsterone (59 Ci/mmol), [9,11-³H] androstane-3α, 17β-diol (56 Ci/mmol) and

[^l4C]UDP-glucuronic acid (285 mCi/mmol) were obtained from NEN Dupont (Boston, MA). [1,2-^] dihydrotestosterone (47 Ci/mmol), [1, 2, 6, 7-'H] testosterone (90

Ci/mmol), α-["P]-dCTP (3000 Ci/mmol) and α-[³²P]-dUTP (3000 Ci/mmol) were obtained from Amersham (Oakville, Canada). Geneticin (G418) and Lipofectin were

obtained from Gibco BRL (Burlington. Canada). Protein assay reagents were obtained from Bio-Rad (Richmond, CA). Restriction enzymes and other molecular biology reagents were obtained from Pharmacia LKB Biotechnology Inc. (Milwaukee. WI),

Gibco BRL (Burlington, Canada), Stratagene (La Jolla, CA) and Boehringer Mannheim (Indianapolis, IN). AmpliTaq DNA polymerase was from Perkin-Elmer Cetus (Branchburg, NJ). Human embryonic kidney 293 cells (HK293) and LNCaP cells were obtained from the American Type Culture Collection (Rockville, MD). Total RNA from human prostate, adrenal, testis. mammary gland, kidney, uterus and lung was purchased from Clontech (Palo Alto, CA).

Human RNA Isolation. Total RNA was isolated from human liver, adipose tissue, skin, placenta, benign prostate hypeφlasia tissue (BPH) and LNCaP cells according to the Tri reagent acid phenol protocol as specified by the supplier (Molecular Research Center Inc.. Cincinnati, OH). The mRNAs obtained from human prostate hypeφlastic tissue (BPH) and LNCaP cells were affinity purified by chromatography through oligo(dT)-cellulose (Pharmacia, Milwaukee, WI). cDNA Isolation. Affinity purified BPH and LNCaP cell mRNAs were used to construct cDNA libraries in the ZAP Express vector as specified by the supplier

(Stratagene, La Jolla, CA). The filters were prehybridized in 40% formamide, 5X

Denhardt's solution, 5X SSPE, 0.1% SDS and 100 mg/ml salmon sperm DNA for 4 hours at 42°C. The hybridization was performed in the same solution for 16 hours at 42°C with 2.0 x 10⁶ cpm/ml of a pool of probes derived from UGT2B7, UGT2B 10 and

UGT2B15 cDNAs. The cDNA probes were radiolabelled by the random primer technique in the presence of [α-¹²P]dCTP. The filters were washed twice in 2X SSC.

0.1 % SDS at 42°C for 15 minutes and then the filters were exposed for two days at -

80°C on XAR5 film with an intensifying screen (Kodak Coφ., Rochester, NY). Approximately 1 x 10" recombinants were screened. Thirty positive clones were isolated from the LNCaP cell library and five positive clones were isolated from the BPH cDNA library. Of these clones, two clones from the LNCaP library and one clone from the BPH library were found to encode UGT2B17. The three cDNA clones were sequenced in both directions using specific UGT oligonucleotides and were found to be identical except for the length of their 5' untranslated region and the number of residues in the poly (A+) tail.

Stable Expression of UGT2B17. Human kidney 293 cells were grown in Dubelcco's modified Eagle^'s medium containing 4.5 g/1 glucose, 10 mM HEPES.

110 μg/ml sodium pyruvate, 100 IU of penicillin/ml. 100 μg/ml of streptomycin and 10% fetal bovine serum (FBS) in a humidified incubator, with an atmosphere of 5%

CO,, at 37°C. 5 μg of pBK-CMV-UGT2B 17 was used to transfect HK293 cells using

Lipofectin and following the manufacturer's instructions (Gibco BRL: Burlington.

Canada). Forty-eight hours after transfection. stable transfectants were selected in a media containing 800 μg/ml G418. After five rounds of selection, a clonal cell line which stably expressed a high level of UGT2B17 was isolated.

