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The Ephx1(d) allele encoding an Arg338Cys substitution is associated with heat lability

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Abstract

Heat lability of the mouse hepatic microsomal epoxide hydrolase 1 enzyme-specific activity (EC 3.3.2.3) is greater for the A/J than the C57BL/6J strain. Analysis of the microsomal epoxide hydrolase 1 cDNA coding sequences shows the C57BL/6J and A/J strains to differ in a single base, a C to T transition at position 1012 from the ATG. This change would predict a substitution of an Arg for a Cys at codon 338. Lyman et al. (J. Biol. Chem 255:8650, 1980) studied 26 inbred mouse strains and assigned each strain to one of two groups based upon functional criteria that included heat lability and pH optima for microsomal epoxide hydrolase 1. The heat-labile strains including A/J were denoted with the Ephx1 d allele, whereas C57BL/6J and other members of the heat-stable strains were denoted with the Ephx1 b allele. We examined those same inbred mouse strains and found complete concordance between the assignment of microsomal epoxide hydrolase 1 allele superscript ``b'' or ``d'' and the wild-type and C1012T polymorphism respectively (Fisher's Exact Test, two-sided p < 0.0001). These data suggest that mouse hepatic microsomal epoxide hydrolase 1 heat lability is associated with the presence of a Cys at residue 338. Genomic samples from the available AXB and BXA recombinant inbred strains were allelotyped for the SNP identified in the Ephx1 gene that distinguishes the A/J and C57BL/6J parental strains and used to map Ephx1 to Chromosome (Chr) 1 at approximately 98.5cM (LOD = 10.0).
The Ephx1
d
allele encoding an Arg338Cys substitution is associated
with heat lability
James K. Hartsfield, Jr.,
1,2
Eric T. Everett
1,3
1
Oral Facial Genetics Program, Department of Oral Facial Development, Indiana University School of Dentistry, 1121 W. Michigan Street, DS270,
Indianapolis, Indiana 46202, USA
2
Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, 46202, USA
3
Department of Dermatology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA
Received: 16 March 2000 / Accepted: 19 May 2000
Abstract. Heat lability of the mouse hepatic microsomal epoxide
hydrolase 1 enzyme-specific activity (EC 3.3.2.3) is greater for the
A/J than the C57BL/6J strain. Analysis of the microsomal epoxide
hydrolase 1 cDNA coding sequences shows the C57BL/6J and A/J
strains to differ in a single base,aCtoTtransition at position 1012
from the ATG. This change would predict a substitution of an Arg
for a Cys at codon 338. Lyman et al. (J. Biol. Chem 255:8650,
1980) studied 26 inbred mouse strains and assigned each strain to
one of two groups based upon functional criteria that included heat
lability and pH optima for microsomal epoxide hydrolase 1. The
heat-labile strains including A/J were denoted with the Ephx1
d
allele, whereas C57BL/6J and other members of the heat-stable
strains were denoted with the Ephx1
b
allele. We examined those
same inbred mouse strains and found complete concordance be-
tween the assignment of microsomal epoxide hydrolase 1 allele
superscript “b” or “d” and the wild-type and C1012T polymor-
phism respectively (Fisher’s Exact Test, two-sided p < 0.0001).
These data suggest that mouse hepatic microsomal epoxide hydro-
lase 1 heat lability is associated with the presence of a Cys at
residue 338. Genomic samples from the available AXB and BXA
recombinant inbred strains were allelotyped for the SNP identified
in the Ephx1 gene that distinguishes the A/J and C57BL/6J paren-
tal strains and used to map Ephx1 to Chromosome (Chr) 1 at
approximately 98.5cM (LOD 10.0).
Introduction
Microsomal epoxide hydrolase (EC 3.3.2.3) is a bifunctional mem-
brane protein that plays a central role in the metabolism of xeno-
biotics and in hepatocyte uptake of bile acids (Lu and Miwa 1980;
Seidegard and DePierre 1983; von Dippe et al. 1993, 1996). On the
basis of differences in pH optimum for enzyme activity, sensitivity
to heat denaturation, cross-strain breeding, and genetic linkage
studies, Lyman et al. (1980) have shown that the mouse micro-
somal epoxide hydrolase 1 (Ephx1, formally Eph-1) gene on Chr
1 is biallelic, with codominant expression in heterozygotes. The
use of superscripts b and d designates the two alleles. C57BL/6J
are homozygous for Ephx1
b
, while A/J are homozygous for
Ephx1
d
.
