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Genetica, Vol. 138(7), pp. 695-708 (2010)
Mammalian Carboxylesterase 3: Comparative Genomics and Proteomics
Roger S Holmes1-4
, Laura A Cox1,2 and John L VandeBerg1,2
1Department of Genetics and 2Southwest National Primate Research Center, Southwest Foundation for
Biomedical Research, San Antonio, TX, USA, and 3School of Biomolecular and Physical Sciences, Griffith
University, Nathan, QLD, Australia
Roger S Holmes, D.Sc.
Department of Genetics
Southwest National Primate Research Center
Southwest Foundation for Biomedical Research
San Antonio, TX, USA 78227
Keywords: Mammals; amino acid sequence; carboxylesterase; evolution; gene duplication.
Running Head: Mammalian CES3: comparative genomics and proteomics
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At least five families of mammalian carboxylesterases (CES) catalyse the hydrolysis or transesterification of a
wide range of drugs and xenobiotics and may also participate in fatty acyl and cholesterol ester metabolism. In
this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures,
and gene locations for CES3 genes and encoded proteins using data from several mammalian genome projects.
Mammalian CES3 genes were located within a CES gene cluster with CES2 and CES6 genes, usually containing
13 exons transcribed on the positive DNA strand. Evidence is reported for duplicated CES3 genes for the chimp
and mouse genomes. Mammalian CES3 protein subunits shared 58-97% sequence identity and exhibited
sequence alignments and identities for key CES amino acid residues as well as extensive conservation of
predicted secondary and tertiary structures with those previously reported for human CES1. The human genome
project has previously reported CES3 mRNA isoform expression in several tissues, particularly in colon, trachea
and in brain. Predicted human CES3 isoproteins were apparently derived from exon shuffling and are likely to
be secreted extracellularly or retained within the cytoplasm. Mouse CES3-like transcripts were localized in
specific regions of the mouse brain, including the cerebellum, and may play a role in the detoxification of drugs
and xenobiotics in neural tissues and other tissues of the body. Phylogenetic analyses demonstrated the
relationships and potential evolutionary origins of the mammalian CES3 family of genes which were related to
but distinct from other mammalian CES gene families.
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At least five families of mammalian carboxylesterases (CES; E.C.18.104.22.168) have been reported (Holmes
et al., 2008a,b; 2009a-d) including CES1, the major liver enzyme (Munger et al., 1991; Shibata et al. 1993;
Ghosh 2000; Holmes et al., 2009a); CES2, the major intestinal enzyme (Langmann et al. 1997; Schewer et al.
1997; Holmes et al., 2009a); CES3, expressed in liver, colon and brain (Sanghani et al. 2004; Holmes et al.,
2009a-c); CES5 (also called CES7 or cauxin), a major urinary protein of the domestic cat (Miyazaki et al. 2003;
2006; Holmes et al., 2008b); and CES6, a predicted CES-like enzyme in brain (Clark et al., 2003; Holmes et al.,
2009d). These enzymes catalyse hydrolytic and transesterification reactions with xenobiotics, anticancer pro-
drugs, and narcotics (Satoh & Hosokawa 1998; 2006; Satoh et al. 2002; Ohtsuka et al. 2003; Redinbo & Potter
2005), and detoxify organophosphates and insecticides (Ahmad & Forgash 1976; Leinweber 1987). CES may
also catalyse several cholesterol and fatty acid metabolic reactions (Ghosh 2000; Tsujita & Okuda 1993; Becker
et al. 1994; Hosokawa et al. 2007; Diczfalusy et al. 2001), the conversion of lung alveolar surfactant (Ruppert et
al. 2006) and assist with the assembly of low density lipoprotein particles in liver (Wang et al. 2007).
