the journal of bio~ical chemistry vol. 269, no. · pdf filemolecular cloning and functional...

THE JOURNAL OF B I O ~ I C A L CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 19, Issue of May 13, pp. 13792-13797, 1994 Printed in U.S.A.

Molecular Cloning and Functional Expression of Human 3-Hydroxyanthranilic-acid Dioxygenase*

(Received for publication, October 27, 1993, and in revised form, January 24, 1994)

Pari Malherbe, Christer Kohler, Most5 Da Prada, Gabrielle Lang, Vivian Kiefer, Robert SchwarczS, Hans-Werner Lahm§, and Andrea M. Cesuran From the Pharma Division, Preclinical Research, Nervous System and $New Technologies, I;: Hoffmann-La Roche Limited, CH-4002 Basel, Switzerland and the $Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21228

Increased cerebral levels of the endogenous excitotoxin quinolinic acid (QUIN) have been speculatively linked to neuronal damage following neurological and inflammatory disorders. 3-Hydroxyanthranilic-acid dioxygenase (3-HAQ 3-hydroxyanthranilate 3,4-dioxygenase, EC 1.13.11.6) is the enzyme that catalyzes the synthesis of QUIN from 3-hydroxyanthranilic acid, and evidence suggests that it could play a role in disorders associated with altered tissue levels of QUIN. In this report, we describe the isolation of a full-length cDNA clone encoding human 3-HA0 (h3-HAO). Degenerate oligonucleotides were designed from the amino acid sequences of tryptic peptides of rat liver 3-HAO, and they were used as primers for reverse transcription-polymerase chain reaction of rat liver RNA. The resulting rat cDNA product was used to screen a human hepatoma cell line (HepG,) cDNA library and to isolate a human 3-HA0 cDNA clone. This clone was found to have an insert of 1276 nucleotides. The deduced primary structure of h3-HA0 is composed of 286 amino acid residues with a predicted molecular mass of -32.6 kDa. The human sequence exhibits high similarity (94%) to the rat partial amino acid sequence deduced from the rat reverse transcription-polymerase chain reaction fragment. Insertion of the h3-HA0 coding sequence into a eukaryotic expression vector yielded relatively high amounts of the active enzyme in human embryonic kidney HEK-293 cells. The K,,, value of 3-HANA for recombinant h3-HA0 (-2 m) was in good agreement with that reported for the native enzyme. Immunoblot analysis of recombinant h3-HA0 revealed a polypeptide with an ap- parent molecular mass of 32 kDa, as predicted from the deduced amino acid sequence. RNA blot analysis of human liver and HepG, cells revealed one major species of h3-HA0 mRNA of -1.3 kilobases.

Interest in the kynurenine pathway for the metabolism of L-tryptophan (Fig. 1) and in the role of quinolinic acid (QUIN)’

* The costs of publication of this article were defrayed in part by the

“aduertisement” in accordance with 18 U.S.C. Section 1734 solely to payment of page charges. This article must therefore be hereby marked

indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted

to the GenBankmIEMBL Data Bank with accession numbeds) 229481. 1 To whom correspondence should be addressed Pharma Div., Pre-

clinical Research, PRPN (70/307b), F. Hoffmann-La Roche Ltd., CH- 4002 Basel, Switzerland. Tel.: 41-61-688-5231; Fax: 41-61-688-4484.

The abbreviations used are: QUIN, quinolinic acid; 3-HAO, 3-hydroxyanthranilic-acid dioxygenase; h3-HA0 and r3-HA0, human and rat 3-hydroxyanthranilic-acid dioxygenases, respectively; 3-HANA, 3-hydroxyanthranilic acid; RT-PCR, reverse transcription-polymerase chain reaction; bis-Tris, 2-[bis(2-hydroxyethyl)aminol-2-(hydroxy- methyl)-propane-1,3-diol; MS, mass spectrometridspectrometry; PAGE,

and other kynurenine metabolites in several neuropathological conditions (for review, see Schwarcz et al. (1992), Schwarcz (19931, and Heyes (1993)) has grown steadily over the last decade. QUIN is an excitotoxin (Schwarcz et al . , 1983; Moroni et al . , 1984) whose toxicity is mediated by its ability to activate glutamate N-methyl D-aspartate receptors (Perkins and Stone, 1983). It has been suggested that increased cerebral levels of QUIN may participate in the pathogenesis of neurological disorders such as epilepsy (Nakano and Kito, 1991; Du et al., 19931, Huntington’s disease (Schwarcz, 1992; Beal et al . , 1990), and damages deriving from brain trauma and hypoxic states (Saito et al . , 1993a). Of considerable interest is the proposed role of QUIN as mediator for neurodegeneration occurring in inflammatory conditions (Heyes et al., 1992a; Heyes, 1993). In fact, increased and possibly neurotoxic levels of QUIN have been detected in the cerebrospinal fluid and serum of humans with infectious diseases (e.g. in patients affected by AIDS and Lyme borreliosis) (Heyes et al . , 1992a; Halperin and Heyes, 1992) and in animal tissues under experimental conditions that activate the immune system (Moroni et al . , 1991; Heyes et al . , 1992b; Saito et al., 1993b). Induction of indoleamine 2,3-dioxygenase, the first enzyme of the kynurenine pathway in extra- hepatic tissues (Takikawa et al., 1986), by cytokines (inter- feron-? and tumor necrosis factor-a) (Takikawa e t al., 1988; Saito et al., 1993~) appears to be the main link between the inflammatory response and the accumulation of QUIN and other kynurenine metabolites. Besides interfering with N-methyl D-aspartate receptors, QUIN has also been reported to have other actions, e.g. inhibition of gluconeogenesis and impairment of cardiac contractility (see Saito et al. (1993b)). 3-Hydroxyanthranilic-acid dioxygenase (3-HAO; 3-hydroxy-