Lyman et al. (1980) had used 2(3)-t-butyl-4-hydroxyanisole
(BHA) to induce hepatic microsomal epoxide hydrolase 1 activity
in the mice prior to evaluating the heat sensitivity of the enzyme.
Citing the attention given to microsomal epoxide hydrolase 1 ac-
tivity as an important factor in phenytoin teratogenicity (Buehler et
al. 1994; Hartsfield et al. 1995a), Hartsfield et al. (1995b) showed
that oral gavage of phenytoin for 14 days induced hepatic micro-
somal epoxide hydrolase 1 activity in both the C57BL/6J and A/J
strains, with the C57BL/6J strain having the greater constitutive
and induced enzyme activity.
The first purpose of this study was to determine and compare
the cDNA coding sequence for the enzyme in the C57BL/6J and
A/J strains. Identification of an underlying molecular difference
between these two alleles could be useful in understanding the
basis of the heat lability. The cDNA coding sequence in the rat
(accession #M26125; Porter et al. 1986), rabbit (M21496; Hassett
et al. 1989), and human (J0518; Jackson et al. 1987; Skoda et al.
1988), and the human genomic sequence (L29766; Hassett et al.
1994b) have been published. Upon finding only one difference in
the coding sequence between the C57BL/6J and A/J strains, the
study was extended to analyze the other 24 inbred mouse strains
assigned either the Ephx1
b
or Ephx1
d
allele by Lyman et al.
(1980). The second purpose was to map the Ephx1 locus by using
an SNP (single nucleotide polymorphism) found to distinguish the
two strains.
Materials and methods
Adult female A/J and C57BL/6J mice were purchased from The Jackson
Laboratory (Bar Harbor, Me.), housed and maintained at the University of
South Florida Medical Center Vivarium, which is fully accredited by the
American Association for Accreditation of Laboratory Animal Care. Total
cellular RNA was prepared from liver samples of C57BL6/J and A/J mice
according to the method of Chomczynski and Sacchi (1987), followed by
first-strand cDNA synthesis with oligo (dT)-12-18 and SuperScript
RNaseH-reverse transcriptase (Gibco/BRL Life Technologies, Gaithers-
burg, Md.). Initial oligonucleotide primers were synthesized based upon
conserved cDNA sequences between rat and human microsomal epoxide
hydrolase. Primers internal to the initial oligonucleotide primers were then
used in 5 and 3 RACE (rapid amplification of cDNA ends) reactions
(Gibco/BRL Life Technologies). A number of RACE products were gen-
erated, gel purified, and subjected to direct DNA sequencing through the
Department of Biochemistry and Molecular Biology Biotechnology Core
Facility, Indiana University School of Medicine. The most 5 and 3 con-
sensus sequences were then used to design a pair of oligonucleotide prim-
ers for PCR-mediated cloning of the entire cDNA encoding microsomal
epoxide hydrolase from C57BL/6J and A/J mice, generating products that
were approximately 1.5 kb for each mouse strain. The PCR products were
gel purified by using QIAEX resin (QIAGEN, Chatsworth, Calif.) and
blunt end cloned into the EcoRV site of pBluescript SKII (Stratagene,
LaJolla, Calif.). At least three individual recombinant clones for each
mouse strain were selected for expansion and DNA sequencing.
Exon 7 of the Ephx1 gene containing the SNP at codon 338 that
distinguishes the A/J and C57BL/6J strains was PCR amplified from ali-
quots of genomic DNA representative of the mouse strains originally ex-
amined by Lyman et al. (1980) with the following oligonucleotide primers:
Correspondence to: J.K. Hartsfield, Jr.; E-mail: jhartsfi@iupui.edu
The nucleotide sequence data for the Ephx1
b
allele reported in this paper
have been deposited in GenBank and assigned the accession number
U89491.
Mammalian Genome 11, 915–918 (2000).
DOI: 10.1007/s003350010169
© Springer-Verlag New York Inc. 2000
Incorporating
Mouse Genome
Ephx1ex7F 5-GCTGTGCTCTGAATGACTCT and Ephx1ex7R 5-
CTCTCCAGGCCTCCATCC. The resulting 109-bp product containing
codon 338 was digested with BsiYI (Boehringer Mannheim, Indianapolis,
Ind.), at 37°C overnight. The digestion products were visualized with a
1:10,000 dilution of Syber-Green (FCM BioProducts, Rockland, Me.) fol-
lowing separation on a 2% agarose TAE gel.