Structures for several human and animal CES genes have been determined, including human (Becker et
al. 1994; Langmann et al. 1997; Ghosh 2000; Marsh et al. 2004) and rodent CES1 and CES2 ‘like’ genes (Ghosh
et al. 1995; Dolinsky et al. 2001; Hosokawa et al. 2007). Predicted gene structures have also been described for
other mammalian CES gene families, including CES1, CES2 and CES5 genes, which are localized in two
contiguous CES gene clusters on human (chromosome 16), horse (chromosome 3), cow (chromosome 18) and
opossum (chromosome 1) genomes (Holmes et al. 2008a; 2009a-c). Mammalian CES genes usually contain 12
to 14 exons of DNA encoding CES enzyme sequences which may undergo exon shuffling generating several
CES isoproteins in each case (Thierry-Mieg and Thierry-Mieg, 2006). There are similarities for the exon
boundaries for each of the mammalian CES genes (Holmes et al., 2008a,b; 2009a-d) as well as significant
sequence identities, especially for key regions previously identified for the major human liver CES1 (Bencharit
et al. 2003; 2006; Fleming et al. 2005). Three-dimensional structural analyses of human CES1 have identified
three major ligand binding sites, including the promiscuous active site, ‘side door’ and ‘Z-site’, where
substrates, fatty acids and cholesterol analogues respectively, are bound; and the ‘gate’, reportedly functioning
in product release (Bencharit et al. 2003; 2006; Fleming et al. 2005).
This paper reports the predicted gene structures and amino acid sequences for several mammalian CES3
genes and proteins, the predicted secondary and tertiary structures for mammalian CES3 protein subunits, and the
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structural, phylogenetic and evolutionary relationships for these genes and enzymes with those for other mammalian
CES gene families.
In silico mammalian CES gene and protein identification.
BLAST (Basic Local Alignment Search Tool) studies were undertaken using web tools from the
National Center for Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al,
1997). Protein BLAST analyses used mammalian CES amino acid sequences previously described (Table 1).
Non-redundant protein sequence databases for several mammalian genomes were examined using the blastp
algorithm, including human (Homo sapiens) (International Human Genome Sequencing Consortium, 2001);
chimp (Pan troglodytes) (Chimpanzee Genome Analysis Consortium, 2005); orangutan (Pongo abelii)
(http://genome.wustl.edu) ; the cow (Bos Taurus) (Bovine Genome Project, 2008); horse (Equus caballus)
(Horse Genome Project, 2008); mouse (Mus musculus) (Mouse Sequencing Consortium, 2002); and opossum
(Monodelphis domestica) (Mikkelsen et al., 2007). This procedure produced multiple BLAST ‘hits’ for each of
the protein databases which were individually examined and retained in FASTA format, and a record kept of the
sequences for predicted mRNAs and encoded CES-like proteins . These records were derived from annotated
genomic sequences using the gene prediction method: GNOMON and predicted sequences with high similarity
scores for human CES6. Predicted CES3-like protein sequences were obtained in each case and subjected to in
silico analyses of predicted protein and gene structures.
BLAT analyses were subsequently undertaken for each of the predicted CES3 amino acid sequences
using the UC Santa Cruz web browser [http://genome.ucsc.edu/cgi-bin/hgBlat] (Kent et al. 2003) with the
default settings to obtain the predicted locations for each of the mammalian CES3 genes, including predicted
exon boundary locations and gene sizes. BLAT analyses were similarly undertaken of CES1, CES2, CES5 and
CES6-like human genes using previously reported sequences for encoded CES subunits in each case (see Table
1). Structures for human CES3 isoforms (splicing variants) were obtained using the AceView website
(http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html?human) to examine predicted gene and
protein structures using the human CES3 cDNA sequence (GenBank NM024922) to interrogate this database of
human mRNA sequences (Thierry-Mieg and Thierry-Mieg, 2006).
BLAT (BLAST-Like Alignment Tool) in silico studies were undertaken using the UC Santa Cruz web
browser [http://genome.ucsc.edu/cgi-bin/hgBlat] (Kent et al. 2003) with the default settings. UniProtKB/Swiss-Prot
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Database [http://au.expasy.org] and GenBank [http://www.ncbi.nlm.nih.gov/Genbank/] sequences for human,
orangutan and mouse CES sequences (Table 1) were used to interrogate human, chimp, orangutan, horse, cow,
mouse and opossum genome sequences. Gene locations, predicted gene structures and CES protein subunit
sequences were observed for each CES examined for those regions showing identity with the respective mammalian
CES gene products.
Predicted Structures and Properties of Mammalian CES Subunits.