anthranilate 3,4-dioxygenase, EC 1.13.11.6) is the enzyme directly involved in the synthesis of QUIN (see Fig. 1). 3-HA0 is a monomeric cytosolic protein belonging to the family of in- tramolecular dioxygenases containing non-heme ferrous iron (Fez+). 3-HA0 catalyzes the cleavage of the benzene ring of 3-hydroxyanthranilic acid (3-HANA), an intermediate in the kynurenine pathway. The product of the reaction is an unstable intermediate, a-amino-P-carboxymuconic acid esemialdehyde, which can either spontaneously rearrange to QUIN or be converted to picolinic acid after decarboxylation. 3-HA0 is widely distributed in peripheral organs, such as liver and kidney, and is also present in low amounts in the central nervous system (Foster et al., 1986). In the rat, the liver and brain enzymes show similar biochemical and immunological properties, sug- gesting that a single protein accounts for cerebral and peripheral 3-HA0 activity (Okuno et al . , 1987). Regarding its cellular compartmentation within rat brain, 3-HA0 appears to be

polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; bp, base pairb).

13792

Cloning and Expression of 3-HA0 13793

L-Tryptophan

23dioxygenose II Z3-dioxygenase L-Tryptophon lndolomine

N-Formylkynurenine

Kynurenine Formomidose omhotronsferose

Kynurenic acid L-Kynurenine

Kynurenine Jj 3-monooxygenose

3-Hydroxykynurenine

Anthranilic acid 1 Kynureninose

3-Hydroxyanthranilic acid

3-Hydroxyonthronilic u acid dioxygenose

PicOlinic acid - - a-amino-0-carboxymuconic acid E-semialdehyde

JJ non-enzymic

Quinolinic acid

I NAD

FIG. 1. Schematic overview of kynurenine pathway for metabolism of L-tryptophan.

mainly located in glial cells (Kohler et al., 1988). An increase in cerebral 3-HA0 has been reported in Huntigton's disease patients (Schwarcz et al., 1988) and after transient cerebral is- chemia in gerbils (Saito et al., 1993b). In a rat model of epilepsy, 3-HAO-positive glial cells have been reported to become highly hypertrophied in brain areas where neurodegeneration oc- curred (Du et al., 1993).

The identification of the amino acid sequence of 3-HA0 and of the transcript encoding this protein is instrumental for clari- fying the function of the enzyme and its possible involvement in disorders associated with increased levels of QUIN. In this report, we describe the molecular cloning of a cDNA encoding human 3-HA0 (h3-HAO) from liver. For this purpose, we used internal amino acid sequences obtained from the rat liver enzyme to construct degenerate oligonucleotide primers for the isolation of the rat cDNA clone by reverse transcription-polymerase chain reaction (RT-PCR) of rat liver RNA. The rat cDNAclone then served as a hybridization probe for the screening of a human hepatoma cell cDNA library.

MATERIALS AND METHODS Assay of 3-HA0 Activity-3-HA0 activity was measured spectropho-

tometrically as previously described (Walsh et al., 1991). The assay mixture consisted of small aliquots (2-25 111) of the enzyme preparation and 3-HANA (0.1 mM, unless otherwise stated) in a 1-ml final volume of 10 mM Hepes, pH 6.5, containing 0.3 mM Fe(NH,SO,),. The reaction was

started by the addition of the substrate, and the increase in absorbance at 360 nm was monitored over 15 s a t 30 "C. For activity calculation, a molar extinction coefficient of 47,500 M" cm" for the reaction product (a-amino-p-carboxymuconic acid esemialdehyde) (Koontz and Shiman, 1976) was used.

Since 3-HA0 is quite unstable following manipulation (possibly due to oxidation and/or iron removal), the activity of the enzyme was also assayed after reactivation of the enzyme essentially as described by Koontz and Shiman (1976). The pH of the enzyme preparation was adjusted to 3.5 with acetic acid; and after incubation for 3 min in the presence of 1 mM Fe(NH,SO,), and 0.5 mM dithiothreitol and subsequent 10-fold dilution in 0.1 M Hepes, pH 6.5, activity was measured as described above.

