Gene, 108 (1991) 289-292 0

1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

289

0378-1119/91/$03.50

06181

Cloning and analysis of a cDNA encoding a human liver carboxylesterase (Oligodeoxyribonucleotides;

active site serine;

Peter W. Riddles a, Lalette J. Richards”,

primary

structure;

mRNA;

recombinant

DNA)

Mark R. Bowles b and Susan M. Pond b

a CSIRO, Division of Tropical Animal Production, Long Pocket Laboratories, Indooroopilly. Queensland 4068 (Australia) and b The University of Queensland, Department of Medicine. Princess Alexandra Hospital, Brisbane. Queensland 4102 (Australia) Tel. /61)72405329 Received by P.A. Manning: 10 May 1991 Revised/Accepted: 22 August/23 August 1991 Received at publishers: 19 September 1991

SUMMARY

A human

liver carboxylesterase

(CE)-encoding

cDNA has been cloned using synthetic

oligodeox~ibonucleotides

(ohgos)

based on the known amino acid (aa) sequences of rabbit and rat liver CEs. The oligos hybridize specifically to DNA encoding liver CEs. The longest cDNA obtained from screening several cDNA libraries encodes about 80% of the protein and translates into an aa sequence which has a high degree of similarity with the sequences of liver CEs from other species. On hybridization to mRNA isolated from human liver, the cDNA gave a single band of about 2.0 kb consistent with its encoding a protein of ~68 kDa. DNA obtained from a number of human livers and probed with the CE cDNA gave identical hybridization patterns. These patterns were moderately complex by comparison with published data.

INTRODUCTION

The CEs (EC 3.1.1.1) are a group of serine-dependent esterases which are found in a wide range of tissues and organisms. Whereas the biological role of some of these enzymes is clearly indicated (e.g., acetycholinesterases and juvenile hormone esterase), the function of the remaining enzymes is not so evident. This is particularly true of the hepatic microsomal CEs which have been studied in some detail (Krisch, 1971; Heymann, 1982). In the broadest

Correspondenceto: Dr. P.W. Riddles, CSIRO, mal Production,

Long Pocket

Laboratories,

Division

of Tropical

Indooroopilly,

Ani-

Queensland

4068 (Australia) Tel. (61)7377-0711;

Fax (61)7870-7034.

Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, mentary to RNA; CE, carboxylesterase; CE, gene(DNA) EtdBr,

ethidium

bromide;

kb, kilobase

DNA compleencoding CE;

or 1000 bp; nt, nucleotid~s);

oligo, oligodeoxyribonucleotide; ORF, open reading frame; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/O.OlS M Na,.citrate pH 7.6; SSPE, 0.18 M NaCl/O.Ol M

Na . phosphate/l

mM EDTA pH 7.7.

sense, one role for CEs appears to be in the detoxification of foreign compounds. In insects, they appear to play a role in the development of resistance to compounds that contain an esterolytic bond such as organophosphorus compounds (Mouches et al., 1986). The study of the liver CE is complex because many isoenzymic forms have been identified in the rat and other laboratory animals (Mentlein et al., 1980; 1987; Heymann, 1982). Evidence for two CEs from human liver has been obtained using purification and kinetic analysis (Ketterman et al., 1989). A report of the identity of two CEs isolated in different laboratories (Mentlein et al., 1985) emphasizes the fundamental impo~ance of the definition of the various CEs/esterases. This has heretofore relied necessarily on substrate and inhibitor studies of the enzyme. Sequence data on different CEs will enable a more discriminating approach to the classification of these enzymes, and will enable the design of specific DNA probes for detailed analysis of the relevant genes. We therefore report the first cloning of a large fragment of human liver CE cDNA using oligos specific for liver CEs.

290 30 10 20 40 50 60 70 CTGAACACTGTCTTTACCTCAATATTTACACTCCTGCAGACTTGACCAAGA~AACAGGCTGCCGGTGATGGTGTGGATCCACG GluHisCysLeuTyrLeuAsnIleTyrThrProAlaAspLeuThrLysLysAsnArgLeuProVal~

80 27

90 100 110 130 140 160 120 150 GAGGGGGGCTGATGGTGGGTGCGGCATCAACCTATGATGGGCTGGCCCTTGCTGCCCATGAA~CGTGGTGGTGGTGACCATTC ~GlyGlyLeuMetValGlyAlaAlaSerThrTyrAspGlyLeuAlaLeuAlaAlaHisGluAsnValValValValThrIle

