GENOMICS

9,

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(lY9l)

Characterization

of a Murine cDNA Encoding a Member Carboxylesterase Multigene Family

M. OVNIC, *” K. TEPPERMAN, D. A. STEPHENSON,§ 5. G.

t 5. MEDDA,~ GRANT) AND

of the

R. W. ELLIOTT,~ R. E. GANSCHOW*

*Institute for Developmental Research, Children’s Hospital Medical Center, E//and and Bethesda Avenues, Ononnat~, Ohio 45229; tDepartment of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221; $Genetics and Biochemistry Branch, National institute of Diabetes, Digestive, and Kidney Diseases, National /nst/tutes of Health, Bethesda, Maryland 20892; SDepartment of Molecular and Cellular Biology, Roswell Park Memorial Institute, New York State Department of Health, 666 E/m Street, Buffalo, New York 14263; and iiDivis/on of Biomedical Sciences, Lawrence Livermore National Laboratory, Univers/ty of Califorma, P.O. Box 5507, Llvermore, California 94550 Received

We have characterized a mRNA sequence containing the entire coding region of a mouse carboxylesterase (EC 3.1.1.1). pEs-N, an 1840-bp composite of five overlapping cDNA clones,contains an open reading frame of 554 amino acids that display a high degree of similarity with rat and rabbit carboxylesterases. Genetic mapping studies place this carboxylesterase in cluster 1 of the esterase region on chromosome 8. Results of blot hybridization analysis of genomic DNA probed with a pEs-N cDNA under both low and high stringency conditions suggest membership in a carboxylesterase multigene family, as would be expected for a nonspecific carboxylesterase. A message size of 18501900 nucleotides was revealed by RNA blot hybridization analysis. S 1 nuclease protection analyses with a probe representing a segment of pEs-N detected message in liver, kidney, and lung, but not in spleen, brain, testes, and submandibular gland, with higher levels in female than in male kidney. Additional Sl nuclease-protected mRNA species were found, suggesting the expression of distinct members of a multigene family. In vitro translation of a full-size transcript of pEs-N resulted in a product of 51.5 kDa. Upon the addition of microsomes, this product was processed into a protein of 60.4 kDa, which is within the size range of monomeric units of mouse carboxylesterases. IL’ 1991 Academic Press. Inc.

INTRODUCTION

isozymes of the mouse (EC are a group of at least nine members encoded by genes within the esterase region on chromosome 8 (Peters, 1982; von Deimling, 1982). This region is composed of two gene subclusters located within 8 CM The X1.1.1)

carboxylesterase

1 To whom

correspondence

should

0888-7643/91 $x00 Copyright !c 1991 by Academic Press. All rights of reproduction in any form

he addressed.

Inc. resemed.

May

2, 1990

of each other (Womack, 1975). The members of clusters 1 and 2 are believed to belong to a distinct multigene family, which arose from the duplication and subsequent. diversification of a single ancestral gene (Eisenhardt and von Deimling, 1982). Several of these esterases have been purified and their physical properties characterized (Eisenhardt and von Deimling, 1982; Otto et al., 1981; Oehm et al., 1982; de Looze et al., 1985). The esterases within each cluster retain a high degree of relatedness, which is demonstrated by similar biochemical and molecular characteristics and immunological cross-reactivities (von Deimling, 1982; Otto et al., 1981). Although they overlap in their properties, the carboxylesterase isozymes within each cluster have been distinguished by electrophoretic mobility, genetic variation among strains, substrate specificity, susceptibility to inhibitors, developmental appearance, and tissue-specific expression (Eisenhardt and von Deimling, 1982; Berning et al., 1985; de Looze et al., 1982; von Deimling et al.. 1983). It is notable that the members of t.his functionally related group of enzymes display considerable heterogeneity in their patterns of developmental and tissue-specific expression, thus retaining a high degree of regulatory flexibility (Eisenhardt and von Deimling, 1982). A common characteristic of the carboxylesterases is their broad range of substrate specificities (Peters, 1982). While t,hey react with carboxyl esters of both endogenous and exogenous origin, their biologic function is not. fully understood (Heymann, 1982). HOWever, at least, one, esterase-6, may be involved in fatty acid utilization (de Looze et al., 1982). Esterase-22 (also known as egasyn) forms complexes with Qglucuronidase through its active site, which results in the localization of bound p-glucuronidase within the