Purification of 3-HA0 from Rat Liver-Rat 3-HA0 (r3-HAO) was purified starting from livers from male albino rats (Fiillinsdorf SPF; 100 9). Livers were cut into small pieces and homogenized by means of an Ultra-Turnax T25 (8500 rpm for -1 min) in Hepes, pH 6.5, containing 0.15 M KCl, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM benzamidine, and 0.3 mM Fe(NH,SO,),. The homogenate was centrifuged a t 800 x g for 10 min at 4 "C, and the supernatant was centrifuged at 49,000 x g for 20 min a t 4 "C. The pH of the collected cytosolic fraction was then adjusted to 5.0 by the addition of 10% acetic acid, and the mixture was stirred for 20 min in ice. After centrifugation a t 12,000 x g for 20 min, the supernatant was collected, and its pH was readjusted to 6.5 with 1 M KOH and then subjected to ammonium sulfate fractionation. The protein pellet resulting from 40-65% (saturation) ammonium sulfate precipitation, containing most of the 3-HA0 activity, was resuspended in 10 ml of 10 mM Hepes, pH 6.5, 1 mM dithiothreitol and dialyzed overnight against the same buffer. After centrifugation at 12,000 x g for 20 min a t 4 "C, the preparation was applied to a DEAE-Sepharose Fast-Flow column (34 x 2.5 cm; Pharma- cia LKB Biotechnology Inc.). The column was eluted at 0.5 mVmin with a linear NaCl gradient (0-0.4 M in 10 h) in the dialysis buffer. Fractions containing 3-HA0 activity were pooled and concentrated by ammonium sulfate precipitation (70% saturation), and the protein pellet was resuspended in 5 ml of 20 mM bis-Tris, pH 6.5, containing 1 mM dithiothreitol. The preparation was applied to a Sephadex G-75 column (150 x 5 cm), which was then eluted at 1 mumin with the above buffer. After concentration of the active fractions in a stirred ultrafiltration cell (PM-10 membrane, Amicon, Inc.), the enzyme was further purified by ion-exchange chromatography on a Mono Q HR 5/5 column (Pharma- cia). 3-HA0 was eluted with a linear NaCl gradient (0-0.2 M in 45 min) in 20 mM bis-Tris, pH 6.5, a t a flow rate of 0.8 mumin.

For mass spectrometric (MS) analysis of rat 3-HAO, the purified protein was injected into a reverse-phase Poros HR perfusion chromatography column (100 x 2.1 mm; PerSeptive Biosystems) and eluted at 1 mL'min with an acetonitrile gradient in 0.1% trifluoroacetic acid. The collected protein was dried in a Speed-Vac (Savant Instruments, Inc.), reconstituted in MS buffer (2.5% acetic acid, 50% acetonitrile in water), and subjected to ion-spray MS.

Protein content was determined by the Pierce bicinchoninic acid protein assay kit according to the manufacturer's instructions. The purity of 3-HA0 after the various purification steps was monitored by SDS-polyacrylamide gel electrophoresis (PAGE).

SDS-PAGE, Isoelectric Focusing, and Immunoblotting Techniques- Analytical SDS-PAGE was performed using a Mini-Protean" apparatus (Bio-Rad) on 12.5% polyacrylamide slab gels (Laemmli, 1970). Isoelec- tric focusing was performed on 2.5% polyacrylamide gels by means of a mini-isoelectric focusing cell (Model 111, Bio-Rad) using 3-10 or 4-6.5 Pharmalyte" (Pharmacia) as carrier ampholytes. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 or with a silver staining kit (Bio-Rad) according to the manufacturer's instructions.

For immunoblotting, proteins were separated by SDS-PAGE and then transferred (1 h at 0.3 A) to nitrocellulose membranes as previously described (Towbin et al., 1979). The nitrocellulose sheet was incubated with rabbit polyclonal antibodies raised against rat liver 3-HA0 (see Okuno et al. (1987)) or with antisera of mice immunized with the rat protein purified in the present experiments, 3-HA0 was then detected, after incubation with a peroxidase-linked second antibody, by the enhanced chemiluminescence detection system (ECL, Amersham Corp.).

Rat Liver 3-HA0 Digestion, Peptide Mapping, and Amino Acid Sequencing-Purified 1-3-HA0 was subjected to SDS-PAGE on a 12.5% minigel and subsequently electroblotted onto a polyvinylidene difluoride membrane (Immobilon-P"q, Millipore Corp.) for 30 min at 0.5 A. In the SDS-PAGE step, piperazine diacrylamide was used as cross-linker instead of N,N"methylenebisacrylamide, and the running buffer in the

13794 Cloning and Expression of 3-HA0 TABLE I

Summary of the purification steps for 3-HA0 fiom rat liver Activity was measured with 0.1 m 3-HANA for 15 s a t 30 "C. The

amount of product formed was calculated using a molar extinction coefficient ( E ) of 47,500 cm" (Koontz and Shiman, 1976). The purification factor over the starting material after each step is shown in parentheses.

Purification step Total Total Specific activity

prokin actlvlty Nonactivated ActivatedD

mg pmollmin pmollminlmgprotein

Homogenate 750 105 0.14 0.19

(NH,),SO, 141 39.6 0.28(2.0) 0.54(2.8) DEAE-Sepharose 12.7 19.8 1.56(11.1) 3.09U6.3) SephadexG-75 4.6 17.6 3.79(27.0) 5.30(28.0) Mono Q 0.4 3.6 8.95(63.9) 53.80 (283)

supernatant

"Materials and Methods." a The activation procedure was performed as described under

upper reservoir contained 0.002% thioglycolic acid. This should mini- mize protein residue modification during electrophoresis. After transfer, the membrane was quickly stained with Ponceau S (0.1% in 1% acetic acid) and destained with several changes of 1% acetic acid and water. The band corresponding to 3-HA0 was then cut into small pieces and placed in a 2-ml polypropylene tube. After treatment with 0.2% polyvinylpyrrolidone (30 kDa) in methanol, digestion was performed in 200 pl of 0.1 M Tris, pH 8.5 (containing 5% isopropyl alcohol), by two subsequent additions of 1 pg of modified trypsin (Promega). The procedure used for the digestion of the blotted protein and the subsequent extrac- tion of the resulting peptides was essentially that described by Bauw et al. (1989).

r3-HA0 peptides were separated by reverse-phase HPLC on a Vydac C,, column (250 x 2.1 mm, 5-pm particles). The HPLC system consisted of a Waters 625 LC pump (1-ml sample loop) with a Model 994 photo- diode array detector. Peptides were eluted with an acetonitrile gradient in water, 0.05% trifluoroacetic acid a t a flow rate of 250 pl/min. Peaks were manually collected and directly submitted to sequence analysis by means of an Applied Biosystems Model 475A protein sequenator with on-line phenylthiohydantoin-derivative detection. The sequencing pro- tocols for pulsed liquid chemistry were essentially those recommended by the manufacturer. The sequence of some peptides was further con- firmed by ion-spray MS or tandem MS.