Activity of the UGT2B17 Enzyme. The UGT2B17 protein was

expressed by excising the cDNA encoding the UGT2B17 enzyme into a pBK-CMV

vector. The pBK-CMV-UGT2B 17 construct was then transcribed in vitro using T3 polymerase and the resulting transcript was translated using a rabbit reticulocyte system.

The expressed protein was found to have a molecular weight of 53 kilodaltons.

Glucuronidation Assay Using Cell Homogenates. HK293 ceils expressing UGT2B17 were suspended in a Tris buffered saline containing 0.5 mM DTT and were homogenized using a Brinkman polytron. Enzyme assays were performed using [^UC] UDP-glucuronic acid (UDPGP), 500 μM of the various aglycones and 150

μg of protein from cell homogenates in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 100 μg/ml phosphatidylcholine and 8.5 mM saccharolactone in a final volume of 100 μl.

The enzyme assays were terminated by adding 100 μl of methanol. and the tubes were centrifuged at 14.000 g for 1 minute to remove the precipitated proteins. 100 μl of the aqueous phase was applied onto thin layer chromatography (TLC) plates (0.25 mm thick silica gel, 60 F₂₅₄ S) (EM Science, Gibbstown. NJ) and then was chromatographed in a solvent of toluene:methanol:acetic acid in the proportion of 7:3:1. The TLC plates were exposed for four days and the extent of glucuronidation was measured by Phosphorimager (Molecular Dynamics).

Assays were conducted to screen for substrates which react with

UGT2B17 in which the substrate was exposed to 6μM of [^,4C]UDPGA and 94μM of unlabelled UDPGA for 16 hours at 30°C. The compounds which demonstrated reactivity to UGT2B17 in the screening assay were subsequently reassayed to determine the amount of enzyme activity. In the second assay, the substrate was exposed to 6 μM [14C]

UDPGA and 494 μM of unlabelled UDPGA for 15 minutes at 30°C. The enzyme reaction is linear for 30 minutes at these conditions when the Km of UDPGA is 200 μM. No glucuronidation activity was detected in nontransfected HK293 cells.

As described above, the enzymatic activity of UGT2B17 was evaluated by transfecting HK293 cells with vectors which included the gene encoding UGT2B17. As shown in Figure 3, the HK293 cell homogenate containing the stably expressed UGT2B17 was analyzed for aglycon specificity using TLC. As shown in Table 1, over sixty endogenous and exogenous substances were tested for activity and of these, it was found that 25 compounds were glucuronidated by UGT2B17. There was no glucuronidation ofthe compounds in the control HK293 cell homogenates which did not contain the UGT2B17 protein.

The following compounds, which are 3α and 17β hydroxyandrogens, were identified as the major endogenous substrates: Dihydrotestosterone Testosterone

Androsterone

Androstane-3α, 17β-diol

Androstane-3β. 17β-diol Androst-5-ene-3β. 17β-diol

Further, as set forth in Table 1 , of the androgens tested, testosterone and its 5α-reduced metabolites, DHT. 3α-DIOL and ADT are the preferred substrates for UGT2B 17 glucuronidation.

A kinetic analysis was also performed to further characterize the specificity of UGT2B 17 for ADT. DHT. 3α-DIOL and testosterone. As shown in Figures 4A and 4B. the affinity of the enzyme for ADT. DHT,

3α-DIOL and testosterone is similar based on their Km values.

Figure 5 provides a Southern blot of a specific reverse transcriptase polymerase chain reaction (RT-PCR) analysis and shows some ofthe tissues in which the UGT2B 17 transcript is detectable.

As such, the conversion of endogenous and exogenous compounds to glucuronide derivatives can be used to control the levels of substrates. Further, since the levels of UGT2B17 correlate with levels of androgen. a measure ofthe concentration of UGT2B17 can be used to

indicate the levels of androgenic activity in a tissue.