Predicted secondary and tertiary structures for human and other mammalian CES-like subunits were
obtained using the PSIPRED v2.5 web site tools provided by Brunel University
[http://bioinf.cs.ucl.ac.uk/psipred/psiform.html] (McGuffin et al. 2000) and the SWISS MODEL web tools
[http://swissmodel.expasy.org/], respectively (Guex & Peitsch 1997; Kopp & Schwede 2004). The reported tertiary
structure for the rabbit CES1 4-piperidino-piperidine complex (Bencharit et al., 2003) served as the reference for the
predicted human CES3 tertiary structure, with a modeling range of residues 34 to 553. Theoretical isoelectric points
and molecular weights for mammalian CES3 subunits were obtained using Expasy web tools
(http://au.expasy.org/tools/pi_tool.html). SignalP 3.0 web tools were used to predict the presence and location of
signal peptide cleavage sites (http://www.cbs.dtu.dk/services/SignalP/) for each of the predicted mammalian CES
sequences (Emanuelsson et al 2007). The NetNGlyc 1.0 Server was used to predict potential N-glycosylation sites
for human CES and mammalian CES3 subunits (http://www.cbs.dtu.dk/services/NetNGlyc/). Predictions of
subcellular locations for mammalian CES3 ‘like’ enzymes and for human CES1, CES2, CES5 and CES6 were
conducted using PSORT 11 (http://psort.ims.u-tokyo.ac.jp/form2.html) (Horton & Nakai, 1997).
Mouse Brain CES3 Expression
The Allen Mouse Brain Atlas was interrogated for cellular transcription of the mouse CES3 gene using the
GenBank BC061004 sequence (see Table 1) (Lein et al., 2007). Data for sagittal sections of mouse brain were
examined and recorded for mouse CES3 transcripts using web tools available on the Allen Brain Atlas web site
Phylogenetic Studies and Sequence Divergence
Phylogenetic trees were constructed using an amino acid alignment from a ClustalW-derived alignment of
CES protein sequences, obtained with default settings and corrected for multiple substitutions (Chenna et al. 2003)
[http://www.ebi.ac.uk/clustalw/]. An alignment score was calculated for each aligned sequence by first calculating a
pairwise score for every pair of sequences aligned. Alignment ambiguous regions, including the amino and carboxyl
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termini, were excluded prior to phylogenetic analysis yielding alignments of 453 residues for comparisons of
mammalian CES1, CES2, CES3, CES5 and CES6 sequences (Table 1). Pairwise scores were calculated using the
number of identities in the best alignment divided by the number of residues compared. Scores were initially
calculated as percent identity scores and were converted to distances by dividing by 100 and subtracting from 1.0 to
give the number of differences per site. The extent of divergence for the mammalian CES3-like subunits, and the
human CES1, CES2, CES5 and CES6 subunits were determined using the SIM-Alignment tool for Protein
Sequences [http://au.expasy.org/tools/sim-prot.html] (Schwede et al. 2003).
Results and Discussion
Alignments of Human CES Subunits and Mammalian CES3 Amino Acid Sequences.
The deduced amino acid sequences for horse, cow and mouse CES3-like subunits are shown in Figure 1
together with the previously reported sequences for human CES1 (Munger et al. 1991; Shibata et al. 1993); human
CES2 (Langmann et al. 1997; Schewer et al. 1997); human CES3 (Sanghani et al. 2004 ); human CES5 (Holmes et
al., 2008b); and human CES6 (Holmes et al., 2009d) (Table 1). Alignments of the human and other mammalian
CES3 subunits examined in this figure showed between 68-77% sequence identities, suggesting that these CES
protein subunits are products of the same family of genes, whereas sequence alignments of mammalian CES3
subunits with human CES1, CES2, CES5 and CES6 subunits exhibited lower levels of sequence identities with
human CES3 (47%, 50%, 48% and 45%, respectively), indicating that these are members of distinct CES families
The amino acid sequence for human CES3 contained 571 residues whereas other predicted mammalian
CES3 subunits contained fewer amino acids: 570 residues for cow and horse CES3; 554 residues for mouse CES3;
and 568 residues for mouse CES3L (Figure 1). Previous studies on human CES1 have identified key residues which
contribute to the catalytic, subcellular localization, oligomeric and regulatory functions for this enzyme (sequence