Preparation of RNA-Total RNA was extracted from human and adult rat livers and from the human hepatoma HepG, cell line (ATCC HB8065) by the guanidinium isothiocyanatdcesium chloride method (Chirgwin et al., 1979). Poly(A)* RNA was obtained by two purification cycles on oligo(dT)-cellulose columns (Sambrook et al., 1989).

Isolation of Rat 3-HA0 cDNA by RT-PCR-Reverse transcription reactions were carried out in 10 m Tris-HC1, pH 8.3,50 m KC1,5 m MgCl,, 1 m dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dITp, 20 units of RNase inhibitor, 100 pmol of antisense primer, 1 pg of rat liver poly(A)+ RNA, and 50 units of cloned Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer) in a final volume of 20 pl at 42 "C for 60 min. PCR was performed in a final volume of 100 pl containing 20 pl of reverse transcription reaction, 10 m Tris-HC1, pH 8.3, 50 m KC], 2 mM MgCl,, 0.2 m dATP, 0.2 m dCTP, 0.2 mM dGTP, 0.2 m d'ITP, 100 pmol of sense primer, and 2.5 units of AmpliTaq DNA polymerase (Per- kin-Elmer). Cycling was performed a t 95 "C (1 rnin), 56 "C (1 rnin), and 72 "C (1 min) for 35 cycles. The final cycle had an extension time of 10 min.

The final PCR product was extracted with phenollchloroform and then precipitated with ethanol. PCR products were electrophoresed on a 1% agarose gel, and the major band of 470 base pairs (bp) was cut from the gel and subcloned into the SmaI site of M13mp19 replicative form DNA by blunt-end ligation. Degenerate oligonucleotide primers were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX).

Construction and Screening of cDNA Library-Human single- stranded cDNA was synthesized from 10 pg of HepG, poly(AY RNA and converted to double-stranded cDNA by RNase H and DNA polymerase I using the Amersham cDNA synthesis kit (RPN 1256). EcoRI meth- ylation of double-stranded cDNA, addition of EcoRI linkers, ligation to h g t l l DNA, and in uitro packaging were carried out according to the Amersham h g t l l cDNA cloning kit (RPN 1280). The cDNA library was screened following previously described procedures (Huynh et al., 1984). In brief, the filters were hybridized with a 32P-labeled nick- translated 470-bp rat liver cDNAprobe for 24 h at 42 "C in 6 x NET (1

A kDa 1

98 -I 45 66 1 31 4- 22 -I

2oo 1

FIG. 2. A, SDS-PAGE analysis of purified r3-HA0 (Mono Q column eluate, -4 pg of protein). The gel lane was stained with Coomassie Brilliant Blue R-250. The molecular mass scale shown on the ordinate was obtained using the Bio-Rad SDS-PAGE marker proteins (low range of 14-97 ma) . B, immunoblot analysis of purified r3-HA0 and recombinant h3-HA0 expressed in HEK-293 cells. Lane 1, purified r3-HA0 (-4 pg); lane 2, cytosolic proteins from nontransfected HEK-293 cells (-40 pg); lane 3, cytosolic proteins from transfected cells (-40 pg). Proteins blotted onto nitrocellulose membranes were incubated overnight with rabbit anti-r3-HA0 polyclonal antibodies (Okuno et al., 1987). Bound immunoglobulins were detected, after incubation with a peroxidase-linked secondary antibody, using the Amersham ECL detection system. The molecular mass scale was obtained using Rainbow' marker proteins (12-200 kDa; Amersham Corp.).

0.06T

0.04 k I \ I I

0 . 0 0

40 50 60 70 80

Time (min)

FIG. 3. Reverse-phase C,, HPLC analysis of rat liver 3-HA0 tryptic digest. The polyvinylidene difluoride-blotted protein was digested, and the generated peptides were extracted as described under "Materials and Methods." Chromatography was performed with 3% acetonitrile in 0.05% trifluoroacetic acid as initial buffer and with 80% acetonitrile in 0.05% trifluoroacetic acid as buffer B. The column was

buffer B in 70 min. The flow rate was 250 pl/min, and absorbance was eluted isocratically for 20 min and then with a linear gradient to 60%

monitored at 214 nm. The tryptic peptides subjected to sequence analysis are indicated. AU, absorbance units.

x NET = 150 m NaC1, 15 mM Tris-HC1, pH 8.3, 1 m EDTA), 1 x Denhardt's solution (1 x Denhardt's solution = 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 0.1% SDS, 1 m Na,P,O,, 100 m ATP, 20% deionized formamide, and 500 pg/ml salmon sperm DNA and were washed twice a t 42 "C for 1 h in 2 x SSC (1 x SSC = 150 m NaCI, 15 m sodium citrate, pH 7.4), 0.1% SDS.