Production and purification of a UGT2B17 fusion protein to make antisera. For the expression of a 29-kDa protein from amino acid sequence between 57 to 300 of UGT2B17 enzyme. HincII-SpcI fragment from UGT2B17 cDNA was subcloned into the pET23a (Novagen. WI) prokaryotic expression vector. The E. coli BL21 cells (Novagen. WI)

harboring the recombinant vectors were grown at 37°C in 1 liter of

terrific broth medium supplemented with ampicillin (100 μg/ml). When the absorbance at 600 nm reached 0.5-0.6 OD U. the production of the fusion protein was achieved by adding 1 mM isopropyl β-p- thiogalactopyranoside (IPTG) fir 2 h at 37"C. The cells were harvested by centrifugation at 4°C, for 10 min at 5000 x g. The bacterial pellets were resuspended in 25 ml of a lysis buffer (125 mM Tris-HCL. pH 8.0, 4.6% SDS, 10% β mercaptoethanol and 20% glycerol) and sonicated

until homogeneity using an ultrasound sonicator. The proteins were separated on a preparative 12% polyacrylamide gel in the presence of

SDS-PAGE and followed by two rounds of dialyses for 4 h at room temperature and 16 h at 4°C using 50 mM Tris-HCl pH 7.5 and 50mM

Tris-HCl, pH 7.5. 150 mM NaCl.

Immunization procedure. Rabbits (Charles River Inc., Quebec,

Canada) were kept in separate cages in an environmentally controlled room. They were injected at multiple sites with 500 μl of a total of 100 μg of purified fusion protein in phosphate buffered saline, in the presence of 500 μl of complete freund's adjuvant. Two booster injections were given at 6-week intervals with the same quantity of protein in the presence of incomplete Freund's adjuvant. The production of antibodies was checked 12 days after each injection on blood collected by ear puncture.

Immunoblot analysis, to gain information concerning the novel anti-UGT2B17 antibodies, microsomes from the HK293 cells, the stable

HK293-UGT2B17 cells and from treated LNCaP cells were purified using a standard method, ten micrograms of each microsomal protein and one hundred nanograms of the E. cυli BL21 pLys S (Novagen) strain

expressing or not the fusion protein were separated on a 12% SDS- PAGE gel. The gel was transferred onto a nitrocellulose filter and probed with a dilution 1 :2000 of the rabit antiserum. Antirabbit IgG horse peroxidase conjugates (Amersham. Oakville. Canada) was used as secondary antibodies, and the recognized proteins were then visualized using enhanced chemiluminescence (Renaissance, Quebec. Canada) and exposed on a hyperfilm for 1 h (Kodak Corp.. Rochester. NY). One hundred nanograms of E. coli BL21 (pLys S) cell lysate containing the recombinant UGT2B17 fusion protein were used to demonstrate the reactivity ofthe polyclonal antibody.

Reducing androgen activity with UGT2B17. LNCaP cells were transfected with UGT2B17 in the presence of a reporter construct controlled by an androgen response element so as the expression of the reporter gene is indicative of androgenic activity. The presence of the transfected UGT2B17 lead to a decreased expression ofthe reporter gene thereby indicating that UGT2B17 conjugates androgens and terminates the androgen response.

Although the present invention has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will be apparent to those skilled in the art.

SEQUENCE LISTING

(1) GENERAL INFORMATION: d) APPLICANT: Endorecherche Inc. BEAULIEU, Martin LEVESQUE, Eric HUM, Dean BELANGER, Alain di) TITLE OF INVENTION: CHARACTERIZATION AND USE OF AN ISOLATED URIDINE DIPHOSPHO-GLUCURONOSYLTRANSFERASE (m) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Marks & Clerk

(B) STREET: P.O. Box 957, Station B (C) CITY: Ottawa

(D) STATE: Ontario

(E) COUNTRY: CA

(F) ZIP: KIP 5S7 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vm) ATTORNEY/AGENT INFORMATION:

(A) NAME: Mitchell, Richard J.

