THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 6, Issue of February 25, pp. 3256-3262, 1990 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

An Alternatively Processed mRNA Specific for y-Glutamyl Transpeptidase in Human Tissues*

(Received for publication, November 29, 1988)

Andre! Pawlak, Edward H. Cohen+, Jean-Nob1 Octaves, Rene SchweickhardtS, Shi-Jun Wu, Fr6d6rique Bulle, Naima Chikhi, Ja-Hyun Baik, Sylvie Siegrist, and Georges Guellaen From the Znstitut National de la Sant6 et de la Recherche Mkdicale Unite? 99, H6pital Henri Mondor, 94010 Cr&eil, France, ilnteerated Genetics. Framineham. Massachusetts 01701, and the §Uniuersiti Catholique de Louucin, Neurochimie UCL 53-59, ku. Ey Mounier 120d Bruxelles, Belgium

Human y-glutamyl transpeptidase (GGT)’ is com- posed of two subunits derived from a single precursor (Nash, B., and Tate, S. S. (1984) J. Biol. Chem. 259, 6’78-685; Finidori, J., Laperche, Y., Tsapis, R., Bar- ouki, R., Guellaen, G., and Hanoune, J. (1984) J. Biol. Chem. 259,4687-4690) consisting of 569 amino acids (Laperche, Y., Bulle, F., Aissani, T., Chobert, M. N., Aggerbeck, M., Hanoune, J., and Guellai+n, G. (1986) Proc Natl. Acad. Sci. U. S. A. 83, 937-941). In the present study we report the cloning of an altered form of this precursor from human liver. We have isolated two clones, one 2,632 base pairs (bp) long from a fetal liver cDNA library and one 926 bp long from an adult liver cDNA library, each containing a 22-bp insertion that introduces a premature stop codon and shortens the open reading frame to 1,098 bp when compared with known human cDNA sequences specific for GGT. Sequence analysis of a human genomic GGT clone shows that this insertion of 22 bp is generated by a splicing event involving an alternative 3’-acceptor site. By polymerase chain reaction experiments we demonstrate that the alternatively spliced mRNA is present in polysomes from the microsomal fraction of a human hepatoma cell line (Hep G2) and thus could encode an altered GGT molecule of 39,300 Da (366 amino acids) encompassing most of the heavy subunit which is normally 41,500 Da (380 amino acids). The altered mRNA is detected in various human tissues including liver, kidney, brain, intestine, stomach, pla- centa, and mammary gland. This report is the first demonstration of an alternative primary sequence in the mRNA coding for GGT, a finding that could be related to the presence of some inactive forms of GGT detected in human tissues.

y-Glutamyl transpeptidase ((Lglutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2) (GGT)’ is an enzyme involved in the metabolism of glutathione and other y-glu- tamyl compounds (l-3). It is a glycosylated heterodimer; the light subunit (Mr 30,000) contains the y-glutamyl binding site and is noncovalently linked to the heavy subunit (Mr 50,000)

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505235.

1 The abbreviations used are: GGT, y-glutamyl transpeptidase; SDS, sodium dodecyl sulfate; bp, base pairs.

that anchors the enzyme in the membrane. Both subunits are synthesized from a single mRNA encoding a 64-kDa peptide (4,5). GGT is mainly distributed in tissues exhibiting absorp- tive and secretory processes (1). The highest activity is found in kidney and intestinal cells (l-3). In normal adult liver, the GGT activity is low and mainly located in bile ducts and canalicular membranes of the hepatocytes (6). In this organ, increases in activity are observed under various physiological and pathological conditions (7, 8). Several experiments have demonstrated that these increases are often associated with structural changes in the sugar chains of the enzyme, as evidenced by a variation in the pattern of GGT isoforms in serum (9, 10). Antibodies, specific for GGT isoforms from a human primary hepatoma, have been shown to be useful for the diagnosis of some neoplastic diseases (11). In human serum (12) or in rat liver (13) such antibodies recognize reactive species which are not directly correlated with GGT activity, suggesting the presence of altered forms of GGT.

Recently we have cloned the cDNA encoding the rat kidney GGT (14), and using this cDNA we have demonstrated that several genes or pseudogenes are present in the human ge- nome (15) on chromosome 22 (16). According to the recent cloning of GGT mRNA from human placenta (17), fetal liver (18), or the human hepatoma cell line Hep G2 (19), this multigene family codes for the same GGT precursor.

In the present report we describe the isolation and sequence of two cDNAs from human adult and fetal liver libraries. These cDNAs have a 22-bp insertion as compared with other known sequences (17-19). This insertion, apparently gener- ated by alternative splicing, induces a frameshift resulting in an open reading frame that could code for an altered GGT.