DNA Sequencing-The cDNA clones were digested with a number of restriction enzymes. The resulting fragments were subcloned into the M13mp18 sequencing vector in both orientations. Sequencing was performed by the dideoxy chain termination method (Sanger et al., 1980) using deoxyadenosine 5'-[a-35Slthiotriphosphate (Amersham Corp.) and the Sequenase Version 2.0 system (U. S. Biochemical Corp). All ambigu- ous regions were sequenced using synthetic oligonucleotide primers. Sequence analysis was performed on a VAX computer using the GCG program (Genetics Computer Group, University of Wisconsin, Madison, WI).

Production of Recombinant Human 3-HA0 in HEK-293 Cells-The


A,

.. 0)

5

c. 2 n - N

Peptide sequence I I 1

T7: ELOAGTSLSLFGDSYETQVIAXGO -

T9: SDYHIEEGEEVFYQLEGDMVLR

B. Degeneraie oligonucleotides

S-T7: S'TTYGGIGAYWSITAYGARACICARGT 3'

A-T7: SACYTGIGTYTCRTAISWRTCICCRAA 3'

S-T9: STTYTAYCARYTIGARGGIGAYATGCT 3'

A-T9: SACCATRTCICCYTCIARYTGRTARAA 3'

FIG. 4. Isolation of r3-HAO cDNA fragment by RT-PCFL A, the amino acid sequences of two tryptic peptides from purified rat liver 3-HA0 are shown in single-letter code. The regions of peptides that were selected for the synthesis of degenerate oligonucleotides are ouerlined. B, degenerate sense ( S ) and antisense (A) oligonucleotide primers corresponding to the peptide sequences inA are indicated. Y, CT; W, AT; S, GC; R, AG; I, inosine. C, two combinations of sense and antisense oligonucleotides (lane 1, sense-T7/antisense-T9; lane 2, sense-T9/antisense-T7) were used in RT-PCRs of rat liver poly(AY RNA. RT-PCR products from each reaction (lanes I and 2 ) were resolved by agarose gel electrophoresis and visualized after staining with ethidium bromide. The arrow points to a 470-bp fragment containing the 1-3-HA0 protein sequence.

cDNA encoding h3-HA0 was subcloned into the blunt-ended BamHI site of the expression vector pBC/CMV as described previously (Bertocci et al., 1991). This vector allows the high level expression of a foreign

bryonic kidney fibroblast cells (HEK-293 cell line, ATCC CRL1573) gene under the control of the cytomegalovirus promoter. Human em-

were transfected at their exponentially growing stage by the lipopoly- amine-mediated method using Transfectam (IBF Biotechnics) as described (Loeffler et al., 1990). Two days after transfection, the cells were harvested, centrifuged, and washed twice with phosphate-buffered sa- line. The cell pellet was then resuspended in 2 ml of 10 mM Hepes, pH 6.5, containing 10 mM 2-mercaptoethanol and 0.3 mM Fe(NH,S04), and homogenized in a glass-Teflon homogenizer. After centrifugation at 28,000 x g for 15 min, the resulting cytosolic fraction was assayed for 3-HA0 activity or subjected to immunoblot analysis.

Northern Blot Analysis-Poly(AY RNA (6 pg) was fractionated on a 1.1% agarose gel containing formaldehyde, vacuum-blotted (PosiBlot, Stratagene) onto an Amersham Hybond-N filter (RPN 1520N), and cross-linked by W irradiation. The RNA blot was hybridized for 20 h at 42 "C with a 32P-labeled nick-translated hHAOF, cDNA probe (1 x lo6 cpdml) in 50% formamide, 5 x SSC, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% SDS, 10% dextran sulfate, and 50 pg/ml salmon sperm DNA. The filters were washed once in 2 x SSC at room temperature for 30 min and then once in 1 x SSC, 0.5% SDS a t 55 "C for 1 h.

RESULTS AND DISCUSSION Purification of 3-HA0 from Rat Liver and Peptide Se-

quencing-To obtain partial amino acid sequences of 3-HAO, the protein was purified from rat liver using a simplified version of previously published methods (Koontz and Shiman, 1976; Okuno et al., 1987). As shown in Table I, the purification factor was markedly influenced by the reactivation procedure (partial acid denaturation and renaturation in the presence of Fe2+ and dithiothreitol), indicating, as previously reported (Ves- cia and di Prisco, 1962; Koontz and Shiman, 19761, that the enzyme is progressively inactivated during the purification procedure. K,,, values of 3-HANA were unchanged in crude and purified enzyme preparations (6.8 2 2.0 and 5.6 2 2.5 p ~ , respectively). The reactivation of the enzyme did not significantly alter the affinity of the substrate 3-HANA. After the final Mono