Aliquots of genomic DNA representative of both panels of AXB and
BXA recombinant inbred strains were obtained from the DNA Resource at
The Jackson Laboratory. Using the described difference between the A/J
and C57BL/6J alleles for Ephx1, we allelotyped genomic samples from the
available AXB and BXA recombinant inbred strains. Statistical analyses of
the experimental results were performed with the program, Map Manager
QT version 3.0b16 (Manly and Olson 1999).
Results
Cloning of Mouse mEH cDNAs. The cDNA consisting of the en-
tire coding region for the C57BL/6J Ephx1
b
allele is shown in Fig.
1. Alignment of the nucleotide sequences obtained from C57BL/6J
and A/J cDNAs identified a single base difference at position 1055
(1012 from the start of the coding region), which is predicted to
occur within codon 338. This transition CGT (C57BL/6J) to TGT
(A/J) would predict a change from an arginine to a cysteine.
BLASTN (Altschul et al. 1990) search through the NCBI by using
the C57BL/6J sequence as a query found the mouse cDNA se-
quence to be nearly identical to the rat (M26125) microsomal
epoxide hydrolase cDNA at 93%, similar and highly homologous
to human (J03518), rabbit (M21496), and pig (AB000883) micro-
somal epoxide hydrolase cDNAs at 84%, 83%, and 83%, respec-
tively. Several previous reports indicate that the gene for micro-
somal epoxide hydrolase 1 (human, rat, and rabbit) is transcribed
and procesed as a single mRNA (Falany et al. 1987; Hassett et al.
1989, 1994a; Wilson and Omiecinski 1989). We have identified
through 5 RACE and cDNA cloning an mRNA transcript that
diverges in sequence at seven bases upstream from the start codon
at the junction between exon 1 and exon 2. A search of the Gen-
Bank database for sequence similarities found significant homol-
ogy with the reported identification of multiple mRNAs for mEH
by alternative splicing in the rat (Honscha et al. 1991), but not with
those reported for humans by Gaedigk et al. (1994). Comparisons
of translated microsomal epoxide hydrolase 1 protein sequences
are shown in Fig. 2. A survey of the 26 inbred mouse strains (Table
1), originally reported by Lyman et al. (1980), shows all of the
strains categorized as having Ephx1
d
allele to carry the C1012T
polymorphism, whereas those strains having the Ephx1
b
allele all
carry the cytosine at nucleotide 1012 (Fisher’s Exact Test, two-
sided p < 0.0001).
Mapping of Ephx1 using the 338 polymorphism. Analysis of the
AXB and BXA RI sets combined maps the Ephx1 gene proximal
to Pmv24 (polytropic mouse leukemia virus locus) and distal to the
D1Mit456 marker with a LOD score of 10.0 (mapping data de-
posited with the Mouse Genome Informatics at The Jackson Labo-
ratory, Accession ID:MGI:1337851). Pmv24 and D1Mit456 have
been mapped to Chr 1 at 95.8 cM and 98.7 cM, respectively
(Frankel et al. 1989, 1991; Higgins and Paigen 1997). This present
Fig. 1. Mouse (C57BL/6J, liver) microsomal epoxide hydrolase 1
(Ephx1
b
) mRNA, complete coding sequence with translation. This se-
quence has been deposited in GenBank with accession number U89491.
Cytosine (bold and double underline) at nucleotide 1055 (1012 from the
start of the coding sequence) and an arginine (R, bold and double under-
lined) codon 338. Start the stop codons are shown underlined. Additional
3 untranslated sequence (underlined) and the 5 untranslated sequence that
diverges from exon 1 sequence previously reported for rat (M26125) and
human (J0518) is shown with a dashed underline.
Fig. 2. Microsomal epoxide hydrolase 1 peptide sequence comparison.
Mouse (U89491) sequence for the Ephx1
b
allele aligned with rat (P07687),
human (P07099), porcine (1840391), and rabbit (P04068) peptide se-
quences. Symbols are the following: (*) complete identity; (+) conserva-
tive substitution; (−) nonconservative substitution; and at position 338 the
R is changed toaCintheEphx1
d
allele.