EXPERIMENTAL PROCEDURES

Materials

Escherichia coli DNA polymerase I and Klenow fragment were obtained from New England Biolabs. Restriction endonucleases were purchased from New England Biolabs, Boehringer Mannheim, or Genofit S.A. TaqI polymerase was obtained from Stratagene. Radio- labeled nucleotides, cDNA synthesis kit, human placental ribonucle- ase inhibitor, and nylon membrane (Hybond N) were from Amersham Radiochemical Centre. All the other reagents were of analytical grade (Sigma, Merck, or BDH). The human genomic library has been already described (15).

Methods

Construction and Screening of the Human Fetal Liver cDNA Li- brary-The construction of the Xgtll human fetal liver cDNA library has been described before (20). Two DNA fragments generated in an indirect fashion were used to screen this library. Four oligonucleotides based on the published rat GGT cDNA sequence (14) were synthe- sized. They consisted of nucleotides 384-420, 1334-1363, 1464-1505, and 1817-1849, respectively, of the rat cDNA sequence. All four

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oligonucleotides were kinased and used to screen a rat kidney Xgtll library (Clontech). Procedures employed were essentially those used in screening the adult liver library (see next paragraph) except the hybridization temperature was 45 “C and washing conditions were 1 x SSC, 0.1% SDS at 65 ‘C. Plaques hybridizing with all four probes were purified, and four were characterized using restriction enzymes. An insert in one of the Xgtll clones appeared to be nearly as long as the published rat cDNA sequence and was subcloned into pUC18 as two separate EcoRI DNA fragments. The two fragments were then used as probes to screen a human adult kidney XgtlO cDNA library obtained from Clontech. One clone to which both probes hybridized was partially sequenced and shown to be highly homologous to the rat GGT cDNA sequence. This clone in turn was subcloned into pUC18 as two separate EcoRI DNA fragments. These two subcloned fragments were then used as probes to screen 6.3 X lo6 clones from the human fetal liver cDNA library.

Construction and Screening of the Human Adult Liver cDNA Li- brary-A liver was recovered from a donor with confirmed brain death. Ventilation and hemodynamic parameters were maintained during hepatectomy. 2 g of tissue were immediately frozen in liquid nitrogen and stored at -80 “C until the preparation of the RNA. This was done essentially by the method described by Chirgwin et al. (21). Poly(A+) RNA was isolated using oligo(dT)-cellulose chromatography as previously described (22).

The adult human liver cDNA library was constructed in the hgtll vector described by Young and Davis (23). Double-stranded cDNA (250 ng) was synthesized in the presence of reverse transcriptase, ribonuclease H, and DNA polymerase, according to Amersham pro- cedures. After methylation of the cDNA with EcoRI methylase, addition of EcoRI linkers, digestion with EcoRI, and size fractionation on a Bio-Gel A-50m column, the cDNA fragments were inserted in the EcoRI site of the Xgtll vector (2 rg). After in uitro packaging, 530,000 independent clones were obtained of which 95% were recom- binant.

Approximately 3 x lo6 clones were screened according to the method of Benton and Davis (24) using the 1.6-kilobase-long insert from the rat kidney cDNA clone Psp 64/39-l as a probe (16). The probe was purified by electroelution from agarose gel and radiolabeled by nick translation to a specific activity of 5 X 10’ cpm/rg. Filters were prehybridized in 6 x SSC (1 X SSC: 0.15 M NaCl, 15 mM sodium citrate), 1 x Denhardt’s (0.2 g/liter Ficoll, 0.2 g/liter polyvinylpyrrol- idone, 0.2 g/liter bovine serum albumin (fraction V)) containing 100 rg/ml of denatured salmon sperm DNA for 5 h at 68 ‘C; the hybrid- ization was performed overnight at 68 “C in 2 X SSC, 1 X Denhardt’s, 0.05% SDS,-25 mM NaH,PO,, pH 7.2,2 mM EDTA, pH 8, containing 100 Da/ml denatured salmon sperm DNA and 50 ng of the probe. The -. filters were washed in 0.1 X SSC, 0.1% SDS at 58 ‘C and autoradi- ographed overnight at -80 “C on Amersham hypertilm MP using an intensifying screen.

DNA Sequencing-The inserts of the cDNA or genomic clones were subcloned in M13mp18 or M13mp19 and sequenced at least twice in both directions by the dideoxy nucleotides method (25) after generation of independent overlapping clones (26). In addition, oli- gonucleotide primers were synthesized on an Applied Biosystems DNA synthesizer and used in sequencing certain regions of the fetal liver cDNA clone.