55

170 190 180 200 210 220 230 240 AATATCGCCTGGGCATCTGGGGATTCTTCAGCACAGGGGATG~CACAGCCGGGGGAACTGGGGTCACCTGGACCAGGTGGCTG GlnTyrArgLeuGlyIleTrpGlyPhePheSerThrGlyAspGluHisSerArgGlyAsnTrpGlyHisLeuAspGlnValAla

250 83

270 280 260 290 300 310 320 330 CCCTGCGCTGGGTCCAGGACAACATTGCCAGCTTTGGAGGGAACCCAGGCTCTGTGACCATCTTTGGAGAGTCAGCGGGAGGAG AlaLe~ArgTrpValGlnAspAsnIleAlaSerPheGlyGlyAsnProGlySerValThrIle VGly

111

340 350 360 370 380 390 400 410 AAAGTGTCTCTGTTCTTGTTTTGTCTCCATTGGCCAAGAACCTCTTCCACCGGGCCATTTCTGAGAGTGGCGTGGCCCTCACTT GluSerValSerValLeuValLeuSerProLeuAlaLysAsnLeuPheHisArgAlaIleSerGluSerGlyValAlaLeuThr

420 139

440 450 460 470 490 430 480 CTGTTCTGGTGAAGAAAGGTGATGTCAAGCCCCTTGAACCACCACCTCTGCTG SerValLeuValLysLysGlyAspValLysProLeuAlaGluGlnIleAlaIleThrAlaGlyCysLysThrThrThrSerAla

500 167

520 530 550 560 570 580 510 540 CTATGGTTCACTGCCTGCGACAGAAGACGGAAGAGGAGGAGCTCTTGGAGACGACATTGAAAATTGGAAATTCTTATCTCTGGACTT AlaMetValHisCysLeuArgGlnLysThrGluGluGluLeuLeuGluThrThrLe~LysIleGlyAsnSerTyrLe~~TrpThr 640 660 590 600 610 620 630 650 ACAGGGAGACCCAGAGAGAGTCAACCCTTCTGGGCACTGTGATTGATGGGATGCTGCTGCTGAAAACACCTGAAGAGCTTCAAC TyrArgGluThrGlnArgGluSerThrLeuLeuGlyThrValIleAspGlyMetLeuLeuLeuLysThrProGluGluLeuGln

195 670 223

750 680 690 700 710 720 730 740 GTGAAAGGAATTTCCACACTGTCCCCTACATGGTCGGAATTAACAAGCAGGAGTTTGGCTGGTTGATTCC~TGCAGTTGATGA ArgGluArgAsnPheHisThrValProTyrMetValGlyIleAsnLysGlnGluPheGlyTrpLeuIleProMetGlnLeuMet

251

760 830 770 780 790 800 I310 820 GCTATCCACTCTCCGAAGGGCAACTGGACCAGAAGACAGCCATGTCACTCCTTGGAAGTCCTATCCCCTTGTTTGCCATTGCTA SerTyrProLeuSerGluGlyGlnLeuAspGlnLysThrAlaMetSerLeuLeuGlySerProIleProLeuPheAlaIleAla

840 279

870 890 910 850 860 880 900 AGGAACTGATTCCAGAAGCCACTGAGAAATACTTAGGAGGAACAGACGACACTGTCAA~AGAAAGACCTGATCCTGGACTTGA LysGluLeuIleProGluAlaThrGluLysTyrLeuGlyGlyThrAspAspThrValLysLysLysAspLeuIleLeuAspLeu

920 307

980 1000 940 950 960 970 990 930 TAGCAGATGTGATGTTTGGTGTCCCATCTGTGATTGTGGCCCGGAACCACAGAGATGCTGGAGCACCCACCTACATGTATGAGT IleAlaAspValMetPheGlyValProSerValIleValAlaArgAsnH~sArgAspAlaGlyAlaProThrTyrMetTyrGlu

335

1050 1060 1080 1090 1010 1020 1030 1040 1070 TTCAGTACCGTCCAAGCTTCTCATCAGACATGAAACCCCAAGACGGTGATAGGAGACCACGGGGATGAGCTCTTCTCCGTCTTTG PheGlnTyrArgProSerPheSerSerAspMetLysProLysThrValIleGlyAsp WSerValPhe

363

1170 1140 1150 1160 1110 1120 1130 1100 GGGCCCCATTTTTAAAAGAGGGTGCCTCAGAAGAGGAGATCTTTGCTC GlyAlaProPheLeuLysGluGlyAlaSerGluGluGluIleArgLeuSerLysMetValMetLysPheTrpAlaAsnPheAla