CHARACTERIZATION

OF

A cDNA

endoplasmic reticulum (ER) of tissues that express esterase-22 (Medda et al., 1986, 1987). No other carboxylesterase is known to exhibit this funct,ion. Recently, two laboratories have reported partial cDNA sequences corresponding to nonspecific carboxylesterases in the mouse (Genetta et al., 1988) and rat (Long et al., 1988). We now report the isolation and characterization of a cDNA that contains the entire coding region of a murine carboxylesterase. This clone, originally identified utilizing antisera to egasyn/esterase-22, encompasses the majority of the 5’ and the entire 3’ untranslated regions. It maps to cluster 1 of t,he esterase region of chromosome 8. It is related, but not identical, to egasyn/esterase-22, and thus represents a distinct cluster 1 carboxylesterase. MATERIALS

AND

METHODS

Materials

Six 12-week-old male and female C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Male animals from recombinant inbred (RI) strains were purchased from The Jackson Laboratory. RI strains were constructed by brother-sister matings commencing at the F, generation from crosses of the progenitor strains. The 26 BXD RI strains were derived from a cross between C57BL/6J (B) and DBA/2J (D) (Taylor et al., 1975). The 12 BXH RI strains were derived from a cross between C57BL/6J and C3H/HeJ (H) (Watson et al., 1977). The interspecies backcross was performed in the laboratory of Dr. Verne Chapman (Mullins et al., 1988). (C57BL/GJRos X Mus spretus)F, females were mated to C57BL/GJRos males. In this study only male progeny were analyzed. Es-N cDNA clone A was isolated from a Xgtll C57BL/6 liver cDNA library, provided by A. Lusis, utilizing goat antiserum (kindly provided by R. Swank) prepared against a purified preparation of egasyn/esterase-22. This clone was rescreened utilizing rabbit antiserum prepared by A. Lusis (Lusis et al., 1976) and made available to us by R. Swank. Additional cDNA clones (B-E) were isolated from XgtlO C57BL/6 and XZAP C57BL/6 X CBA F, liver cDNA libraries (both from Stratagene, Inc.) by colony hybridization utilizing random primer-labeled probes (Feinberg and Vogelstein, 1983). Total RNA was prepared from various tissues of C57BL/6J mice according to the method of Chirgwin et al. (1979). Poly(A)+ RNA was prepared by oligo(dT)-cellulose chromatography (Aviv and Leder, 1972). DNA

Sequence

Analysis

cDNA clones were sequenced by the quasi-end-labeling adaptat.ion of the dideoxy chain termination method (Duncan, 1985). Primer extension analysis

ENCODING

345

A CARBOXYI,ESTERASE

was performed as described (Degen et al., 1987). DNA sequences were recorded and analyzed using the Microgenie DNA sequence analysis program from Beckman Instruments (Queen and Korn, 1984). DNA

Blot

Hybridization

Analysis

Restriction enzyme-digested liver DNA was transferred onto Nytran (Schleicher & Schuell) following electrophoresis through agarose gels. pEs-N clone A was purified twice through low-melting-temperature agarose and random primer labeled. Hybridizations in 50% formamide, 5X SSPE (20X SSPE is 3.6 M NaCl, 200 mM NaH,PO,, pH 7.4,20 mM EDTA, pH 7.4) were carried out at 39 or 42°C. Filters were washed in the following manner: twice for 15 min at RT in 2X SSC, 0.1% SDS, once for 30 min at RT in 0.1X SSC, 0.1% SDS, and once for 30 min at, 65°C in 0.1x SSC, 0.1% SDS. RNA Blot Hybridization/Solution Sl Nuclease Analysis

Hybridization

and

Total liver RNA (20 pg) was fractionated on 1.2% formaldehyde/agarose gels (Maniatis et al., 1982) and blotted onto Nytran filters. cRNA probes were generated according to the manufacturer’s instructions (Stratagene, Inc.). Prior to the use of all cRNA probes, full-length transcripts were electrophoresed and isolated by phenol/chloroform extraction from low-melting-temperature agarosegels (FMC BioProducm). 3”P-labeled cRNA (l-2 X lo5 cpm/ml of hybridization buffer) was added. Hybridizations and washes were performed according to manufacturer’s instructions (Stratagene, Inc.). Total RNA (100 pg) from various tissues was hybridized in 50% formamide at 65’C for 6 h with l-2 X lo6 cpm of “‘P-labeled cRNA probe. Following hybridization, reactions were digested for 1 h at 37°C with 800 units of Sl nuclease (Boehringer Mannheim). The samples were ethanol-precipitated and electrophoresed through 5% nondenaturingpolyacrylamide gels. In Vitro