Polysome Preparation-Polysomes bound to the microsomal frac- tion of Hep G2 cells were prepared according to Cory et al. (27) with minor modifications. 10s cells were resuspended in 18 ml of ice-cold 0.8 M sucrose in buffer A (25 mM KCl. 5 mM MaCl,. 50 mM Tris- HCl, pH 7.5, 0.1 mM glutathione, 0.1 mg/ml heparin, 0.05 mg/ml cycloheximide, 50 units/ml human placental ribonuclease inhibitor) and homogenized with 6 strokes of a loose A pestle then 6 strokes of a tight B pestle in a Dounce glass homogenizer. After dilution with 1 volume of 0.57 M sucrose in buffer A, the homogenate was spun down at 12,500 rpm for 10 min in a Sorvall SS34 rotor at 4 ‘C. Microsomes were recovered from the postmitochondrial supernatant (Sl) by sedi- mentation for 20 min at 30,000 rpm (Beckman type 50 rotor) through a cushion of 5.75 ml of 0.8 M sucrose, 100 mM KCI, 5 mM MgCl,, 50 mM Tris-HCl, pH 7.5. The supernatant (S2) was extracted twice with phenol and chloroform before ethanol precipitation. The pellet was resuspended at 4 “C in 2 ml of 150 mM NaCl, 2.5 mM MgCl,, 35 mM Tris-HCl, pH 7.5, 50 units/ml human placenta nuclease inhibitor, 0.5% Nonidet P-40. 0.5% sodium dodecvl sulfate. 20 mM EDTA. 1 mg/ml proteinase K, and incubated for 10 min at ‘20 “C. The sample was then extracted and precipitated as for the S2 supernatant. Both ethanol precipitates were resuspended in 40 ~1 of sterile water.

Lactate dehydrogenase activity of each fraction was assayed (28)

during the purification in order to assess the contamination of the microsomes by the supernatant.

RNA Amplification by Polymeruse Chain Reaction Using TaqZ Polymerase-Poly(A+) RNAs were prepared from human adult kid- ney, placenta, brain, liver, human fetal liver and kidney, and Hep G2 cells as described for the adult liver cDNA librarv. with two cvcles of oligo-dT columns at the end.

Amplification of the mRNA sequences was done according to Simpson et al. (29) with minor modifications. The synthesis of the first cDNA strand was done according to the Amersham cDNA synthesis system using l-5 fig of poly(A+) mRNA or 5 ~1 of the polysomal samples, oligo-dT as primer, and reverse transcriptase. For the polymerase chain reaction amplification, 20% of the first strand cDNA was amplified with TaqI DNA polymerase in 10 mM Tris-HCl, PH 8.3. 50 mM KCl. 3 mM M&l,, 0.01% gelatin containing 200 LLM _ _ . of each deoxynucleotide and 70-100 ng of the two synthetic oligonu- cleotide primers. After the addition of 2 units of TaoI DNA polym- erase, the solution was mixed and covered with mineral oil. The DNA was heat-denatured at 94 “C (1 min) and allowed to cool at 55 “C (1 min) for primer hybridization. Synthesis was then performed at 72 ‘C (1.5 min). 30 and 60 runs of denaturation, hybridization, and synthesis were performed on mRNA isolated from tissues or polysome, respec- tively. After the amplification reaction, 20% of the samples were loaded on a 3% agarose low melting gel in TBE buffer (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA). The DNAs were then transferred onto a Hybond N membrane. The membrane was prehybridized and then hybridized overnight at 37 ‘C in 6 X SSC, 5 X Denhardt’s, 0.1% SDS, 5 mM EDTA containing 100 rg/ml denaturated salmon sperm DNA. 100 ng of 5’-end-labeled synthetic oligonucleotides (“5 X lo7 cpm/50 ml) were used as probe.

The blots were washed 3 times for 1 h at 53 “C in 1 liter of 6 X SSC, 0.1% SDS and autoradiographed overnight at -80 “C on Amer- sham hyperfilm MP using two intensifying screens. All oligonucleo- tides used were synthesized as for DNA sequencing.

RESULTS

Cloning of the Human Fetal Liver cDNA Specific for GGT- Only two positive clones were obtained after screening 6.3 X 10s clones from the human fetal liver cDNA library as de- scribed under “Methods.” The longer of the two clones is 2632 bp exclusive of a short run of A residues at the 3’-end (Fig. 1). There is a 743-bp-long 5’-untranslated region in front of one open reading frame of 366 amino acids. A second open reading frame is observed beginning at position 1055 and could encode a protein of 225 amino acids encompassing the complete sequence of the small GGT subunit. In the 3’- noncoding region (160 bp) the polyadenylation signal is found 43 bp upstream from the poly(A) tail.

