Mammalian carboxylesterase 3: comparative genomics and proteomics

Abstract

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.

Fig. 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 (MTSmicrosomal (endoplasmic reticulum) targeting sequence) (in red bold); N-glycosylation residues at 79NAT (Human CES1) and potential N-glycosylation sites (in blue bold); active site (AS) triad residues Ser; Glu; and His (in shaded pink). ‘Side door’, ‘Gate’ residues and Cholesterol binding Gly residue (Z site) for human CES1 (in shaded dark green); Disulfide bond Cys residues for human CES1 (in shaded blue): S–S. Charge clamp residues identified for human CES1 (in shaded purple); Helix (Human CES1) or predicted helix (in shaded yellow); Sheet (Human CES1) or predicted sheet (shaded light grey);. Bold font shows known or predicted exon junctions. Exon numbers refer to human CES1 gene. Color designatations were not available for the printed version.

Fig. 2

Tertiary structure for human CES1 subunit and predicted tertiary structure for human CES 3 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) (Color figure online)

Fig. 3

Nucleotide sequence alignments for mouse CES3 ‘Like’ genes: CES3 (Es31) and CES3L (Es31L): predicted exons 2–4-; and predicted introns 2 and 3. 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

Fig. 4

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

Fig. 5

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

Fig. 6

Phylogenetic tree of mammalian CES3, human CES1, CES2, CES5 and CES6 and fruit fly (Drosophila melanogaster) CES6 amino acid sequences. The tree is labeled with the gene name and the name of the animal. Note the major cluster for mammalian CES3 ‘like’ sequences and the separation of these sequences from human CES1, CES2, CES5 and CES6 sequences, and from the Drosophila melanogaster CES6 (Est6) sequence, which served as the ‘root’ for the tree. Bootstrap values >995 (of 1,000 bootstrap analyses conducted) are shown. Bar, 2 changes per 100 amino acid residues. 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)