(lx) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (613) 236-9561

(B) TELEFAX: (613) 230-8821

(C) email: mitch@markclerk.com

(2) INFORMATION FOR SEQ ID NO:l:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2107 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(lx) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 52..1644

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GGCACGAGGA AAGAAACAAC AACTGGAAAA GAAGCATTGC ATAAGACCAG G ATG TCT

Met Ser

AGC TCT GGG AGT TGT GGA AAG GTG CTG GTG TGG CCC ACA GAA TAC AGC 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 lie Asn Met Lys Thr lie Leu Glu Glu Leu Val Gin 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 lie Val Leu Thr Ser Ser Ala Ser lie 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 lie 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 lie Ser Lys Asn Thr Phe Trp Ser Tyr Phe Ser Gin Leu Gin 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 lie Lys Leu Cys Glu Asp 115 120 125 130

GCA GTT TTG AAC AAG AAA CTT ATG AGA AAA CTA CAA GAG TCA AAA TTT 489 Ala Val Leu Asn Lys Lys Leu Met Arg Lys Leu Gin Glu Ser Lys Phe 135 140 145

GAT GTC CTT CTG GCA GAT GCC GTT AAT CCC TGT GGT GAG CTG CTG GCT 537 Asp Val Leu Leu Ala Asp Ala Val Asn Pro Cys Gly Glu Leu Leu Ala 150 155 160

GAA CTA CTT AAC ATA CCC TTT CTG TAC AGT CTC CGC TTC TCT GTT GGC 585 Glu Leu Leu Asn lie Pro Phe Leu Tyr Ser Leu Arg Phe Ser Val Gly 165 170 175 TAC ACA GTT GAG AAG AAT GGT GGA GGA TTT CTG TTC CCT CCT TCC TAT 633 Tyr Thr Val Glu Lys Asn Gly Gly Gly Phe Leu Phe Pro Pro Ser Tyr 180 185 190

GTA CCT GTT GTT ATG TCA GAA TTA AGT GAT CAA ATG ATT TTC ATG GAG 681 Val Pro Val Val Met Ser Glu Leu Ser Asp Gin Met lie Phe Met Glu 195 200 205 210

AGG ATA AAA AAT ATG ATA TAT ATG CTT TAT TTT GAC TTT TGG TTT CAA 725 Arg lie Lys Asn Met lie Tyr Met Leu Tyr Phe Asp Phe Trp Phe Gin 215 220 225

GCA TAT GAT CTG AAG AAG TGG GAC CAG TTT TAT AGT GAA GTT CTA GGA 777 Ala Tyr Asp Leu Lys Lys Trp Asp Gin Phe Tyr Ser Glu Val Leu Gly 230 235 240

AGA CCC ACT ACA TTA TTT GAG ACA ATG GGG AAA GCT GAA ATG TGG CTC 82= Arα Pro Thr Thr Leu Phe Glu Thr Met Gly Lys Ala Glu Met Trp Leu 245 250 255 ATT CGA ACC TAT TGG GAT TTT GAA TTT CCT CGC CCA TTC TTA CCA AAT 873 lie Arα Thr Tyr Trp Asp Phe Glu Phe Pro Arα Pro Phe Leu Pro Asn 260 265 270

GTT GAT TTT GTT GGA GGA CTT CAC TGT AAA CCA GCC AAA CCC TTG CCT 921 Val Asp Phe Val Gly Gly Leu His Cys Lys Pro Ala Lys Pro Leu Pro 275 280 285 290

AAG GAA ATG GAA GAG TTT GTG CAG AGC TCT GGA GAA AAT GGT ATT GTG 969 Lys Glu Met Glu Glu Phe Val Gin Ser Ser Gly Glu Asn Gly He Val 295 300 305 GTG TTT TCT CTG GGG TCG ATG ATC AGT AAC ATG TCA GAA GAA AGT GCC 1017 Val Phe Ser Leu Gly Ser Met He Ser Asn Met Ser Glu Glu Ser Ala 310 315 320

AAC ATG ATT GCA TCA GCC CTT GCC CAG ATC CCA CAA AAG GTT CTA TGG 1065 Asn Met He Ala Ser Ala Leu Ala Gin He Pro Gin Lys Val Leu Trp 325 330 335

AGA TTT GAT GGC AAG AAG CCA AAT ACT TTA GGT TCC AAT ACT CGA CTG 1113 Arg Phe Asp Gly Lys Lys Pro Asn Thr Leu Gly Ser Asn Thr Arg Leu 340 345 350

GCT ATA ACT CAT GGT GGA ACC AAT GGC ATC TAT GAG GCG ATC TAC 1209 Ala Phe He Thr His Gly Gly Thr Asn Gly He Tyr Glu Ala He Tyr 375 380 385

