Opossum carboxylesterases: sequences,phylogeny and evidence for <Emphasis Type="Italic">CES</Emphasis> gene duplication events predating the marsupial-eutherian common ancestor

Background  

Carboxylesterases (CES) perform diverse metabolic roles in mammalian organisms in the detoxification of a broad range of drugs and xenobiotics and may also serve in specific roles in lipid, cholesterol, pheromone and lung surfactant metabolism. Five CES families have been reported in mammals with human CES1 and CES2 the most extensively studied. Here we describe the genetics, expression and phylogeny of CES isozymes in the opossum and report on the sequences and locations of CES1, CES2 and CES6 'like' genes within two gene clusters on chromosome one. We also discuss the likely sequence of gene duplication events generating multiple CES genes during vertebrate evolution.     

Horse carboxylesterases: Evidence for six CES1 and four families of CES genes on chromosome 3

Carboxylesterases (CES) are responsible for the detoxification of a wide range of drugs and xenobiotics, and may contribute to cholesterol, fatty acid and lung surfactant metabolism. In this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures, and gene locations for horse CES genes and encoded proteins, using data from the recently completed horse genome project. Evidence was obtained for six CES1 genes closely localised on horse chromosome 3, for which the predicted CES1 gene products are ≥ 74% identical. The horse genome also showed evidence for three other CES gene classes: CES5, located in tandem with the CES1 gene cluster; and CES2 and CES3, located more than 9 million base pairs downstream on chromosome 3. Horse CES2, CES3 and CES5 gene products shared 42–46% identity with each other, and with the CES1 protein subunits. Sequence alignments of these enzymes demonstrated key enzyme and family specific CES protein sequences reported for human CES1, CES2, CES3 and CES5. In addition, predicted secondary and tertiary structures for horse CES1, CES2, CES3 and CES5 subunits showed extensive conservation with human CES1. Phylogenetic analyses demonstrated the relationships and potential evolutionary origins of the horse CES sequences with previously reported sequences for human and other mammalian CES gene products. Several CES1 gene duplication events have apparently occurred following the appearance of the ‘dawn’ horse ~ 55 million years ago.     

Bovine carboxylesterases: Evidence for two CES1 and five families of CES genes on chromosome 18

Predicted bovine carboxylesterase (CES) protein and gene sequences were derived from bovine (Bos taurus) genomic sequence data. Two bovine CES1 genes (CES1.1 and CES1.2) were located on chromosome 18 encoding amino acid sequences that were 81% identical. Two forms of CES1.2 were also observed apparently caused by an indel polymorphism encoded at the C-terminus end. Two CES gene clusters were observed on chromosome 18: CES5–CES1.1–CES1.2 and CES2–CES3–CES6. Bovine CES1, CES2, CES3, CES5 and CES6 shared 39–45% identity with each other, but showed 71–76% identity with each of the five corresponding human CES family members. Phylogeny studies indicated that bovine CES genes originated from five ancestral gene duplication events which predated the eutherian mammalian common ancestor. In addition, a subsequent CES1 gene duplication event is proposed during mammalian evolution prior to the appearance of the Bovidae common ancestor ~ 20 MY ago.     

Biochemical Genetics of Opossum Aldehyde Dehydrogenase 3: Evidence for Three ALDH3A-Like Genes and an ALDH3B-Like Gene

Mammalian ALDH3 isozymes participate in peroxidic and fatty aldehyde metabolism, and in anterior eye tissue UV-filtration. BLAT analyses were undertaken of the opossum genome using rat ALDH3A1, ALDH3A2, ALDH3B1, and ALDH3B2 amino acid sequences. Two predicted opossum ALDH3A1-like genes and an ALDH3A2-like gene were observed on chromosome 2, as well as an ALDH3B-like gene, which showed similar intron–exon boundaries with other mammalian ALDH3-like genes. Opossum ALDH3 subunit sequences and structures were highly conserved, including residues previously shown to be involved in catalysis and coenzyme binding for rat ALDH3A1. Eleven glycine residues were conserved for all of the opossum ALDH3-like sequences examined, including two glycine residues previously located within the stem of the rat ALDH3A1 active site funnel. Phylogeny studies of human, rat, opossum, and chicken ALDH3-like sequences indicated that the common ancestor for ALDH3A- and ALDH3B-like genes predates the appearance of birds during vertebrate evolution.     

Assignment of the βB1 Crystallin Gene (CRYBB1) to Human Chromosome 22 and Mouse Chromosome 5

By using primers complementary to the rat βB1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences.CRYBB1was assigned to the group 5 region in 22q11.2–q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products ofCRYBB1were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other β crystallin genes (βB2(−1), βB3, and βA4) have previously been mapped to the same regions in human and mouse. We demonstrate that the βB1 and βA4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major β crystallins genes that are expressed in the bovine lens.     