391

1250 1220 1230 1240 1180 1200 1210 1190 GCAATGGAAACCCCAATGGGAAAGGGCTGCCCCACTGGCCAGAGTACAACCAGAAGGAAGGGTATCTGCAGATTGGTGCCAACA ArgAsnGlyAsnProAsnGlyLysGlyLeuProHisTrpProGluTyrAsnGlnLysGluGlyTyrLeuGlnIleGlyAlaAsn 1290 1300 1270 1280 CCCAGGCGGCCCAGAAGCTGAAGGACAAAGAAGTAGCTTTGCCACCCC ThrGlnAlaAlaGlnLysLeuLysAspLys~

1310

1320

1330

1260 419 1340

snLeuPheAlaLysLysAlaValGluLysProPro

1350 1360 1370 1380 1390 1400 1410 1420 AGACAGACCACATAGAGCTGTGATGAGATCCAGCCGGCCTTGGAGCTGGACGAGGAGC~AGACTGGGGTCTTTTGCGGAAAGG GlnThrAspHisIleGluLeuEndEnd 1430 1440 1450 1460 1470 1480 1490 1500 GATTGCAGGTTCAGAAGGCATCTTACATGCTGGGAATGTCTGGTGGTGGGGGCAGGGGAGAGAGGCCGAGAGGCCATGAAGGAG 1520 1530 1540 1550 1560 1570 GCAAGCTTTTGTATTTGTGACCTCAGCGTTTGGG~GGATCTTTTG~GGCCAA~AA~AAAAAA~AA~A

447

454 1510

1580

Fig. 1. The nt sequence ofclone pLJR1 encoding a human liver CE, and corresponding aa sequence. Two oligos, corresponding to the peptide sequences: MVWIHG, EVAPWT (double-underlined in the figure) and found by comparison of the rat and rabbit aa sequences (see section a), were used to screen a human liver cDNA library in lgtl

1(Clontech).

Conditions

for labeling ofprobe

and transfer

to Hybond

N + were as recommended

by the manufacturer

291 EXPERIMENTAL

AND

DISCUSSION

(a)

(b)

I 2 3

-0

(a) Cloning of human liver CE cDNA by specific hyhridization to oligos The peptide sequences used to design the synthetic oligos are shown in Fig. 1. These regions were selected because they are in conserved domains of the rat and rabbit liver enzymes but do not occur in other related esterases such as the esterase D (Lee et al., 1986a); and cholinesterases (Schumacher et al., 1986; Lockridge et al., 1987). Thus, nt sequences encoding these latter enzymes are unlikely to be selected. The oligos were subsequently used to screen several cDNA libraries. One clone designated, pLJR1, which was obtained from a commercial cDNA library,

9.5 7.5

8.5 7.4 6.1

4.4

4.8

24 1.4

contained a 1.6-kb cDNA. The nt sequence, together with the hypothetical translation of the major ORF, is shown in Fig. 1. The striking similarity of the primary structure to the sequences of other liver CEs identified in rabbit (Korza and Ozols, 1988) and rat (Long et al., 1988) confirms the nature of the cDNA cloned (analysis not shown). Moreover, specific features so far ascribed to microsomal CEs can be identified. In particular, all CEs contain a conserved sequence around the active-site Ser which includes the residues FGESAG (Augusteyn et al., 1969). A domain containing the hypothetical active site His residue can also be located (Fig. 1). The C-terminal sequence HIEL, characteristic of proteins which are retained in the lumen of the microsomes, including other microsomal esterases (Robbi et al., 1990; Andres et al., 1990), is also present. There is little similarity with the sequence of the esterase D enzyme which is also found in human liver (Lee et al., 1986a,b).

2.8

Fig. 2. Hybridization mission

from

analysis

with human

the appropriate

Ethics

liver CE cDNA.

committee

With per-

and the next-of-kin,

human liver was obtained from kidney transplant donors. Genomic DNA and mRNA were isolated from these samples using procedures described by Maniatis labeled

et al. (1982). The cDNA

with [x-32P]dCTP

Origin

insert

of the gels is indicated

as 0. (Panel

Samples of DNA (10 mg) were digested phoresed

on a 0.8% agarose

transferred

to Hybond

prehybridized 50%

bridized

x

pg/ml

was washed

at 42°C

to DNA.

of EtdBr. The DNA was (Fig. 1). The filter was

herring

in the same solution in the presence

The membrane

a) Hybridization

x Denhardt’s

denatured

was

of Amersham.

with Hind111 for 2 h and electro-

gel in the presence SSPEj5

from pLJR1

System

N + filters as described

using [S

formamide/lOO

isolated

using the Multiprime

solution/OS% sperm

DNA]

SDS/ and hy-

of probe at 42°C overnight.

once

with

SDS/2

twice with 0.1 y0 SDS/O.1 x SSC. The filter was exposed

x SSC,

and

to film as de-

scribed (Fig. 1)for seven days (Fig. 2a). (Panel b) Hybridization

to mRNA.