Cloning of the Human Adult Liver cDNA Specific for GGT- In screening the human adult liver cDNA library using a rat cDNA probe specific for GGT, only one positive clone out of 3 X 10s was identified. This clone has an insert of 963 bp. In this sequence, the first 794 bp correspond to the 3’-region of the coding sequence (Fig. 1). They are followed by a 169-bp 3’-untranslated region, and a polyadenylation signal AA- TAAA is found 52 bp upstream of the poly(A) tail (Fig. 2). There is a complete homology between this GGT cDNA isolated from human adult liver and the 3’-part of the human fetal liver cDNA. The 3’-untranslated region is 9 bases longer in the adult liver cDNA than in the fetal liver cDNA.

Comparison with Other Known Human cDNA Sequences Specific for GGT-The sequences, for the two cDNAs we obtained, were compared with sequences coding for GGT isolated from human placenta (17), human fetal liver (18), and Hep G2 hepatoma cells (19) (Fig. 2). The 5’-noncoding sequences are organized differently. They are identical from +1 to -88 except for single base pair differences at positions -23 and -84 in the Hep G2 cDNA relative to the other sequences. A region from -89 to -243 in the human fetal liver cDNA (present study) is observed in the Hep G2 cDNA from position -207 to -361. At position -139 in our clone,

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An Alternative mRNA for Human GGT Sequences

40 50 60 KEPDNHVYTRAAVAADAKQCSKIGRDALRD

MC WA 'XT GAC MC CAT GTG TAC ACC AGG GCT CCC "W CCC GCG CAT CCC MC CA0 TGC TCG AAG ATT CC2.i AGG CAT GCA GIG COG ‘XC

70 80 90 GGSAVDAAIAALLCVGLMNAKSMGIGGGLF

GGTGGCTCTGCGGK: CAT GCAGCCATl'GCAGCC cn;'ITGTCrGI'GooC CTC ATGAATGCCCACAGCATGOOCATCGGG GGTGGCCIC~

100 110 120 LTIYNSTTRKAEVINAREVAPRLAFATMFN

CIV ACC ATC TAC MCACC AU2 ACACCA AAA GCT GAG GTC ATC MCGCC CGC GAG GTC Ccc CCCAM; CTG Ccc TTT GCCACCATGTICAAC

130 140 150 SSEPSQKGGLSVAVPGEIRGYELAHQRHGR

AGC TCG GAG CAG TCC CAG AA0 GGG GGO Cl-0 TCG CI-0 CCC GTG 0.7 GW GAG ATC CGA CCC TAT GAG CTC GCA CAC CAG CGG CAT GGG CCG

160 170 180 LPWARLFQPSIQLARQGFPVGKGLAAALEN

cTGccCToG~ccCcn:m:WrGccCA~ATCcAccrCccCcoC~GccCm:ccCcrCccCMGccCITGccGccAocC~oAAMC

190 200 210 KRTVIEQQPVLCEVFCRDRKVLREGERLTL

AAG OX ACC FLY: ATC GAG CAG CAG WI GTC 'l-E WF GAG GTG l-l"2 'EC CGG GAT AGA AAG GTG CIT Ccc GAG GGG GAG AGA GIG ACC CT'S

220 230 240 PQLADTYETLAIEGAQAFYNGSLTAQIVKD

CCG CAG CTG GCT GAC ACC TAC GAG AW CTG CCC AX GAG GGT CCC CAG KC lTC TAC MC GGC AGC CT'2 ACG G'X CA0 Al-I GTG AAG GAC

250 260 270 IQAAGGIVTAEDLNNYRAELIEKPLNISLG

ATC CAG GCGGCCGGGGGCATI GlVACAGcTGAG OACCTCMCMCTAC COT GCTGAGCTG ATCGAGCACCCGCTG AAC ATCAGCCTGGGA

280 290 300 DVVLYMPSAPLSGPVLALILNILKCYNFSR

CA'2 GTG Gn; CT0 TAC ATCCCC ACT OX OX CTC AGC OCiO CCC GTCCTGGCCCTC An: CICAAC ATC CTC AM GGGTAC AAC lTC TCC CGG

310 320 330 ESVESPEQKGLTYliRIVEAFRFAYAKRTLL

GAG ACC GTG GAG AGC Ccc GAG CAG AAG GGC Cro ACG TAC CAC WC ATC GTA GAG o(;T TIC CGG m CCC TAC Ccc MC AGG ACC CTC 'XT

340 4 350 363 GDPKFVDVTE A; '$i: iis; ::@,yX. j:.$i I:~,i::i-pij;~.:ii::p: .;a‘.:; :;gj ji$j ,:iL 4..:'ir.;yt;;::i&:. i~:-:-.g;iji~: :f a;