numbers refer to human CES1). These include the catalytic triad for the active site (Ser228; Glu345; His458)
(Cygler et al. 1993); microsomal targeting sequences, including the hydrophobic N-terminus signal peptide (von
Heijne 1983; Zhen et al. 1995; Potter et al. 1998) and the C-terminal endoplasmic reticulum (ER) retention sequence
(His-Ile-Glu-Leu) (Robbi & Beaufay 1983); disulfide bond forming residues (Cys95/Cys123 and Cys280/Cys291)
(Lockridge et al. 1987); and ligand binding sites, including the ‘Z-site’ (Gly358), the ‘side door’ (Val424-Met425-
Phe426) and the ‘gate’ (Phe551) residues. Identical residues were observed for each of the mammalian CES3
subunits for the active site triad and disulfide bond forming residues for human CES1although several changes were
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observed for the corresponding residues for CES3 subunits of other key human CES1 residues: the ‘side-door’
identified for human CES1 (Val424-Met425-Phe426) (Bencharit et al., 2003; 2006) has a Val416-Phe417-Ile418
sequence for human CES3; the ‘Z-site’ (Gly358 for human CES1) has been changed to Ser355 for human CES3,
although other mammalian CES3 sequences examined have retained a glycine residue at this position; the
hydrophobic N-terminal sequence for mammalian CES3 sequences has undergone major changes however this
region retains in each case a predicted signal peptide property; the mammalian CES3 C-terminal sequences have
undergone significant changes in sequence (QEDL, QEEL or PEEL) in comparison with human CES1 (HIEL). This
may be responsible for a change in the predicted microlocalization for the CES3 subunits (secreted or extracellular
enzymes), as compared with human CES1, which is retained by the endoplasmic reticulum (Table 1). The ‘gate’
residue has also undergone changes for the mammalian CES3 sequences examined from Phe551 (CES1) to Tyr
(human and horse CES3) or Leu (cow and mouse CES3).
Other key human CES1 sequences included two charge clamps which are responsible for subunit-subunit
interaction, namely residues Lys78/Glu183 and Glu72/Arg193 (Bencharit et al. 2003; 2006; Fleming et al. 2005).
Predicted mammalian CES3 subunit sequences for these sites indicated that only one of the charge clamps would be
retained for human CES3 subunits: Arg88/Glu191 (Figure 1), whereas other mammalian CES3 subunits examined
showed no potential for forming charge clamps at the corresponding sequence positions. Previous reports have
shown that human and baboon CES2 subunits, which lack these charge clamp residues, behave as monomers (Pindel
et al. 1997; Holmes et al., 2009a). With the exception of human CES3, the mammalian CES3 sequences have
undergone amino acid substitutions for those residues which contribute to the oligomeric subunit structure for human
CES1. The identified human CES1 charge clamp residues Lys78/Glu183 and Glu72/Arg193 have other residues at
the corresponding sites (Arg88/191Lys and Gln82/Ala194 for the horse CES3 sequence) (Figure 1), and it is likely
that these mammalian CES3 subunits are also monomeric in subunit structure. This may have a major influence on
the kinetics and biochemical roles for mammalian CES3 since three dimensional studies have suggested that ligand
binding to the human CES1 ‘Z-site’ shifts the trimer-hexamer equilibrium towards the trimer facilitating substrate
binding and enzyme catalysis (Redinbo & Potter 2005). This property is unlikely to be shared by mammalian CES3
which is expected to display distinct kinetic properties to the oligomeric CES1 enzyme.
The N-glycosylation site reported for human CES1 at Asn79-Ala80-Thr81 (Kroetz et al. 1993; Bencharit et
al. 2003; 2006; Fleming et al. 2005) has not been retained for any of the mammalian CES3 sequences, although
additional potential N-glycosylation sites were observed at one or several positions (Table 3). Given the reported
role of the N-glycosylated carbohydrate group in contributing to mammalian CES stability and maintaining catalytic
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efficiency (Kroetz et al. 1993), this property may be shared by the mammalian CES3 subunits as well, especially for
those containing multiple potential sites for N-glycosylation, such as the mouse CES3L sequence, which contains
four such sites.
Predicted Secondary and Tertiary Structures for Mammalian CES3 Subunits.