CGCGGGAGGACAGCGCTGCGAGGAGGCGCCCGGGACAGTCATGGAGCGCCGCCTGGGAGT M E R R L G V I

G A G G G C C T G G G T G A A G G A G A A C C G G G G C T C C T C C C C T C A T R A W V K E N R G S F Q P P V C N K L M

GCACCAGGAGCAGCTCRAAGTCATGTTCGTCGGAGGCCCCAACACCAGGAAGGACTATA H Q E Q L K V M F V G G P N T R K D Y H

CATCGAAGAGGGTGAAGAGGTATTTTACCAGCTGGAGGGAGACATGGTTCTCCGAGTCT S-

I E E G E E V F Y Q L E G D M V L R V L

GGAGCAAGGGRRACACCGGGATGTGGTCATTCGGCAGGGAGAGATATTCCTCCTGCCTGC E Q G K H R D V V I R Q G E I F L L P A

1D

T(u CAGGGTGCCCCACTACCACAGAGGTTTGCCAACACCGTGGGGCTGGTGGTTGAGCGAAG

+ R V P H S P Q R F A N T V G L V V E R R -?.I T4 I-

GCGGCTGGAGACCGAGCTAGATGGGCTCAGGTACTATGTGGGCGACACCATGGACGTTCT R L E T E L D G L R Y Y V G D T M D V L

GTTTGAGAAGTGGTTCTACTGCAAGGACCTCGGCACGCAGTTGGCCCCCATCATCCAGGA F E K W F Y C K D L G T Q L A P I I Q E

GTTCTTCAGCTCTGAGCAGTACAGAACAGGRRAGCCCATCCCTGACCAGCTGCTC~GGA F F S S E Q Y R T G K P 6 P D Q L L K E

GCCACCATTCCCTCTGAGCACACGATCCATCATGGAGCCCATGTCCCTGGATGCCTGGCT X-

P P F P L S T R S I M E P M S L D A W L

G G A C A G C C A C C A C A G G G A G C T G C A G G C A C A C C A C T C D S H H R E L Q A G T P L S L F G D T Y

TGAGACCCAGGTGATCGCCTATGGGCAAGGCAGCAGCGAAGGCCTGAGACAGAATGTGGA E T Q V I A z G Q G S S E G L R & ! Y D

CGTGTGGCTGTGGCAGCTGGAGGGCTCCTCGGTGGTGACAATGGGGGGACGGCGCCTGAG - V F A W Q L E G S S V V T M G G R R L S

" T6h

x-1,+

S T 7 S-

" T R t

CCTGGCCCCTGATGACAGCCTCTGGTGCTAGCTGGGACCTCGTATGCCTGGGAGCGAAC L A P D D S L L V L A G T S Y A W E R T

ACAAGGCTCTGTGGCCCTGTTGTGACCCAGGACCCTGCCTGCAAGAAGCCCCTGGGGTG Q G S V A L S V T Q D P A C K K P L G '

ACCCTCTn;CCATGGCCTGAAGCAGCCACAGGTTGGCCAAGCACCCTCGAGTGCCATCCC TGCCAAACAACTCTCCCAGCCCCCACTACCTCTCTGTGTACTGCCGCTGTGTCCCCCACA GACCTGCACATTGTTGTCACCCACCCTCCTGCCCTTCTCAGCCCAGATGCCATGCCCTGG GCGGGCAGCAGCTCCCCATCTCTCTGGCAGACTCAGCCCACTGCCTTGCCAGT~CC AGGTGGTCTACCCCCGGCCCCGCT~CCATTCCTCTGTCCCTGCAGACTCAGTGCAG CACTTCCACACCAAGAAGGCCCTCAATAAAGGCTTCCTTCCTGAGGAACGC- AAAAAAAAAAAAAAAA

20 7

nn 27

1411 47

200 67

260 x7

3211 I m

3110 127

440 147

500 I 67

S 6 0 I R7

620 207

6R0 227

740 247

800 267

860 286

9211 980

Iwn I I 0 0 IIM

1226 12211

cloned human 3-HAO. The amino acid sequence of the encoded FIG. 5. Nucleotide and predicted amino acid sequences of

polypeptide is shown in single-letter code below the nucleotide sequence and is numbered beginning with the initiating methionine. Nucleotides are numbered in the 5' to 3' direction, and positions are shown to the

ATG. The tryptic peptides (T9, T6a, T4, T6b, T7, and T8) that have been right. Nucleotide 1 is the A residue of the initiating methionine codon

isolated and sequenced from rat liver 3-HA0 are underlined. X denotes amino acids whose identities could not be assessed by Edman degradation. The asterisk denotes the 3"terminal stop codon. The polyadenylation signal is ouerlined.

Q column step, SDS-PAGE analysis of the active fractions showed the presence of a single major protein band with a molecular mass of -33 kDa (Fig. 2 A ) . This value is in the same range as those reported for 3-HA0 from bovine kidney and rat liver (Koontz and Shiman, 1976; Okuno et al., 1987). In West- ern blot analysis, the isolated protein was recognized by rabbit polyclonal antibodies raised against rat liver 3-HA0 (Okuno et al., 1987) (Fig. 2 B ) . Isoeletric focusing analysis of the purified protein indicated that the PI of r3-HA0 was -5.2 (data not shown).

More precise determination of the molecular mass of the purified protein could be achieved by MS analysis after chromatography of the protein on a reverse-phase HPLC column to remove buffer salts and additives. A mass of 32,627 2 3 Da was obtained for r3-HAO.