Tran,slation

cRNA t,ranscripts of pEs-N clone E were made according to manufacturer’s instructions (Stratagene, Inc.). RNA (1 pgper reaction) was translated utilizing a rabbit reticulocyte lysate system (Promega). Canine pancreatic microsomes (Promega) were added for processing and glycosylation of in vitro-translated product,s. Proteins were metabolically labeled with [3”S]methionine residues (New England Nuclear). Products were electrophoresed on 10% SDS-polyacrylamide gels. Sizes were determined by compari-

346

OVNIC

son to those of proteins (Sigma).

of known

molecular

weight

Genetic Mapping Isolation of mouse liver DNAs from RI strain animals and spleen DNAs from backcross animals was performed as described in Mann et al. (1986) and Mullins et al. (1988), respectively. Agarose electrophoresis, Southern blotting, and hybridization were performed as in Mann et al. (1986). The esterase-2 (ES-~) locus was typed as described in Peters and Nash (1977), with the following modifications. Titan III cellulose acetate (Helena) plates were used as the support medium in place of starch. The sample, a 10% homogenate of mouse kidney in 50 mM Tris-HCl, pH 8.0, was applied cathodally three times, and electrophoresis was performed in Tris-glycine buffer at 240 V for 40 min with cooling. Esterase activity was visualized using a solution prepared by adding 0.1 ml CYnaphthyl acetate (1%) in 50% acetone, 0.2 ml Fast BB saturated water solution, and 0.8 ml phosphate butler (pH 7.0) to 2.6 ml water. Probes used in the analysis of the segregation of alleles in the recombinant inbred and backcross animals represent these genes: adenine phosphoribosyltransferase pseuodogene-1 (Aprt-psi, Dush et al., 1986); adenine phosphoribosyltransferase (Aprt-I, Sikela et al., 1983); esterase-N (Es-N clone A, this paper); metallothionein-1 (Aft-l, Searle et al., 1984); metallothionein-2 (Mt-2, Searle et al., 1984); and tyrosine aminotransferase (Tat, Muller et al., 1985). Analysis of the segregation of alleles in RI strains was performed by the method described in Silver (1985). For each map interval in the backcross, map units were estimated as percentage recombination t standard error (Green, 1981).

RESULTS

AND

DISCUSSION

Sequence Analysis and Comparisons Goat antiserum prepared against egasynlesterase22 was utilized to isolate a cDNA, designated pEs-N clone A, from a hgtll mouse liver library. Clone A, 360 bp in length, was also shown to react with a wellcharacterized rabbit anti-egasyn antiserum (Medda et al., 1986; Lusis et al., 1976). A series of overlapping clones was obtained by hybridization of two mouse liver libraries with clone A. Their restriction maps and the sequencing strategy employed to obtain a composite DNA sequence are presented in Fig. 1. Extensive sequence information obtained from these clones, B (924 bp), C (1235 bp), D (1759 bp), and E

ET

AL.