Analyses of predicted secondary structures for mammalian CES3 sequences were compared with the
previously reported secondary structure for human CES1 (Bencharit et al., 2003; 2006) and with those predicted for
human CES2, CES5 and CES6 (Holmes et al., 2009a) (Figure 1). Similar α-helix β-sheet structures were observed
for all of the mammalian CES3 and human CES gene products examined. Consistent structures were apparent near
key residues or functional domains including the α-helix within the N-terminal signal peptide; the β-sheet and α-
helix structures near the active site Ser228 (human CES1) and ‘Z-site’ (Glu354/Gly356 respectively); the α-helices
bordering the ‘side door’ site; and the α-helix containing the ‘gate’ residue (Phe551 for human CES1). In addition,
two random coil regions (residues 51-115 and 169-188 for human CES1) were predominantly retained for all forms
of mammalian CES3 examined which have been shown for human CES1 to contain 2 charge clamps sites:
(Lys79/Glu183 and Glu73/Arg186); an N-glycosylation site at Asn79-Ala80-Thr81; a second potential N-
glycosylation site for human CES2, and one of the disulfide bridges (87Cys /117Cys) reported for human CES1. The
human CES5 secondary structure, however, predicted an additional helix at the hydrophobic C-termini, in
comparison with mammalian CES3 C-terminal sequences. In addition, the predicted 3-D structure for human CES3
is similar to the reported for human CES1 (Bencharit et al., 2003; 2006) (Figure 3). These were based on structures
reported for residues 21-553 of human CES1 and a predicted structure for residues 34-553 of human CES3. The
single helix and following two β-sheet structures at the C-termini (shown in red at the top) for the human CES1 and
CES3 subunits are readily apparent, as are four β-sheet structures at the N-termini of the CES subunits (shown in
blue at the base), in each case. It is apparent that human CES3 is likely to be highly similar in its secondary and
tertiary structure to that reported for human CES1.
Predicted Gene Locations and Exonic Structures for Mammalian CES3 Genes.
Table 1 summarizes the predicted locations for mammalian CES3 genes based upon BLAT interrogations
of several mammalian genomes using the reported sequences for human CES3, mouse CES3 and CES3L, and the
predicted sequences for horse and cow CES3 and the UC Santa Cruz Web Browser (Kent et al. 2003). The predicted
mammalian CES3 genes were located in a CES2/CES3/CES6 gene cluster in each case, although the gene order
underwent changes for different species. In addition, mouse showed evidence of duplicate CES3 genes, with
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predicted CES3 and CES3L genes being located within a four CES gene cluster on chromosome 8: CES2-CES3-
CES3L-CES6 (Table 1). Chimp CES3 also provided evidence for duplicate forms of this gene on chromosome 16
but with a changed order of genes: CES6-CES3L-CES2-CES3 (Table 1). BLAT interrogations of mammalian
genomes with the corresponding CES sequences suggested that the CES2-CES3-CES6 gene cluster was syntenic for
chromosomes 16 (human, chimp and orangutan), 3 (horse), 18 (cow), 8 (mouse) and 1 (opossum), respectively.
With the exception of the opossum CES3 gene, mammalian CES3 ‘like’ genes were apparently transcribed
on the positive strand, as for the human CES2 and CES6 genes. In contrast, human CES1 and CES5 genes were
transcribed on the negative strand (Table 1). Figure 1 summarizes the predicted exonic start sites for cow, horse and
mouse CES3 genes with each having 13 exons, in identical or similar positions to those predicted for the human
CES3 gene (Holmes et al., 2009a). The predicted chimp CES3L gene however exhibited only 11 predicted exons.
Human CES1 (Munger et al. 1991; Shibata et al. 1993), CES2 (Tang et al., 2008), CES5 (Ota et al., 2004) and CES6
(Holmes et al., 2009d) genes contained 14, 12, 13 and 14 exons respectively (Figure 1), in similar positions to those
predicted for the mammalian CES3 genes (Figure 1).
Figure 4 shows the predicted nucleotide sequences for exons 2-4 and introns 2 and 3 of mouse CES3 and
CES3L genes. The sequences for the exons were 98% identical with nearly identical amino acid sequences involving
five amino acid substitutions for these genes, whereas mouse CES3 and CES3L introns 2 and 3 showed distinct
nucleotide sequences which were 85% and 94% identical, respectively. In addition, BLAT analyses of the mouse
genome revealed distinct but closely localized predicted positions for these genes, being separated by ~26 kilobases
of DNA on chromosome 8 (Table 1). These data support an hypothesis for two closely related and localized CES3-
like genes in the mouse, for which the subunits have been previously designated as esterase-31 (also called CES3)
(Aida et al., 1993) and esterase-31L (also called CES3L) (Mouse Genome Consortium, 2004).