(h) Hybridization analysis of the human liver CE cDNA The cDNA, pLJR1, was used to examine human liver genomic DNA obtained from several individuals by hybridization in Southern blots. The level of stringency was set to identify sequences that would have an 80% or greater similarity. A moderately complex pattern of hybridization was observed using Hind111 to digest the DNA (Fig. 2a).

Adelaide,

Other enzymes gave similarly complex patterns (data not shown). However, the hybridization patterns were identical among the individuals examined, which indicates at least a

gross similarity at the chromosomal level. It was suggested that the complex pattern observed with rat liver CE DNA

(Amersham).

Prehybridization

was 42°C

[6

x

SSC, 5

x

Denhardt’s

The mRNA formaldehyde (Amersham) described

obtained

method

(Sanger

Deletions

was subcloned

by exonuclease

coli strain JM83 (Yanisch-Perron containing

A possible

possible

The nt sequence

+ )(Promega,

et al., 1977) using double-stranded

were prepared

both strands.

into pGEM7Zf(

III digestion,

Madison,

for the DNA (panel

a) and the tilter was washed

SDS at 42°C and then with 1 x SSPE/O.l%

and exposed

to film as described

SPP-1

bacteriophage

Australia)

0.5% SDS/l0

followed by treatment

et al., 1985). Oligos were prepared

polyadenylation

active site Ser; HGDEI,

data have been submitted

signal is shown domain

WI) and designated

DNA with a kit supplied

containing

to GenBank

by Promega.

with mung-bean

as DNA primers

double-underlined.

pg/ml denatured

pLJR1.

the accession

with 2 x

SDS at 42°C

sperm

EcoRI

(Bresatec,

Mannheim).

DNA]

and hybridization

1y. Ficoll/O. 1 y0 polyvinylpyrolidone. screens.

The nt sequence

For sequencing,

was determined

are aligned

overlapping

are shown

the largest

by the dideoxy

with corresponding

before ligation and transformation

of the enzyme

No. M65261.

with

(Boehringer

herring

Last digits of numerals nuclease,

digested

and RNA ladder

to ensure that complete

Key features

M

above for 24 h (Fig. 2b). Size markers

DNA

sequence

underlined:

possible active side His. Note the last four aa, HIEL, indicating

and assigned

a 1% agarosej2.2

gel and the RNA was transferred and fixed to Hybond N + as above (Fig. 1). Hybridization was carried out as

overnight in the same buffer at 44°C using both oligos. The 5 x Denhardt’s solution is 0.1 y$, bovine serum albumin/O. The filters were washed with 4 x SSC/O.l% SDS at 42°C and exposed to film (Fuji) at -80°C with two intensifying cDNA

through

SSPE/O.l% were

solution,

(5 Fg) was electrophoresed

was obtained FGESAG,

retention

nt.

into Escherichiu on

domain

in microsomes.

292 evidence for a multigene family (Long et al., 1988). Certainly evidence is accumulating for the existence of more than one form of liver CE. There are two forms of the rabbit CE identified so far (Ozols, 1989; Korza and Ozols, 1988). Primary structure analysis reveals two forms of rat liver enzymes (Takagi et al., 1988; Long et al., 1988; Robbi et al., 1990). This question is yet to be fully resolved for the human enzyme although two forms of CE from human liver have been identified (Ketterman et al., 1990). Plasmid pLJR1 hybridized to one size of mRNA at approx. 2.0 kb (Fig. 3b). This is sufficient to encode a protein of 68 kDa and indicates that about 400 bp of CE cDNA is absent from clone pLJR1. The cDNA libraries were rescreened with pLJR1 at high stringency but failed to identify any additional clones containing the missing sequence. This question of multiple nt sequences and the exact nature of a multigene family for human liver CE may be analysed in detail by examining the genomic DNA clones and cDNAs encoding the CEs. Was

(c) Conclusions (I) Oligos for the specific identification ofhver CEs have

been designed by comparison of available sequence data on these enzymes. The oligos have been used to isolate a cDNA encoding human liver CE for the first time. (2) An analysis of the aa sequence verifies the nature of the CE cDNA by comparison with the published sequences of other serine-dependent CEs. In particular, the domains containing the active-site Ser and His, and a microsomal retention signal were present. (3) Hybridization analysis indicates that the cDNA encodes a protein of ~68 kDa as deduced by the hybridization to a 2.0-kb mRNA. The hybridization pattern with genomic DNA is similar to those patterns previously reported for the rat liver CE gene (Long et al., 1988).