GCXi GAC CCC AAGTIT GlY3 GAT GTG ACl CAG GCC AGCTCT OCCGTC TCGGCAGGTCGT CCGCAACA~~ MTSEFFAAQLR

11 366

:<x$ :, gg. 5 g: . . . CC&ltiTCiCT GAC GAC ACC ACT CAC CCC AI-C TCC TAC TAC AAG CCC GAG 'I-E TAC AC0 CCG GAT GAC GGG GGC ACT GCT CAC CTG TCT GIG AQISDDTTHPISYYKPEFYTPDDCGTAHLSV

20 30 I 40

Gl'C GCA GAG GAC CCC ACT GCT GTG TCC Ccc ACC AGC ACC ATC AAC CTC TAC m GCC TCC AAGGTC CCC TCC CCCGTC AGC GC4i ATC Cl% VAEDGSAVSATSTINLYFGSKVRSPVSGIL

50 60 70

TIT MT MT GAA ATG GAC GAC 7-K AGC TCT CCC ACC ATC ACC MC GAG TIT GGG GTA CCC CCC TCA CCT CCC MT TTC An: CAG ‘3% GGG FNNEnDDFSSPSITNEFGVPPSPANFIQPG

80 90 100

AAG CAGCCG CT-2 TV2 TCC ATGTGC CCC ACG ATC AT0 CTC GCC CAG GAC GGC CAG GTC CGGATG GTGGl-G GGAGCTGCTGGG C4X ACA CAG KQPLSSMCPTIRVGQDGQVRHVVGAAGGTQ

110 120 130

ATC ACC ACG GCC ACT GCA CTG GCC ATC ATC TAC MC CTC TGG TTC GGC TAT GAC GTG AAG CC4 CCC GTC G4G GAG CCC CGG CTG CAC AAC ITTATALAIIYNLWFGYDVKRAVEEPRLRN

140 150 160

CAG c,-I CTC CCC MC GTC AC0 ACA CT-G GAG AGA MC ATT GAC CAG WA Glyj ACT WA CCC CIY: GAG ACC CC0 CAC CAT CAC ACC CA0 ATC QLLPNVTTVERNIDQAVTAALETRHHKTQI

170 180 190

GCG TCC ACC I-l-C ATC G'3 GTG GTC CM CCC AT'2 GTC Ccc ACG GCT CGT CCC TGG GCA GCT GCC TCG GAC TCC AGG AAA Ccc GGG GAG CCT ASTFIAVVQAIVRTAGGWAAASDSRKGGEP

200 210 220

GCC GGC TAC tgegtgctccaggaggaceaggctgacaagcaatccag~acaagatactcacc~gaccaggaaggggactctgggggaccggcttcccctgtgegcagcagagca A G y l . .

225

FIG. 1. Sequence of the human adult and fetal liver cDNA specific for GGT. The adult liver cDNA starts at base 936 (arrow) and is exactly homologous to the human fetal liver sequence except for nine additional bases at the 3’-end of this sequence which are shown in Fig. 2. The 22-bp insertion is underlined and the changes in protein sequence, as compared with other human cDNA sequences (17-19), are boxed. The protein sequences, corresponding to both open reading frames, are represented above and below the nucleic acid sequence. The uertical bar represents the probable NHZ-terminal part of the small GGT subunit (33).

1;;; -477 -358 -239 -120

-1

180

270

360

450

540

630

720

810

9oo

990

1088

1180

1270

1360

1450

1540

1630

1720

1836

1898

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LlVCP CAC~\TRAATGACCCC*LTCTCCC*CCCTCCAGCIGGCL

FIG. 2. Comparison of the sequence of the different human cDNA clones specific for GGT. 1, analysis of the 5’-untranslated region. W represents identical sequences among fetal liver (A), Hep G2 (B) (19), and placenta (C) (17) cDNAs. 0 represents specific sequences for each cDNA. Hatched boxes represents homologous sequences between two clones. The bases are numbered from the first base of the initiation codon. 2, sequence in the coding region of the 22-base pair insertion (A) relative to other known human sequences (B) (17-19). 3, comparison of the different 3’untranslated regions of Hep G2 (19), human fetal liver (A) (18), placenta (17), and of the human fetal (B) and adult liver characterized in the present study. The polyadenylation signals are underlined.

there is a G instead of an A in the homologous Hep G2 position. A part of this sequence -174 to -243 from human fetal liver is found in the human placenta sequence at -185 to -253. However, no other homologies were detected between our human fetal liver cDNA and the other 5’-sequences so far analyzed. It should be noted that some other homologous regions exist between Hep G2 cDNA and placenta cDNA specific for GGT in the 5’-part (Fig. 2).