Mouse Brain CES3 and CES3L Expression.
Figure 5 shows a sagittal section of mouse brain examining the distribution of CES3 and CES3L transcripts
(GenBank mRNA BC061004 and BC019047) within different regions of the brain (provided by the Allen Institute
for Brain Science) (Lein et al., 2007). The cerebellum folds exhibited highest staining levels for both transcripts
although staining was observed throughout the brain, including the hippocampus, amygdalar nuclei, the olfactory
bulb, the cortex, the pons and the medulla regions.
The differential tissue and subcellular distributions of CES family members may assist in determining the
differential roles of CES family members within mammalian organisms. Mammalian liver is predominantly
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responsible for drug and xenobiotic clearance from the body with CES1 and CES2 (with CES1 > CES2) playing
major roles, following absorption of drugs and xenobiotics into the circulation (Pindel et al. 1997; Imai 2006).
Mammalian intestine (with CES2 > CES1) is predominantly responsible for first pass clearance of several drugs and
xenobiotics, with the activity occurring mostly in the ileum and jejunum and processed via CES2 (Imai et al. 2003).
CES1 and CES2 also serve different roles in prodrug activation, as shown for the anti-cancer drug irinotecan (CPT-
11) which is converted to its active form SN-38 predominantly by CES2 (Humerickhouse et al. 2000; Xu et al.
2002). In contrast with CES1 and CES2, mammalian CES5 is predominantly expressed in peripheral tissues,
including brain, kidney, lung and testis (Thierry-Mieg and Thierry-Mieg, 2006), and in cat, is a secreted form of
CES enzyme appearing in cat urine (Miyazaki et al. 2003; 2006). Mammalian CES5 may serve in two major roles
within mammalian fluids and peripheral tissues, including regulating the production of a pheromone precursor in
urine (Miyazaki et al. 2003; 2006) and contributing to lipid and cholesterol transfer processes within male
reproductive fluids (Ecroyd et al. 2005).
The metabolic role(s) for mammalian CES3 has not been extensively investigated although the enzyme is
capable of activating prodrugs such as irinotecan (Sanghani et al., 2004) and the gene is expressed as distinct
isoforms in tissues of the body, including the colon, placenta and neural tissues, such as the cerebellum and
hippocampus (Figure 2; Table 4) (Thierry-Mieg and Thierry-Mieg, 2006). Human CES3 mRNAs encode isoforms
which are predicted to be either secreted or extracellular (CES3a-c), cytosolic (CES3d) or extracellular (CES3e-u),
and may play distinct roles in carboxyl-ester metabolism in the body. A comparison of the predicted cellular
locations for mammalian CES3 reveals that it is likely to be extracellular (including the cell wall) in primate (human,
chimp and orangutan) and mouse tissues, whereas horse and bovine CES3 are predicted to be localized in the
endoplasmic reticulum, which is comparable with human CES1 and CES2. The extracellular locations predicted for
human and mouse CES3 may reflect distinct roles for this enzyme in drug metabolism within peripheral tissues of
Phylogeny and Divergence of CES3 and Other Mammalian CES Sequences.
A phylogenetic tree (Figure 6) was calculated by the progressive alignment of human, chimp, orangutan,
horse, cow, mouse and opossum CES3-like amino acid sequences with human CES1, CES2, CES5 and CES6
sequences. The phylogram showed clustering of all of the CES3-like sequences which were distinct from the other
human CES families. In addition, the CES3 and CES3L chimp and mouse sequences showed clustering with the
primate and mouse CES3-like genes, respectively which is consistent with these genes being products of recent
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duplication events during mammalian evolution. Holmes et al (2008a) have previously demonstrated that the gene
duplication events which generated the ancestral mammalian CES1, CES2, CES3, CES5 and CES6 genes predated
the common ancestor for marsupial and eutherian mammals [estimated at ~ 173-193 million years ago by
Woodburne et al (2003)], and appear to coincide with the early diversification of tetrapods approximately 350-360
million years ago ((Donoghue & Benton, 2007). The mammalian CES3 family of genes may therefore be considered
to be of an ancient origin, well prior to the appearance of mammals in evolutionary time.