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids

Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC₅₀ = 33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC₅₀ = 8.1 μM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (K_iapp = 10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (K_iapp = 1.7 μM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo.     

The Gene for Death Agonist BID Maps to the Region of Human 22q11.2 Duplicated in Cat Eye Syndrome Chromosomes and to Mouse Chromosome 6

Cat eye syndrome (CES) is associated with a duplication of a segment of human chromosome 22q11.2. Only one gene,ATP6E, has been previously mapped to this duplicated region. We now report the mapping of the human homologue of the apoptotic agonistBidto human chromosome 22 near locus D22S57 in the CES region. Dosage analysis demonstrated thatBIDis located just distal to the CES region critical for the majority of malformations associated with the syndrome (CESCR), as previously defined by a single patient with an unusual supernumerary chromosome. However,BIDremains a good candidate for involvement in CES-related mental impairment, and its overexpression may subtly add to the phenotype of CES patients. Our mapping of murineBidconfirms that the synteny of the CESCR and the 22q11 deletion syndrome critical region immediately telomeric on human chromosome 22 is not conserved in mice.Bidand adjacent geneAtp6ewere found to map to mousechromosome 6, while the region homologous to the DGSCR is known to map to mouse chromosome 16.     

The human LFA-3 gene is located at the same chromosome band as the gene for its receptor CD2

Lymphocyte function-associated antigen 3 (LFA-3) is a widely distributed cell surface glycoprotein that has been assigned a role in cell-cell adhesion on the basis of its capacity to bind to the T-lymphocyte CD2 antigen. The amino acid sequences of the extracellular domains of these two antigens, predicted from their cDNA sequences, show significant similarities, and both are members of the immunoglobulin supergene family. In this communication, a probe prepared from LFA-3 cDNA has been used in Southern blot analyses of somatic cell hybrids and in in situ hybridization to assign the LFA-3 gene to the human chromosome band 1p13. This is the same location previously assigned to CD2. Thus the LFA-3 and CD2 genes have probably arisen by duplication of a common evolutionary precursor. These genes therefore represent a further instance in which related members of the immunoglobulin superfamily are located in adjacent regions of the genome.     

Mammalian carboxylesterase (CES) releases GPI-anchored proteins from the cell surface upon lipid raft fluidization

Mammalian carboxylesterase (CES) is well known as a biotransformation enzyme for prodrugs and xenobiotics. Here, we purified CES as a GPI-anchored protein (GPI-AP)-releasing factor (GPIase) that releases such protein from the cell surface. All five isoforms of CES showed this activity to various degrees. When the serine residue of the catalytic triad for esterase was replaced by alanine, esterase activity was completely disrupted, while full GPIase activity remained, suggesting that these two activities are exhibited via different mechanisms. CES6, a new class of mammalian CES, exhibited the highest GPIase activity and released specific GPI-APs from the cell surface after lipid raft fluidization. The released product contained a GPI component, indicating that GPI-AP was released by cleavage in GPI. These results revealed for the first time that CES recognizes and catalyzes macromolecule GPI-AP as well as small molecules.     

Sequences variation and classification of B-hordein genes in hull-less barley from the Qinghai-Tibet Plateau

The goal of this study is to understand the evolution relationship of the members of the B-hordein gene family in hull-less barley by analysis of their structure and to explore their utility in grain quality improvement. Six copies of the B-hordein gene (Hn1-Hn3, Hn7-Hn9) were cloned from six hull-less barley cultivars collected from Qinghai-Tibet Plateau and molecularly characterized. Comparison of their predicted polypeptide sequences with the published data suggested that they all share the same basic protein structures. In addition, we found that the C-terminal end sequences of all B-hordeins shared a similar feature. In the six clones and the other three published genes (Hn4, Hn5, and Hn6) from hull-less barley, Hn2 and Hn7 contained the identical C-terminal end sequence DIMPVDFWH. Hn3, Hn4, Hn5, Hn8 and Hn9 also shared the common sequence DIMPPDFWH, which was similar to that of a B-hordein reported previously. Both Hn1 and Hn6 exhibited differences in their C-terminal end sequences, and they clustered into different subgroups. The B-hordeins with identical C-terminal end sequences were clustered into the same subgroup, so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide. Phylogenetic analysis also indicated that there is a relatively weak identity between our predicted B-hordeins and those reported from H. chilense and H. brevisubulatum. All of our nine predicted B-hordeins were clustered together and other B-hordeins formed another cluster. The possible use of these genes in relation to barley quality is discussed. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 1, pp. 63–70. The text was submitted by the authors in English     

Identification of regulatory variants of carboxylesterase 1 (CES1): A proof-of-concept study for the application of the Allele-Specific Protein Expression (ASPE) assay in identifying cis-acting regulatory genetic polymorphisms