To obtain information on its amino acid sequence, purified 1-3-HA0 was blotted onto a polyvinylidene difluoride membrane and submitted to either direct NH,-terminal sequencing or in situ proteolytic digestion. Unfortunately, no sequence data could be obtained from direct Edman degradation, even though the amount of protein estimated by the intensity of the Ponceau S staining should have yielded a clear signal. This indicated that the protein was NH,-terminally modified either post- translationally or during the purification procedure. After in situ trypsin digestion of the polyvinylidene difluoride mem-

13796 Cloning and Expression of 3-HA0

r3-HAO: h3-HAO:

r3-HAO: h3-HAO:

r3-HAO: h3-HAO:

r3-HAO: h3-HAO:

h3-HAO:

T9: SDYHIEEGEEVFYQLEG FYQLEG 6

MERRLGVRAWVKENRGSFQPPVCNKLMHQEQLKVMFVGGPNTRKDYHIEEGEE~~~~;~~ 60

DMVLR DMVLRVLEQGEHRDWIRQGEIFLLPARVPHSPQRFANTMGLVIERRRMETELDGLRYYV 66 DMVLRVLEQGKHRDWIRQGEIFLLPARVPHSPQRFANTVGL~RRRLETELDGLRYYV 120 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T6a: QGEIFLLPAR T 4 : FANTMGLVIER

GDTEDVLFEKWFHCKDLGTQLAPIIQEFFHSEQYRTGKPNPDQLLKEPPFPLSTRSVMEP 126 GDTM~VLFEKWFYCKDLGTQLAPIIQEFFSSEQYRTGKPIPDQLLKEPPFPLSTRSIMEP 180 *** . . . . . . . . . . . . . . . . . . . . . . . . . * * * * * * * * * . . . . . . . . . . . . . . . . . . . .

T6b: TGTPNPDQLLXEPPFXLL

MSLKAWLESHSRELQAGTFLSLFCDSYETQ MSLDAWLDSHHRELQAGTPLSLFGDTYETQVIAYGQGSSEGLRQNVDVWLWQLEGSSWT 240 *** . *** . ** * * * * * * * * * * * * . * * * *

MGGRRLSLAPDDSLLVLAGTSYAWERTQGSVALSVTQDPACKKPLG 286

T7: ELQAGTSLSLFGDSYETQVIAXGQ T 8 : XXXDVPLXQLEGS 156

FIG. 6. Comparison of h3-HA0 primary structure with r3-HA0 partial sequence. The predicted partial amino acid sequences of r3-HAO have been aligned with the human amino acid sequence using the PILE UP program of the Genetics Computer Group sequence analysis software. Amino acid sequences obtained from the rat liver 3-HA0 peptides are shown over the predicted rat sequence. Amino acid identities (*) and conservative substitutions (.) are indicated.

. .

brane-blotted protein and peptide separation on a reverse- phase C,, column (Fig. 3), the collected peptides were submitted to Edman degradation. Peaks T4 and T6 were also analyzed by MS or tandem MS. The analyzed peptide peaks yielded unambiguous sequences, which ranged in size from 10 to 24 residues (see Figs. 5 and 6), with the exception of peptide T8, in which several residues could not be identified. No significant matches were found when these sequences were compared to those in the SwissProt and Protein Identification Resource Pro- tein Data Banks.

Isolation of Rat Liver 3-HA0 cDNA-Using the amino acid sequences of the analyzed tryptic peptides of purified rat liver 3-HAO, we were able to obtain the cDNA for r3-HA0 using RT-PCR. Two peptide sequences from the least degenerate regions of peptides T7 and T9 were selected to construct synthetic degenerate oligonucleotides. Because the relative order of the two tryptic peptides within the protein was not known, degenerate sense and antisense oligonucleotides corresponding to the tryptic peptide sequences were synthesized (Fig. 4, A and B) . Poly(A)+ RNA extracted from rat liver was reverse-transcribed using each degenerate antisense oligonucleotide as a primer, and the resulting cDNA was used as a PCR template with each possible combination of degenerate sense and antisense oligonucleotides from the two tryptic peptides. After 35 rounds of amplification, one distinct PCR product was detected by agarose gel electrophoresis for the primer combination sense-T9/ antisense-T7 (Fig. 4C, lane 2). DNA sequence analysis revealed that the 470-bp fragment contained an open reading frame coding for a polypeptide of 156 amino acids, starting with part of peptide T9 and ending with the T7 sequence. Moreover, this cDNA fragment encoded the tryptic peptides T6a, T4, and T6b (see Fig. 61, confirming that it was indeed a partial cDNA for r3-HAO.

Molecular Cloning of Human 3-HA0 cDNA-Northern blot analysis of HepG, mRNA with the 470-bp rat partial cDNA probe demonstrated a single mRNA species of -1.3 kilobases (data not shown), indicating that these cells express 3-HAO. Therefore, the HepG, poly(A)+ RNA was used to construct a A g t l l cDNA library. An initial screening of 1.2 x lo6 recombi- nants at moderate stringency with the rat partial cDNA probe revealed 18 positive clones with inserts ranging from 2700 to 700 bp. The three clones giving the strongest signal, designated AhHAOF,, AhHAOF,, and AhHAOF,, were chosen for further characterization. DNA sequence analysis revealed that all three clones were identical, except that clone AhHAOF, contained 30 additional nucleotides in the 5"untranslated region.

m

a z E

I I I

kb

4.40 -

2.37 -

1.35 -

0.24 -

FIG. 7. Northern blot hybridization of poly(A)+ RNAs from human liver and HepG, cells. The hybridization probe was the 32P- labeled nick-translated cDNA insert from clone AhHAOF,. Numbers on the left indicate kilobases as determined from RNA size markers (Life Technologies, Inc.). The blot was exposed to autoradiography film for 14 h.