In conclusion, the results of the present study indicate that mammalian CES3 genes and encoded CES3
enzymes represent a distinct CES gene and enzyme family which share key conserved sequences and structures that
have been reported for human CES1 and have family specific sequences consistent with the oligomeric and
monomeric subunit structures for CES1 and CES3, respectively. This study also reports that chimp and mouse
genomes contain at least two CES3 ‘like’ genes, which are located in tandem with the CES2 and CES6 genes, and
with the more distantly located CES1 and CES5 genes on chromosomes 16 and 8, respectively. Predicted secondary
structures and tertiary structures for mammalian CES3 subunits showed a high degree of conservation with human
CES1. Phylogeny studies using several mammalian CES3 subunits (human, chimp, orangutan, mouse, horse, cow
and opossum) indicated that CES3‘ like’ genes have apparently appeared during mammalian evolution well prior to
the eutherian and marsupial common ancestor more than 170 million years ago, which is consistent with previous
studies (Holmes et al, 2008a).
This project was supported by NIH Grants P01 HL028972 and P51 RR013986. In addition, this investigation was
conducted in facilities constructed with support from Research Facilities Improvement Program Grant Numbers 1
C06 RR13556, 1 C06 RR15456, 1 C06 RR017515.
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Figure 1: Amino Acid Sequence Alignments for Mammalian CES3 and Human CES1,
CES2, CES5 and CES6 Subunits
See Table 1 for sources of CES sequences; * shows identical residues for CES subunits; :
similar alternate residues; . dissimilar alternate residues; Residues involved in endoplasmic
reticulum processing at N- (Signal peptide) and C- termini (MTS-microsomal (endoplasmic
reticulum) targeting sequence); N-glycosylation residues at 79NAT (Human CES1) and potential
N-glycosylation sites; active site (AS) triad residues Ser; Glu; and His. ‘Side door’, ‘Gate’
residues and Cholesterol binding Gly residue (Z site) for human CES1 Disulfide bond Cys
residues for human CES1: S---S. Charge clamp residues identified for human CES1; Helix (Human
CES1 or predicted helix; Sheet (Human CES1) or predicted sheet. Bold font shows known or
predicted exon junctions. Exon numbers refer to human CES1 gene.
Page 18 of 26
Figure 2: Gene Structures and Splicing Variants for the Human CES3
(NM024922) and the Mouse CES3 (BC061004) Genes.
Derived from AceView website (Thierry-Mieg and Thierry-Mieg,2006)
http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/ Mature isoform variants (a,b,c etc) are
shown with capped 5’- and 3’- ends for the predicted mRNA sequences. Gene COesterase 1 refers
to human CES3 gene; Gene Es31 refers to mouse CES3 gene.
Page 19 of 26
Figure 3: Tertiary Structure for Human CES1 Subunit and Predicted Tertiary
Structure for Human CES3 Subunit.
The structure for human CES1 is taken from Bencharit et al, 2003. Predicted human CES3 3-D
structure was obtained using the SWISS MODEL web site
http://swissmodel.expasy.org/workspace/index.php? And the reported amino acid sequence
(Sanghani et al.,2004). The rainbow color code describes the 3-D structures from the N- (blue)
to C-termini (red color).
Page 20 of 26
Figure 4: Nucleotide Sequence Alignments for Mouse CES3 ‘Like’ Genes: CES3
(Es31) and CES3L (Es31L): Predicted Exons 2-4; and Predicted Introns 2 and
Exon sequences are in CAPITALS and intron sequence in lower case. Note the different
percentages of sequence identities for exon 2-4, (98%); introns 2-3 (92%). Nucleotide
substitutions are shaded. The deduced amino acid sequence for exons 2-4 of mouse CES3 is shown
as well as any deduced amino acid substitutions observed for CES3L.
Page 21 of 26
Figure 5: Sagittal section of mouse brain showing the distribution of mouse
CES3 and CES3L transcripts.