The coding sequences of all the clones so far analyzed are identical except for two differences. The first difference is minor; a T instead of a C is found at position 815 in our fetal liver cDNA clone changing in alanine codon to a valine codon. The second difference, and by far more important, is a 22-bp insertion found in both our fetal and adult liver cDNAs beginning at positions 1020 and 85, respectively. The insertion modifies the reading frame and introduces a stop codon at position 1099 in the fetal liver cDNA clone and at a similar position in the adult liver cDNA clone.

The 3’-untranslated region starts at the same position for all the cDNAs. The main difference is a microheterogeneity in the length of the sequence preceding the poly(A) tail. There are three nucleotide differences between placenta cDNA spe- cific for GGT and the other sequences. Since the insertion of 22 bp in the coding sequence necessarily alters the product of this mRNA, we further focused our work on this additional sequence.

Analysis of the 2Bbp Insertion at the Gene Level-Four genomic clones corresponding to the different subclasses of human GGT genes (15) were digested by BamHI and hybrid-

ized with a labeled oligonucleotide corresponding to the 22- bp insertion found in fetal and adult liver cDNAs. The result of the Southern blot is shown in Fig. 3. Only the clones corresponding to the subclass F15 and F30 hybridized with this oligonucleotide. The faint band on clone F19 disappeared at slightly higher stringency (data not shown).

The human liver cDNA sequences were compared with the sequence of one of these genomic clones (F15) (Fig. 4). This analysis revealed that in the gene the 22-bp domain is located at the 3’-part of an intron. This sequence is bordered at each end by a 3’-acceptor consensus sequence necessary for a correct splicing (30). Therefore, the cDNAs that we charac- terized reflect the fact that, during maturation of the corre- sponding mRNAs, the internal 3’-acceptor site was used.

Detection of the 22-Base Insertion in Polysomal RNA from Hep G2 Cells and mRNA from Human Tissues-Using the oligonucleotides corresponding to the 22-bp insertion and to the control sequence as probe, we were unable to detect any GGT-specific sequence in the human liver mRNAs tested by Northern blot analysis. In order to increase the chances of detecting mRNAs containing the 22-base insertion, we used the polymerase chain reaction technique. Two oligonucleo- tides, designated oligo-A and oligo-B, were selected as de- scribed in Fig. 5A and used as primers for the amplification procedure. Amplified products using RNAs from Hep G2 polysomes and supernatant as starting materials were sub- jected to Southern blot analysis using the GGT-specific se-

24 15 11 30 19

- 1 - 3.5 Kb

FIG. 3. Hybridization of the series of human genomic clones corresponding to GGT with the oligo-22 (see Fig. 5 for se- quences of this oligonucleotide). Four different classes of human genomic clones have been already described: F15, Fll, F30, and F19 (15). The DNA of these clones was restricted using the enzyme BarnHI. Following electrophoresis and Southern blotting, the nylon membrane was hybridized with the labeled oligonucleotide 22. Clone 24 is a negative control.

FIG. 4. Comparison of the cDNA sequence specific for GGT from human tissues with the genomic sequence of clone F15. The two possible splicing events are represented. A, sequences found in Hep G2 (19), placenta (17), and human fetal liver (18). B, sequence of the human genomic clone F15 in the region involved in the splicing. The respective consensus sequences for the 5’-donor and 3’-acceptor sites are underlined. C, sequences determined on the adult and fetal livers in the present study. The 22-base pair insertion is in boldface. The corresponding protein sequences are above and below the cDNA sequences A and B, respectively. The change in protein sequence, due to the 22-bp insertion is boxed.