ACKNOWLEDGEMENTS

This work was supported by the National Health and Medical Research Council of Australia, the Queensland and Northern New South Wales Lions Kidney and Medical Research Foundation and the CSIRO, Division of Tropical Animal Production.

REFERENCES Andres, D.A., Dickerson, I.M. and Dixon, J.E.: Variants ofthe carboxylterminal KDEL sequence direct intracellular retention. J. Biol. Chem. 265 (1990) 5952-5955.

Augusteyn, RX., de Jersey, J., Webb, E.C. and Zerner, 3.: On the homology of the active site peptides of liver carboxylesterases. Biochim. Biophys. Acta 171 (1969) 128-137. Heymann, E.: Hydrolysis of carboxylic esters and amides. In: Jakoby, W.B., Bend, J.R. and Caldwell, J. (Eds.), Metabolic Basis of Detoxication. Academic Press, New York, 1982, pp. 229-245. Ketterman, A.J., Bowles, M.R. and Pond, SM.: ~ri~catian and characterization of two human liver carboxylesterases. Int. J. Biochem. 21 (1989) 1303-1312. Korza, G. and Ozols, J.: Complete covalent structure of 60-kDa esterase isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced rabbit microsomes. J. Biol. Chem. 263 (1988) 3486-3495. Krisch, K.: Carboxyhc ester hydrolysis. In: Boyer, P. (Ed.), The Enzymes, Vol. 5. Academic Press, New York, 1971, pp. 43-69. Lee, E.Y.-H.P, and Lee, W.-H.: Molecular cloning of the human esterase D gene, a genetic marker of retinoblastoma. Proc. Nat]. Acad. Sci. USA 83 (1986a) 6337-6341. Lee, W.-H., Wheatley, W., Benedict, W.F., Huang, C-M. and Lee, E.Y.-H.: Purification, biochemieai characterization, and biological function of human e&erase D. Proc. Natl. Aead. Sci. USA 83 (1986b) 6790-6794. Lockridge, O., Bartels, C.F., Vaughan, T.A., Wong, C.K., Norton, S.E. and Johnson, L.L.: Complete amino acid sequence of human serum chohnesterase. J. Biol. Chem. 262 (1987) 549-557. Long, R.M., Satoh, H., Martin, B.M., Kimura, S., Gonzalez, F.J. and Pohl, L.R.: Rat liver carboxylesterase: cDNA cloning, sequencing, and evidence for a multigene family. Biochem. Biophys. Res. Commun. 156 (1988) 866-873. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Mentlein, R., Heiland, S. and Heymann, E.: Simultaneous purification and comparative characterization of six serine hydrolases from rat liver microsomes. Arch. Biochem. Biophys. 200 (1980) 547-559. Mentlein, R., Ronai, A., Robbi, M., Heymann, E. and von Deimling, 0.: Genetic identification of rat liver carboxylesterases isolated in different laboratories. Biochim. Biophys. Acta 913 (1987) 27-38. Mouches, C., Pasteur, N., Berge, J.B., Hyrien, O., Raymond, M., De Saint Vincent, B.R., De Silvestri, M. and Georghiou, G.P.: Amplification of an esterase gene is responsible for insecticide resistance in a Californian Culex mosquito. Science 233 (1986) 778-780. Ozols, J.: Isolation, properties and the complete amino acid sequence of a second form of 60-kDa glycoprotein esterase. Orientation of the 60 kDa proteins in the microsomal membrane. J. Biol. Chem. 264 (1989) 12533-12545. Robbi, M., Beaufay, H. and Octave, J.-N.: Nucleotide sequence ofeDNA coding for rat liver ~16.1 esterase (ES-1 O),a carboxylesterase located in the lumen of the endoplasmic reticulum. J. Biochem. 269 (1990) 451-458. Sanger, F., Nicklen, S. and Co&on, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 560-564. Schumacher, M., Camp, S., Ma&et, Y., Newton, M., MacPhee-Quigley, K., Taylor, S.S., Friedman, T. and Taylor, P.: Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence. Nature 3 19 (1986) 407-409. Takagi, Y., Morohashi, K., Kawabata, S., Go, M. and Qmura, T.: Motecular cloning and nucleotide sequence of cDNA of microsomal carboxyesterase El of rat liver. J. Biochem. 104 (1988) 801-806. Yanisch-Perron. C., Vieira, J. and Messing, J.: improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33 (1985) 103-I 19.