The two distinct regions of the mouse brain (C57BL6/6J strain) included the cerebellum (right
and the cerebrum (left). Note major staining of CES3 and CES3L transcripts in the cerebellum
folds and the hippocampus and amygdalar nuclei regions of the cerebrum. The sections were
obtained from the Allen Brain Atlas web site (http://www.brain-map.org) (Lein et al., 2007)
using the GenBank IDs BC061005 and BC019147, respectively.
Page 22 of 26
Figure 6: Phylogenetic Tree of Mammalian CES3 and Human CES1, CES2, CES5
and CES6 Amino Acid Sequences.
The tree is labeled with the gene name and the name of the mammal. Note the major cluster for
mammalian CES3 ‘like’ sequences and the separation of these sequences from human CES1, CES2,
CES5 and CES6 sequences. The gene duplication events generating the five distinct gene
families (CES1, CES2, CES3, CES5 and CES6) have been previously estimated to have occurred ~
328-378 million years ago (Holmes et al., 2008a).
Page 23 of 26
Mammal CES GenBank ID UNIPROT No of Chromosome Strand
⁴CES Gene order
Gene *RefSeq NCBI ID Amino Location Size Exons
Accession No. Acids (bps)
BC053670 Q6UWW8 571
*XP_511224 571 Positive 13,879 13 5.7 6
CES3L *XP_001173132 479 16: 5,892,348-5,905,878
Positive 13,531 11 6.6 5
CR857194 Q5RCL7 569 Positive
CES3-CES2-CES6 23,714 13 6.4 6
*XP_001496251 570 3: 17,417,834-17,428,268
Positive CES2-CES3-CES6 10,434 13 6.3 6
BC061004 Q63880 554
9,429 13 5.9 6
BC019147 568 Positive 9,476 13 5.7 6
*XP_001372405 571 Negative ^CES2-CES3-CES6 53,315 13 5.6 6
L07765 P23141 567
Negative CES4-CES1-CES5 30,311 14 6.2 6
BX538086 O00348 559 Positive
12 5.7 6
BC069501 Q6NT32 575 Negative
28,767 13 6 6
FLJ37464 Q6UX55 545 Positive
22,269 14 9.4 6
Table 1: Mammalian CES3 and Human CES1, CES2, CES5 and CES6 Genes and
*RefSeq NCBI Sequence; ^the mouse and opossum genomes contain multiple contiguous CES2
genes; ¹ extracellular and/or cell wall location; ² ER: endoplasmic reticulum location; ³ pI
refers to predicted isoelectric point. ⁴ CES gene in bold refers to the specific gene among
the contiguous CES genes.
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Table 2: Percentage identities for mammalian CES3 and human CES1, CES2,
CES5 and CES6 subunit amino acid sequences.
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Table 3: Predicted N-glycosylation sites for mammalian CES3 and human CES1,
CES2, CES5 and CES6 subunits.
The identified N-glycosylation site is for human CES1 (Kroetz et al., 1993). Amino acid
residues are shown for potential N-glycosylation sites: N-Asn; A-Ala; T-Thr; S-Ser; M-Met; L-
Leu; D-Asp; G-Gly; F-Phe; I-Ile.
Page 26 of 26 Download full-text
N-signal Predicted Tissue Distribution Predicted
Peptide Subcellular (mRNA Frequency) ID
Secreted or extracellular colon (8), breast, trachea (4) brain, placenta, rectum (2) yes NM024922
Secreted or extracellular colon (10) trachea (6) brain, breast (5) placenta (4) testis (3) yes BC053670
Secreted or extracellular colon (10) trachea (6) brain breast (5) placenta (4) testis (3) yes CR618303
no Cytosol ovary, teratocarcinoma, stomach, trachea (1) yes *DB232966
no Extracellular colon (5), nerve tumor, uterus, brain (2) yes AK025389
Extracellular bladder, uterus (1) yes DA040317
Extracellular trachea (1) yes DB215224
no Extracellular stomach (1) no AW813514
Table 4: Tissue distribution of human mRNA transcripts and predicted
encoded CES3 isoforms.
Predicted CES3 gene sizes (kilobases); CES type B domain refers to the presence of essential
amino acids at the CES active site; N-signal peptide location refers to the predicted site of
hydrolysis for the N-terminal peptide involved in intracellular CES3 processing. Note the
higher frequencies for CES3a, CES3b and CES3c expressed in colon, trachea and breast tissue.
* refers to a partial mRNA sequence.