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3260 An Alternative mRNA for Human GGT Sequences

A A 22 CONTROL II 7 p m

999 F

1020 ,024 1040 1059 1078 1085 ,101

B

103bp, Elbp-

103bp-

s paLfLfK H aKB I S PM 1 2 ‘ST

Oligo A and B, control probe

s p aLfLfKH aKB ISP M 12

Oligo A and B, probe 22

FIG. 5. Detection of specific sequences in the mRNA using oligonucleotides and the polymerase chain reaction amplifi- cation method. A, the relative position is represented, with respect to the first ATG, of the four different oligonucleotides, A, 22, control, and B. Their respective sequences are: A, CAAGTTTGTGGATGT- GACTGAG; 22, AGCTCTGGGGTCTCGGC; control, CCTCCGA- GTTCTTCGCTGCC; B, TCAGAGATCTGGGCCCG. The mRNA from polysome preparation (s, supernatant; p, polysomes) and various tissues (aL, adult liver; fL, fetal liver; jK, fetal kidney; H, Hep G2; UK, adult kidney; B, brain; I intestine; S, stomach; P, placenta; M, mammary gland) were amplified using oligo-A and -B as described under “Experimental Procedures” and blotted. B, the same series of blots was successively hybridized with the control probe (upperpanel) or the oligo-22 (lower panel). The figure is a composite of different autoradiograms obtained from different blots hybridized simultane- ously and processed under the same conditions. The size of both bands was estimated using DNA marker and from amplified cDNA of Hep G2 GGT clone (I ) or human adult liver GGT clone (2) containing the 22-bp insertion.

quences oligo-control and oligo-22, defined in Fig. 5A. If only the normal GGT mRNA is present in the polysomal fraction, we would expect only one band to be detected with a size corresponding to 81 bp when the oligo-control is used as a probe. If the mRNA with the 22-base insertion is also present in the polysomal fraction, a second band should be detected with a size corresponding to 103 bp when the oligo is used as a probe. As can be seen in Fig. 5B (upper panel, lane p) both bands are detected. Using the oligo-22 as a probe, we would expect no detectable band at 81 bp if only the normal GGT mRNA is present since it does not contain the complementary sequence to oligo-22. If the mRNA with the 22-base insertion is also present in the polysomal fraction, a band should be detected with a size corresponding to 103 bp. Such a band is present in Fig. 5B (lower panel, lane p). Contamination of the

polysomal fraction by the supernatant is highly unlikely since (i) no bands were detected in the supernatant fraction (Fig. 5B, upper panel, lane s) and (ii) the measurement of lactate dehydrogenase in the supernatant amounted to 94% of the activity measured in the homogenized cells, whereas only 0.4% was found in the microsomes. Our results strongly support the idea that the 22-base insert-containing mRNA is trans- lated in Hep G2 cells.

We have looked for both types of GGT mRNAs in a number of different human tissues using the same amplification pro- cedures and probes. As evidenced by the presence of both the 81- and 103-bp band when oligo-control is used as a probe, and the 103-bp band when oligo-22 is used as a probe, both types of mRNAs are present in fetal liver, kidney, brain, intestine, stomach, placenta, mammary gland as well as in Hep G2 cells (Fig. 5B). The 103-bp band is also detected in adult liver following a longer exposure (data not shown).

DISCUSSION

We report here the cloning and characterization of two GGT-related cDNAs from human adult and fetal livers. These cDNAs exhibit some differences when compared with the other published cDNA sequences from human placenta (17), human fetal liver (18), or Hep G2 cells (19). The most striking difference is the presence of a 22-bp insertion in the GGT clones we have isolated. The insertion modifies the reading frame and alters the predicted translation product. Analysis of the sequence of the fetal liver cDNA reveals three large open reading frames starting with a methionine. Only two of these are surrounded by a consensus sequence usually ob- served for correct initiation of translation (31). We focused our attention on the first one since it corresponds to the ATG described for the heavy subunit of GGT. In this reading frame, the 22-bp insertion introduces a stop codon at position 1099. The putative truncated protein of 39,287 Da would consist mainly of the GGT heavy subunit differing only in the last 26 amino acids of the carboxyl-terminal portion. This protein would be devoid of any GGT activity since the catalytic activity is associated with the light subunit (32). The second open reading frame, out of phase with the first one, starts at position 1045 and would encode a protein of 24,108 Da (225 amino acids). This protein would encompass the complete sequence of the small GGT subunit since the NH,-terminal part of the light chain of GGT (determined from human kidney (33)) corresponds to amino acid 37 on this protein (see Fig. 1). Nevertheless, although the initiator ATG is in a favorable context for translation (31) we have no evidence that this second open reading frame is utilized in uiuo.