CAT GGG ATC CCT ATG GTG GGC ATT CCC TTG TTT GCG GAT CAA CAT GAT 1257

His Gly He Pro Met Val Gly He Pro Leu Phe Ala Asp Gin His Asp 390 395 400

AAC ATT GCT CAC ATG AAA GCC AAG GGA GCA GCC CTC AGT GTG GAC ATC 1305

Asn He Ala His Met Lys Ala Lys Gly Ala Ala Leu Ser Val Asp He 405 410 415

AGG ACC ATG TCA AGT AGA GAT TTG CTC AAT GCA TTG AAG TCA GTC ATT 1353

Arg Thr Met Ser Ser Arg Asp Leu Leu Asn Ala Leu Lys Ser Val He 420 425 430

AAT GAC CCT ATC TAT AAA GAG AAT ATC ATG AAA TTA TCA AGA ATT CAT 1401

Asn Asp Pro He Tyr Lys Glu Asn He Met Lys Leu Ser Arg He His 435 440 445 450

CAT GAT CAA CCG GTG AAG CCC CTG GAT CGA GCA GTC TTC TGG ATT GAG 1449

His Asp Gin Pro Val Lys Pro Leu Asp Arg Ala Val Phe Trp He Glu 455 460 465

TTT GTC ATG CGC CAT AAA GGA GCC AAG CAC CTT CGG GTC GCA GCC CAC 1497

Phe Val Met Arα His Lys Gly Ala Lys His Leu Arg Val Ala Ala His

470 475 480

TTT TGT TTC CGA AAG CTT GCC AAA ACA GGA AAG AAG AAG AAA AGG GAT 1641 Phe Cvs Phe Arg Lys Leu Ala Lys Thr Gly Lys Lys Lys Lys Arg Asp 515 ^" 520 525 530

TAG TTATATCAAA AGCCTGAAGT GGAATGACCA AAAGATGGGA CTCCTCCTTT 1694

ATTCCAGCAT GGAGGGTTTT AAATGGAGGA TTTCCTTTTT CCTGCGACAA AACGTCTTTT 1754

CACAACTTAC CCTGTTAAGT CAAAATTTAT TTTCCAGGAA TTTAATATGT ACTTTAGTTG 1614

GAATTATTCT ATGTCAATGA TTTTTAAGCT ATGAAAAATA ATAATATAAA ACCTTATGGG 1874

CTTATATTGA AATTTATTAT TCTAATCCAA AAGTTACCCC ACACAAAAGT TACTGAGCTT 1934 CCTTATGTTT CACACATTGT ATTTGAACAC AAAACATTAA CAACTCCACT CATAGTATCA 1994 ACATTGTTTT GCAAATACTC AGAATATTTT GGCTTCATTT TGAGCAGAAT TTTTGTTTTT 2054 AATTTTGCCA ATGAAATCTT CAATAATTAA AAAAAAAAAA AAAAAAAAAA AAA 2107

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 531 ammo acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ser Leu Lys Trp Met Ser Val Phe Leu Leu Met Gin Leu Ser Cys

1 5 10 15

Tyr Phe Ser Ser Gly Ser Cys Gly Lys Val Leu Val Trp Pro Thr Glu 20 25 30

Tyr Ser His Trp He Asn Met Lys Thr He Leu Glu Glu Leu Val Gin 35 40 45

Arg Gly His Glu Val He Val Leu Thr Ser Ser Ala Ser He Leu Val 50 55 60

Asn Ala Ser Lys Ser Ser Ala He Lys Leu Glu Val Tyr Pro Thr Ser 65 70 75 80

Leu Thr Lys Asn Asp Leu Glu Asp Phe Phe Met Lys Met Phe Asp Arg 85 90 95

Trp Thr Tyr Ser He Ser Lys Asn Thr Phe Trp Ser Tyr Phe Ser Gin 100 105 110

Leu Gin Glu Leu Cys Trp Glu Tyr Ser Asp Tyr Asn He Lys Leu Cys 115 120 125

Glu Asp Ala Val Leu Asn Lys Lys Leu Met Arg Lys Leu Gin Glu Ser 130 135 140 Lys Phe Asp Val Leu Leu Ala Asp Ala Val Asn Pro Cys Gly Glu Leu 145 150 155 160