The nucleotide and deduced amino acid sequences of clone AhHAOF, are shown in Fig. 5. The nucleotide sequence of clone AhHAOF, (nucleotides -40 to 1236) contained the largest open reading frame, starting a t nucleotides 1-3 and terminating at nucleotides 859-861 with a stop codon (TGA). This 858-bp open reading frame codes for a polypeptide of 286 amino acid residues with a predicted molecular mass of 32,589 Da, a value very close to the mass obtained by MS analysis of purified r3-HA0 (see above). The first initiation codon (nucleotides 1-3) is embedded in the sequence AGTCATG, which does not match perfectly with the consensus sequence CCACCATG frequently found for eukaryotic translation initiation (Kozak, 1984). Clone AhHAOF, contains 40 and 346 nucleotides in the 5'- and 3'- untranslated regions, respectively.

A consensus sequence for the polyadenylation of mRNA tran-


TABLE I1 Kinetic parameters for recombinant h3-HA0 transiently expressed in

HEK-293 cells Activity was measured in the presence of 10 different concentrations

of 3-HANA (1-50 p ~ ) for 15 s at 30 "C. No activity could be measured in nontransfected HEK-293 cells.

K, vmax w nmol imin i mg protein

Nonactivated 1.91 2 0.21 130 2 14 Activated 2.34 * 0.14 185 -c 21

scripts of higher eukaryotes (AATAAA) is found a t nucleotides 1185-1190, which is 17 nucleotides downstream from the poly(A) tail. The amino acid sequences of the rat tryptic peptides T9, T6a, T4, T6b, T7, and T8 were all found in the predicted protein sequence of h3-HAO. No potential N-linked gly- cosylation site (Asn, Xaa, SerPThr) (Marshall, 1972) was found in the predicted amino acid sequence of h3-HAO.

Comparison of the 3-HA0 partial sequence deduced from the rat liver cDNA (amino acids 1-156) with the deduced amino acid sequence of h3-HA0 (amino acids 1-286) showed that r3-HA0 and h3-HA0 exhibit 94% similarity (Fig. 6). The extent of the sequence identity of h3-HA0 to r3-HA0 was 90%. Nucle- otide sequence identity between h3-HA0 and r3-HA0 was found to be 87%. No significant similarity was found between h3-HA0 and any nucleotide sequences stored in the GenBankTM/EMBL Data Bank using the FASTA program (Pearson and Lipman, 1988).

Blot Hybridization of RNA-A Northern blot of poly(A)+ RNAs isolated from human liver and HepG, cells is shown in Fig. 7. By this analysis, one major species of h3-HA0 mRNA was detected using 32P-labeled nick-translated hHAOF, cDNA. This mRNA species is -1.3 kilobases in size, indicating that our cloned cDNA (AhHAOF,) represents most of the h3-HA0 RNA transcript.

Transient Expression of Human 3-HA0 in HEK-293 Cells- To confirm that the isolated cDNA encoded 3-HAO, the human 3-HA0 cDNA was subcloned into the eukaryotic expression vector pBC/CMV (Bertocci et al., 1991). HEK-293 cells were then transfected with the resulting construct, and 3-HA0 activity was assayed in the cytosolic fraction of the lysed cells. Whereas in the nontransfected HEK-293 cells, no enzymatic activity could be detected, the cells transfected with the pBC/ CMV-h3-HAO construct exhibited high 3-HA0 activity (Table 11). Kinetic analysis of the enzymatic activity in the transfected cells revealed a K, value for 3-HANA in the low micromolar range, similar to the values obtained by us for native r3-HA0 and by other authors for the liver and brain enzymes in both rats and humans (Foster et al., 1986; Schwarcz et al., 1988). Similar to the native enzyme from rat liver (see Table I), partial acid denaturation and renaturation of the recombinant human enzyme in the presence of Fez+ and a reducing agent increased the enzymatic activity by -40% without significantly affecting the substrate K,,, value. The activity of the recombinant enzyme (measured with 50 p~ substrate) could be inhibited by m-hy- droxybenzoic acid (a weak inhibitor of 3-HAO) (Vescia and di Prisco, 1962) with an IC,, of 0.48 mM.

Immunoblot Analysis of Human 3-HA0 Expressed in HEK- 293 Cells-To characterize further recombinant h3-HA0 expressed in HEK-293 cells, cytosolic proteins from h3-HAO- transfected cells were subjected to immunoblotting using rabbit anti-r3-HAO polyclonal antibodies (Okuno et al., 1987). As shown in Fig. 2B, a major immunoreactive band in the 32-33-kDa range, in accordance with the predicted molecular mass of the cloned human protein, was detected in h3-HAO- transfected cells, but not in the nontransfected cells. By using antisera of mice immunized with our purified r3-HA0, identi-

cal results were obtained (data not shown). Although the rabbit anti-r3-HAO polyclonal antibodies used have been reported not to cross-react with the human enzyme in Ouchterlony double- diffusion test (Okuno et al., 1987), these antibodies do appear to stain denatured h3-HA0 in immunoblot analysis. This can be reasonably expected from the high similarity between the rat and human amino acid sequences.

The availability of the human cDNA for 3-HA0 will be instrumental for the study of 3-HA0 function, including the regu- lation of its expression and the role played by this enzyme in pathological conditions.

Acknowledgments-We express our gratitude to the following per- sons at F. Hoffmann-La Roche Ltd. (Basel, Switzerland): Drs. Grayson Richards and Deborah Hartman for critical reading of the manuscript, Francis Vilbois and Urs Rothlisbertger for MS analysis and sequencing of peptides, and Martine Wdonwicki and Brigitte Fritz for invaluable secretarial work.

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