The translation of the first reading frame is highly likely due to the following lines of evidence. First, we demonstrate that this mRNA is found in microsomal polysomes. Second, a doublet has been already described for the heavy subunit of human kidney GGT following immunoprecipitation (34). These bands correspond to a glycosylated form of the protein with a M, of 53,000 and 50,000, respectively. The light species has been attributed to proteolytic degradation of the normal GGT heavy subunit (53,000). According to the present work, one can hypothesize that the core proteins are generated from the normal mRNA (M, 41,239) and from the insert containing mRNA (M, 39,287). Of particular relevance to our study is the fact that immunohistochemistry and histoenzymology of human GGT in liver (13) and in serum (12) do not exactly correlate. It has been proposed that either an inhibitor de- creases the GGT activity (12) or that an altered form of GGT exists in human liver or in serum.

Altered proteins have already been observed in other sys-

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An Alternative mRNA for Human GGT Sequences 3261

terns such as in the case of human apolipoprotein B (35). A premature stop codon generates a protein of 250 kDa instead of the native form of 512 kDa. For GGT, a high molecular weight antigen with no activity has been observed in the rat (36). It has a molecular mass between 85 and 95 kDa and could not result from a truncated protein if one excludes an aggregation process. Such an aggregation has been observed for the purified small subunit of the rat kidney GGT (37).

We have no explanation for the fact that we have prefer- entially cloned cDNAs containing an extra 22 base pairs in the coding region. Although it has been shown in several cases that nonsense mutations correlate with a decrease in the steady state level of mRNA (38,39), the aberrant GGT mRNA might be as abundant as the normal one. This means that the detected levels of mRNA might not be reflective of true protein (enzymatic) levels.

On the basis of sequencing results of a genomic clone, mRNAs containing the extra 22 bases could result from the use of an alternative 3’-acceptor site during splicing (Fig. 4) as already described for other systems (40, 41). In our case the on/off splicing regulation mechanism is not tissue-specific as demonstrated by polymerase chain reaction amplification. Both forms of mRNA are detected in the hepatoma cell line Hep G2, kidney, liver, brain, mammary gland, intestine, and stomach.

The 5’-noncoding region of our human fetal cDNA clone is unusually long (744 bp). In fact, most of the leader sequences on vertebrate mRNAs fall in the range of 20-100 nucleotides (31) if one excepts proto-oncogenes. Long leader sequences are not incompatible with efficient translation, provided that any upstream open reading frame initiated on an ATG in a favorable context for initiation does not overlap with the main open reading frame. In the 5’-untranslated region of the fetal liver GGT mRNA, two ATG (-709, -316) are susceptible to open two short reading frames (18 and 16 amino acids) which terminate at positions -655 and -268, respectively, thus without any sparing effect on GGT mRNA translation.

The 5’-noncoding region characterized in the present study differs from those of the human placenta (17) and Hep G2 cells (19). In this region, homologous sequences are detected among the different cDNAs but at different positions with respect to the ATG. In humans we have characterized a multigene family for GGT (15), and it is possible that the different mRNAs are encoded by different genes. Neverthe- less, such an organization in the 5’-noncoding region has already been observed in other systems where only one gene is active (42). Since it is not known whether these clones are full-length, there is insufficient information to conclude whether there might be a unique promoter or multiple pro- moters. A similar observation has been made for the unique rat GGT gene encoding GGT mRNAs varying in their 5’- untranslated region (43). Concerning the 3’-noncoding region, there are only differences in length between the polyadenyl- ation signal AATAAA and the poly(A) tail. Such a microhet- erogeneity has been described for other mRNAs (44) and has not yet been linked to any regulatory process.

Our results demonstrate a possible complex regulation of GGT genes in humans. Until now, only one modification in the processing of GGT has been described for Hep G2 cells (45) in which the precursor is not cleaved into two subunits. This is not due to an alteration in the primary sequence (19) but rather in a modification of the processing factors. We are now exploring the possibility that the human multigene family specific for GGT encodes altered primary polypeptide struc- tures under different physiopathological conditions.

Acknowledgments-We thank Dr. R. Barouki for the synthesis of

the different oligonucleotides and his advice. We thank Dr. P. H. Romeo and J. C. Kouyoumdjan for supplying mRNAs from human stomach, intestine, and mammary gland. We thank Drs. Y. Laperche and J. Hanoune for their most helpful comments, K. Hehir for her help and guidance with some of the DNA sequencing, Dr. R. Wydro for some helpful suggestions, and L. Rosario and E. Auhenas for their skillful secretarial assistance.

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Baik, S Siegrist and G GuellaënA Pawlak, E H Cohen, J N Octave, R Schweickhardt, S J Wu, F Bulle, N Chikhi, J H

human tissues.An alternatively processed mRNA specific for gamma-glutamyl transpeptidase in

1990, 265:3256-3262.J. Biol. Chem. 

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