It is challenging to study regulatory genetic variants as gene expression is affected by both genetic polymorphisms and non-genetic regulators. The mRNA allele-specific expression (ASE) assay has been increasingly used for the study of cis-acting regulatory variants because cis-acting variants affect gene expression in an allele-specific manner. However, poor correlations between mRNA and protein expressions were observed for many genes, highlighting the importance of studying gene expression regulation at the protein level. In the present study, we conducted a proof-of-concept study to utilize a recently developed allele-specific protein expression (ASPE) assay to identify the cis-acting regulatory variants of CES1 using a large set of human liver samples. The CES1 gene encodes for carboxylesterase 1 (CES1), the most abundant hepatic hydrolase in humans. Two cis-acting regulatory variants were found to be significantly associated with CES1 ASPE, CES1 protein expression, and its catalytic activity on enalapril hydrolysis in human livers. Compared to conventional gene expression-based approaches, ASPE demonstrated an improved statistical power to detect regulatory variants with small effect sizes since allelic protein expression ratios are less prone to the influence of non-genetic regulators (e.g., diseases and inducers). This study suggests that the ASPE approach is a powerful tool for identifying cis-regulatory variants.     

Three novel partial deletions of the low-density lipoprotein (LDL) receptor gene in familial hypercholesterolemia

Summary The low-density lipoprotein (LDL) receptor genes from 18 unrelated Japanese heterozygotes and 1 homozygote with classical familial hypercholesterolemia were analyzed by Southern blot hybridization using fragments of the human LDL receptor cDNA as probes. Four different deletion mutations were detected among 20 mutant LDL receptor genes (20%); they were characterized by restriction mapping. None of these mutations has previously been reported in Caucasian patients with FH: three of the mutations were novel and one was similar to the detetion mutation of FH-Tonami described previously in Japanese patients. In three of the four deletion mutations, the rearrangements were related to intron 15 of the LDL receptor gene, in which many Alu sequences exist. The data suggest that a wide range of molecular heterogeneity exists even in major rearrangements resulting in deletions in the LDL receptor gene. The data also support the hypothesis that there are preferential sites within the LDL receptor gene for major rearrangements resulting in deletions. The possibility that a higher frequency of deletion mutations occurs in classical FH than previously suspected is discussed.     

Comparative analysis of the three major histocompatibility complex-linked heat shock protein 70 (Hsp70) genes of the rat

The organization of the three major histocompatibility complex (Mhc)-linked heat shock protein 70 (Hsp70) genesHsp70-1, Hsp70-2, andHsp70-3, and the nucleotide sequences of these genes, are presented for the rat.Hsp70-1 andHsp70-2 gene products are identical at the amino acid level. From the pattern of sequence similarity of the orthologous Mhc-linkedHsp70 genes of rat, human, and mouse, it is concluded that the gene duplications leading to the three-gene cluster occurred before the separation of the primate and rodent lines and that theHsp70-1 andHsp70-2 genes of rat and human might have undergone homogenization of their sequences.     

Vertebrate endothelial lipase: comparative studies of an ancient gene and protein in vertebrate evolution

Endothelial lipase (gene: LIPG; enzyme: EL) is one of three members of the triglyceride lipase family that contributes to lipoprotein degradation within the circulation system and plays a major role in HDL metabolism in the body. In this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures, and gene locations for LIPG genes and encoded proteins using data from several vertebrate genome projects. LIPG is located on human chromosome 18 and is distinct from other human ‘neutral lipase’ genes, hepatic lipase (gene: LIPC; enzyme: HL) and lipoprotein lipase (gene: LPL; enzyme: LPL) examined. Vertebrate LIPG genes usually contained 10 coding exons located on the positive strand for most primates, as well as for horse, bovine, opossum, platypus and frog genomes. The rat LIPG gene however contained only 9 coding exons apparently due to the presence of a ‘stop’ codon’ within exon 9. Vertebrate EL protein subunits shared 58–97% sequence identity as compared with 38–45% sequence identities with human HL and LPL. Four previously reported human EL N-glycosylation sites were predominantly conserved among the 10 potential N-glycosylation sites observed for the vertebrate EL sequences examined. Sequence alignments and identities for key EL amino acid residues were observed as well as conservation of predicted secondary and tertiary structures with those previously reported for horse pancreatic lipase (PL) (Bourne et al. 1994). Several potential sites for regulating LIPG gene expression were observed including CpG islands near the LIPG gene promoter and a predicted microRNA binding site near the 3’-untranslated region. Promoter regions containing functional polymorphisms that regulate HDL cholesterol in baboons were conserved among primates but not retained between primates and rodents. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate LIPG gene subfamily with other neutral triglyceride lipase gene families, LIPC and LPL. It is apparent that the triglyceride lipase ancestral gene for the vertebrate LIPG gene predated the appearance of fish during vertebrate evolution >500 million years ago.     

Cloning and characterization of a third type of human α-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human α-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229–235; Tomita et al., Gene 76 (1989) 11–18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human α-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively.Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197–1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5′ region; hence the promoter lies far upstream relative to the other two AMY genes.