Leu Ala Glu Leu Leu Asn He Pro Phe Leu Tyr Ser Leu Arg Phe Ser 165 170 175

Val Gly Tyr Thr Val Glu Lys Asn Gly Gly Gly Phe Leu Phe Pro Pro 180 185 190

Ser Tyr Val Pro Val Val Met Ser Glu Leu Ser Asp Gin Met He Phe 195 200 205

Met Glu Arg He Lys Asn Met He Tyr Met Leu Tyr Phe Asp Phe Trp 210 215 220 Phe Gin Ala Tyr ASD Leu Lys Lys Trp ASD Gin Phe Tyr Ser Glu Val

225 ^" 230 ^' 235 240

Leu Gly Arg Pro Thr Thr Leu Phe Glu Thr Met Gly Lys Ala Glu Met 245 250 255

Trp Leu He Arg Thr Tyr Trp Asp Phe Glu Phe Pro Arα Pro Phe Leu 260 265 ^' 270

Pro Asn Val Asp Phe Val Gly Gly Leu His Cys Lys Pro Ala Lys Pro 275 280 285 Leu Pro Lys Glu Met Glu Glu Phe Val Gin Ser Ser Gly Glu Asn Gly 290 295 300

He Val Val Phe Ser Leu Gly Ser Met He Ser Asn Met Ser Glu Glu 305 310 315 320

Ser Ala Asn Met He Ala Ser Ala Leu Ala Gin He Pro Gin Lys Val 325 330 335

Leu Trp Arg Phe Asp Gly Lys Lys Pro Asn Thr Leu Gly Ser Asn Thr 340 345 350

Arg Leu Tyr Lys Trp Leu Pro Gin Asn Asp Leu Leu Gly His Pro Lys 355 360 365

Thr Lys Ala Phe He Thr His Gly Gly Thr Asn Gly He Tyr Glu Ala 370 375 380

He Tyr His Gly He Pro Met Val Gly He Pro Leu Phe Ala Asp Gin 365 390 395 400

His Asp Asn He Ala His Met Lys Ala Lys Gly Ala Ala Leu Ser Val 405 410 415

Asp He Arg Thr Met Ser Ser Arg Asp Leu Leu Asn Ala Leu Lys Ser 420 425 430

Val He Asn Asp Pro He Tyr Lys Glu Asn He Met Lys Leu Ser Arg 435 440 445

He His His Asp Gin Pro Val Lys Pro Leu Asp Arg Ala Val Phe Trp 450 455 460

He Glu Phe Val Met Arg His Lys Gly Ala Lys His Leu Arg Val Ala 465 470 475 480

Ala His Asn Leu Thr Trp He Gin Tyr His Ser Leu Asp Val He Ala

485 490 495

Phe Leu Leu Ala Cys Val Ala Thr Met He Phe Met He Thr Lys Cys

500 505 510

Cys Leu Phe Cys Phe Arg Lvs Leu Ala Lys Thr Gly Lys Lys Lys Lys 515 520 525

Arg Asp 530

Claims

CLAIMS We claim:

1. An isolated nucleotide sequence encoding uridine diphospho- glucuronosyltransferase 2B17, said sequence being sufficiently homologous to SEQ ID No. 1 or a complement thereof to hybridize under stringent conditions to the coding region of SEQ ID No. 1 or a complement thereof and said sequence encoding an enzyme which catalyzes the conversion of androsterone to androsterone-glucuronic acid.

2. The nucleotide sequence, as recited in claim 1. wherein said sequence includes the coding region of SEQ ID No. 1.

3. A recombinant expression vector comprising a promoter sequence and a nucleotide sequence in accordance with claim 1.

4. A recombinant expression vector comprising a promoter sequence and a nucleotide sequence in accordance with claim 2.

5. A recombinant host cell, transformed or transfected with the vector of claim 4.

6. The recombinant host cell of claim 5. wherein said host cell is a eukaryotic cell.

7. A recombinant host cell, transformed or transfected with the vector of claim 3.

8. The recombinant host cell of claim 7. wherein said host cell is a eukarvotic cell.

9. The recombinant host cell of claim 8. wherein a nucleotide sequence that hybridizes under stringent conditions with SEQ ID No. 1 or its complement is integrated into the genome of said host cell.