(1840 bp), indicated that each clone is identical in sequence to the others and therefore implies that clones A-E represent the same gene. pEs-N is a composite cDNA sequence obtained from the analysis of the individual clones. Sequence obtained from a second murine cluster 1 carboxylesterase (Ovnic et al., manuscript in preparation) reveals sequence similarity to pEs-N of between 75 and 85%, again suggesting that pEs-N is composed of clones originating from a single cDNA. The composite DNA sequence reveals an open reading frame (ORF ) of 1679 bp beginning at position 1 (Fig. 2). The ORF is followed by 161 bases of 3’ untranslated sequence containing a poly(A) addition site at bases 1808-1814. A poly(dA) stretch begins 22 bp after the addition site. Primer extension analysis of the longest clone, E, indicates that the terminal 5-6 bp of the 5’ untranslated region are absent (data not shown). pEs-N, beginning with an inframe start codon, encodes 554 amino acids. The ATG is preceded by the pentamer, CCAAC, which conforms to the eukaryotic translation initiation consensus sequence (Kozak, 1984). Beginning at residue 19, the predicted amino acid sequence, His-Ser-Leu-Leu-Pro-Pro-Val-Val, shows strong similarity to the amino-terminal sequence of a rabbit carboxylesterase, His-Pro-SerAla-Pro-Pro-Val-Val (Korza and Ozols, 1988). From this we infer that residues 1-18, which are highly hydrophobic in nature and conform to expected configurations of signal sequences (von Heijne, 1983), comprise the signal sequence for the murine carboxylesterase. The deduced amino acid sequence reveals five potential N-glycosylation sites and four cysteine residues. Amino acids Ser2’l and His455are residues that, among carboxylesterases from other species, have been shown to be active site residues by labeling with radioactive esterase inhibitors (Augusteyn et al., 1969; Ozols, 1987). These active site residues are framed by stretches of conserved amino acids. A notable difference exists between the carboxylesterase encoded by pEs-N and other carboxylesterases in the region encompassing the active site serine residue. One residue in our mouse sequence encompassing this active site region differs from that found in horse, ox, pig, sheep, rabbit, rat., and chicken (Augusteyn et al., 1969; Korza and Ozols, 1988; Long et al., 1988). The sequence of these esterases around the active site is Gly-Glu-Ser*-Ala-Gly, whereas the sequence surrounding our putative active site is Gly-Glu-Ser*-SerGly. At present, we do not know whether this change affects the esterase activity of the protein represented by the composite sequence. A comparison between the deduced amino acid se-

CHARACTERIZATION 0 ,I

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FIG. 1. Restriction map and sequencing strategy of’five overlapping pEs-N clones, A-E. The composite of the pEs-N clones is depicted as a box. The shaded open reading frame is flanked by a 3’ unt.ranslated region and a poly(dA) tail. The direction and extent of each sequence determination are indicated by the arrows. A partial restriction enzyme map for each clone is depicted by short vertical lines. Restriction sites were verified by sequencing.

quence derived from pEs-N and that of a rat cDNA clone (Long et al., 1988) is graphically summarized in Fig. 3 and aligned by amino acids in Fig. 4. Since the rat cDNA begins within the mouse signal sequence, the first 15 residues of the mouse sequence could not be evaluated. The similarity between the mouse and the rat amino acid sequences over the entire region of comparison is relatively high, at 81%. All five potential glycosylation sit,es and 4 cysteine residues are conserved between the two sequences, as are the active site residues. The overall similarity between the amino acid sequence of a rabbit carboxylesterase (Korza and Ozols, 1988) and our composite sequence is 64.5%. Over the region compared, the cysteine residues are conserved between the mouse and the rabbit esterases, although only two of five potential glycosylation sites in t.he mouse sequence are present in the rabbit esterase. In comparisons of sequences between species, it must be noted that pEs-N represents one of a family of murine esterases. Rat and rabbit esterases are also a heterogeneous group (Mentlein et al., 1984; Ozols, 1989). Therefore, sequence comparisons between species may describe species differences for a particular carboxylesterase, variations between one carboxylesterase and anot her, or both.

In addition to a high degree of similarity in amino acid sequence to rat and rabbit carboxylesterases, pEs-N has similarities, over its entire length, to electric ray acetylcholinesterase precursor (38.2%, Schumacher et al., 1986), human cholinesterase (36.0%, Lockridge et al., 1987), rat thyroglobulin (32.8%, Di Lauro et al., 1985), bovine thyroglobulin (35.7%, Mercken et al., 1985), and a second rabbit esterase (47.2%, Ozols, 1989). Two peptide fragments isolated and sequenced from a highly purified preparation of egasynlesterase-22 reveal that the protein represented by the composite sequence is not identical to egasyn/esterase-22 (R. Swank, personal communication). A comparison of either fragment to the corresponding region in our composite clone shows an amino acid identity of approximately 50% (data not shown). Therefore, the anti-egasyn/esterase-22 antiserum contained antibodies that reacted with a carboxylesterase distinct. from ES-22. The original isolate, the 360-bp clone A, contains just 203 bp of coding sequence, which may encode an epitope not normally revealed in the mature, folded protein. Alternatively, the epitope expressed by pEs-N is shared with egasyn/esterase-22, and the isolation of pEs-N resulted from antibody cross-reactivity.

348

OVNIC

E’I-

AL.

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