J.K. Hartsfield, Jr., E.T. Everett: Microsomal epoxide hydrolase heat lability polymorphism916
mapping of Ephx1 is consistent with earlier linkage studies local-
izing the gene to 98.5 cM (Lyman et al. 1980; Simmons et al.
1985; Mullick et al. 1995).
Discussion
The arginine at codon 338 appears to be highly conserved among
mouse, rat, human, rabbit, and pig. Unlike soluble epoxide hydro-
lase, where Asp333, Asp495, and His523 form the catalytic triad
(Arand et al. 1996), the catalytic domain of microsomal epoxide
hydrolase 1 is thought to be composed of Asp226, Glu404, and
His43133. The amino terminus of microsomal epoxide hydrolase 1
containing the membrane anchor sequence appears not to be es-
sential for the catalytic activity of the protein (Friedberg et al.
1994). His431 and Glu404 appear to be essential for catalytic
activity (Bell and Kasper 1993; Arand et al. 1999). Therefore, the
structure function effect of having a Cys at position 338 is not
clearly known. Based upon the association of increased heat labil-
ity of specific activity, the addition of a cysteine may alter the
structure of the protein. Among different mouse strains studied,
two groups can be formed (Table 1) from microsomal epoxide
hydrolase 1 optimum activity pH, sensitivity to heat denaturation,
and the presence of the C1012T polymorphism.
Microsomal epoxide hydrolase 1 has been shown to have ubiq-
uitous tissue expression, furhter underscoring its importance in
cellular metabolism (Seidegard and DePierre 1983). In addition to
the widespread distribution of activity, levels of microsomal ep-
oxide hydrolase 1 enzymes activities vary among organs and dif-
ferent cell types within tissues (Hernandez and Bend 1982; Jef-
coate 1983). The molecular basis for this observed variation in
enzyme activity has been examined only recently. Genetic poly-
morphisms in the coding regions, specifically involving codon
113, appear to result in decreased enzyme activity when tested in
an in vitro system (Hassett et al. 1994a). There is also a nonuni-
form pattern of microsomal epoxide hydrolase 1 induction among
different rat liver lobule regions (Baron and Kawabata 1983).
Polymorphisms in the 5 flanking sequence (Gaedigk et al. 1997;
Raaka et al. 1998) are also likely to effect enzyme activity in
humans. Finally, the use of multiple untranslated exons and tissue-
specific promoters are involved in the regulation of the micro-
somal epoxide hydrolase 1 gene expression in different tissues
(Honscha et al. 1991; Gaedigk et al. 1997). In determining the
mouse cDNA sequence and the association of the C1012T poly-
morphism and functional changes of the enzyme, we also noted the
homology of the mouse 5 untranslated sequence to one of the
alternatively spliced rat cDNAs, suggesting that mouse micro-
somal epoxide hydrolase 1 may be regulated by multiple untrans-
lated exons and tissue-specific promoters as well.
Our data confirm the location of Ephx1 on mouse Chr 1. This
and our earlier localization of the human EPHX1 gene to 1q42.1
(Hartsfield et al. 1998) add an additional locus to the region of
linkage conservation between human Chr 1q42.1 and the distal
region of mouse Chr 1. This follows earlier mapping of the human
gene, EBAF, endometrial bleeding-associated factor (left-right de-
termination, factor A; transforming growth factor beta superfam-
ily) that maps to 1q42.1 (Kothapalli et al. 1997) and the mouse
homolog, Ebaf (formerly TgfB4 and identical to left-right deter-
mination, factor A found in humans) localized to mouse chromo-
some band F (Meno et al. 1997).
Acknowledgments. This work was supported by grants from the National
Institute of Dental Research Physician Scientist Award K11 DE00243 (J.K.
Hartsfield) and FIRST Award R29 DE11280 (J.K. Hartsfield). J.K. Harts-
field gratefully acknowledges the time and assistance given, as well as the
patience shown, by Kristen Hinds-Frey, Robert N. Haire, Rhonda Litman,
and Gary W. Litman when he began to work on this project as a part of his
Ph.D. thesis. We also thank Dr. Beverly J. Paigen for kindly reviewing the
AXB/BXA mapping data.
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C57BR/cdJ SJL/J DBA/1J
C57/LJ SWR/J
HRS/J 129/J
LG/J
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