10. The recombinant host cell of claim 9. wherein said nucleotide sequence is located on a recombinant vector.

1 1. The recombinant host cell, as recited in claim 8. wherein said host cell is capable of expressing a biologically active uridine diphospho-glucuronosyltransferase

2B17.

12. An isolated nucleotide sequence comprising at least thirty consecutive nucleotides identical to thirty consecutive nucleotides in the coding region of SEQ ID No. 1. or the complement thereof.

13. A nucleotide sequence comprising nucleotides 52 to 927 of SEQ ID No. 1.

14. A nucleotide sequence comprising nucleotides 204 to 723 of SEQ ID

No. 1.

15. An isolated nucleotide sequence comprising SEQ ID No. 1.

16. An isolated uridine diphospho-glucoronosyltransferase 2B17 enzyme having the same amino acid sequence as the amino acid sequence shown in SEQ ID NO.

2.

17. An isolated nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 2.

18. A method for producing uridine diphospho-glucuronosyltransferase 2B17, comprising the steps of: preparing a recombinant host transformed or transfected with the vector of claim 3; and culturing said host under conditions which are conducive to the production of uridine diphospho-glucuronosyltransferase 2B17 by said host.

19. A method for producing uridine diphospho-glucuronosyltransferase 2B 17. comprising the steps of: preparing a recombinant host transformed or transfected with the vector of claim 4; and culturing said host under conditions which are conducive to the production of uridine diphospho-glucuronosyltransferase 2B17 by said host.

20. A kit for detecting antibodies to uridine diphospho- glucuronosyltransferase 2B17. said kit comprising: an immobilized antigenic composition comprising uridine diphospho- glucuronosyltransferase 2B17 or an antigenic fragment thereof; means for detecting a complex of said immobilized antigen and an antibody to said antigen.

21. A kit for detecting uridine diphospho-glucuronosyltransferase 2B 17. said kit comprising: immobilized antibodies to uridine diphospho-glucuronosyltransferase

2B17 or an antigenic fragment thereof; means for detecting a complex of said immobilized antibodies and uridine diphospho-glucuronosyltransferase 2B17.

22. Antisera to purified or recombinant diphospho-glucuronosyltransferase 2B17.

23. A method of altering the concentration of an androgenic compound in a tissue comprising the step of administering uridine diphospho-glucuronosyltransferase 2B17.

24. The method of claim 23, wherein said androgenic compound is dihydrotestosterone.

25. A method of altering androgenic activity in a tissue comprising the step of administering uridine diphospho-glucuronosyltransferase 2B17.

26. A method for detecting a localized concentration of uridine diphospho- glucuronosyltransferase 2B17 comprising the steps of: administering labelled antibodies to said uridine diphospho- glucuronosyltransferase 2B17; and thereafter detecting said label.

27. A method of blocking the synthesis of uridine diphospho- glucuronosyltransferase 2B17. comprising the step of introducing a nucleotide sequence of at least 30 consecutive nucleotides in the coding region of SEQ ID No. 1. or the complement thereof.

28. A method for detecting an alteration in the level of androgenic activity in a sample comprising the step determining a concentration of uridine diphospho- glucuronosy .transferase 2B17.

29. A method of detecting uridine diphospho-glucuronosyltransferase 2B17 in a test sample comprising contacting said test sample with antibodies to uridine diphospho-giucuronosyltransferase 2B17 and measuring formation of an immunocomplex between said antibodies and said uridine diphospho- glucuronosyltransferase 2B17.

30. A method of detecting antibodies to uridine diphospho- glucuronosyltransferase 2B17 in a test sample comprising contacting said test sample with uridine diphospho-glucuronosyltransferase 2B17 and measuring formation of an immunocomplex between said antibodies and said uridine diphospho- glucuronosy .transferase 2B17.