Gene_locus Report for: ratno-lmcxe

The involvement of carboxylesterase, acetylcholinesterase, butyrylcholinesterase and cholesterol esterase in pharmacology and toxicology are well recognized. However, there are few papers concerning the comparative studies of these serine hydrolases in terms of molecular level. Recently, we have studied various aspects of carboxylesterases using cDNAs of carboxylesterase isozymes purified from 9 animal species and human liver microsomes, and found that there is high homology of the N-terminal amino acid sequences of the isozymes tested. On the other hand, we compared the amino acid sequences at the active site of the individual esterases and found that the sequences of all esterases tested are strictly conserved. These results strongly suggest that the esterases involved are classified into the serine hydrolase super family.

Using polymerase chain reaction (PCR), we have isolated a cDNA that encodes a rat liver carboxylesterase. This novel enzyme, designated hydrolase C, is structurally very similar to hydrolase B, a microsomal carboxylesterase expressed in rat liver and kidney. Hydrolase B and C are 96% identical in nucleotide sequence and 93% identical in deduced amino acid sequence. Both enzymes have an 18-amino-acid signal peptide at the N-terminus. The C-terminus of hydrolase B and C contains an HXEL consensus sequence for retaining proteins in the endoplasmic reticulum. As expected, when the cDNA encoding hydrolase C was expressed in a baculovirus/Sf21 cell system, the recombinant enzyme was localized in the endoplasmic reticulum. Hydrolase B and C both have putative N-linked glycosylation sites at Asn1 and Asn61. The active site of hydrolase B and C appears to be composed of a nucleophile, Ser203, a basic residue, His448, and an acidic residue, either Asp97 or Glu228. Based on cloning experiments, restriction endonuclease mapping and Northern blotting, hydrolase B is expressed in both rat liver and kidney, whereas hydrolase C is expressed predominantly, perhaps exclusively, in liver. When expressed in Escherichia coli, hydrolase C was catalytically inactive and unstable, but when expressed in the baculovirus/Sf21 cell system hydrolase C it was stable and catalytically active toward 1-naphthylacetate and esters of para-nitrophenol. Hydrolase C is the fourth member of the rat carboxylesterase family to be cloned and sequenced. In terms of nucleotide and deduced amino acid sequence, hydrolase C is highly similar to hydrolase B, but differs from hydrolase B in terms of its catalytic activity and tissue distribution. Recombinant hydrolase C has properties similar to those described for esterase RL2, which was purified from rat liver microsomes by Hosokawa et al. (Arch. Biochem. Biophys. 277, 219-227, 1990), although additional studies will be required to establish conclusively the identity of this enzyme. The high degree of sequence identity (96%) between hydrolase B and C, particularly in the 3' untranslated region, suggests that the genes encoding these two carboxylesterases evolved by duplication and divergence of a common ancestral gene.

We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase B, that catalyzes the hydrolysis of para-nitrophenylacetate with low affinity (Km approximately 400 microM) and is relatively insensitive to the inhibitory effects of phenylmethylsulfonyl fluoride. A carboxylesterase with identical properties is also present in rat kidney microsomes, at levels comparable to those in liver microsomes. The kidney enzyme is immunochemically indistinguishable from hydrolase B by Western immunoblotting and Ouchterlony double diffusion analysis. This study describes the cloning and sequencing of hydrolase B. A 1809-base pair (bp) cDNA was isolated from a rat kidney cDNA library screened with antibody against hydrolase B. Screening the same cDNA library by two-step polymerase chain reaction with external and internal primers based on the sequence of the 1809-bp cDNA and a primer based on the sequence of the adjoining lambda gt11 arm yielded a 279-bp cDNA that overlapped by 179 bp with the 1809-bp-sequence. Together these two cDNAs spanned a 1909-bp sequence with an opening reading frame encoding 561 amino acids, which includes all 543 amino acid residues in the mature protein plus an 18-amino acid signal peptide at the N terminus. The mature protein encoded by this kidney cDNA matches perfectly the N-terminal amino acid sequence of purified hydrolase B for 30 amino acid residues, as determined by automated Edman degradation. The mature protein contains 5 cysteine residues, two potential N-linked glycosylation sites, and a C-terminal tetrapeptide (His-Asn-Glu-Leu) that matches the HXEL consensus sequence for retaining proteins in the lumen of the endoplasmic reticulum. Based on alignment of conserved amino acid sequences in several mammalian carboxylesterases, and based on the mechanism of catalysis of serine proteases, the catalytic triad in hydrolase B is apparently composed of the nucleophile Ser203, the basic amino acid His448, and the acidic amino acid Asp97 or Glu228. Northern blots probed with the 1809-bp cDNA identified high levels of a approximately 2-kilobase mRNA for hydrolase B in liver and kidney. Little or no mRNA for hydrolase B was detected in testis, lung, prostate, brain, and heart, which confirms the tissue distribution of hydrolase B based on catalytic activity and Western immunoblotting. Immunocytochemical studies established that hydrolase B is localized in the centrilobular region of the liver and in the proximal tubules of the kidney, where it presumably plays a role in the metabolism of xenobiotics and possibly endogenous lipids, although a precise physiological role for hydrolase B remains to be determined.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

The involvement of carboxylesterase, acetylcholinesterase, butyrylcholinesterase and cholesterol esterase in pharmacology and toxicology are well recognized. However, there are few papers concerning the comparative studies of these serine hydrolases in terms of molecular level. Recently, we have studied various aspects of carboxylesterases using cDNAs of carboxylesterase isozymes purified from 9 animal species and human liver microsomes, and found that there is high homology of the N-terminal amino acid sequences of the isozymes tested. On the other hand, we compared the amino acid sequences at the active site of the individual esterases and found that the sequences of all esterases tested are strictly conserved. These results strongly suggest that the esterases involved are classified into the serine hydrolase super family.

Using polymerase chain reaction (PCR), we have isolated a cDNA that encodes a rat liver carboxylesterase. This novel enzyme, designated hydrolase C, is structurally very similar to hydrolase B, a microsomal carboxylesterase expressed in rat liver and kidney. Hydrolase B and C are 96% identical in nucleotide sequence and 93% identical in deduced amino acid sequence. Both enzymes have an 18-amino-acid signal peptide at the N-terminus. The C-terminus of hydrolase B and C contains an HXEL consensus sequence for retaining proteins in the endoplasmic reticulum. As expected, when the cDNA encoding hydrolase C was expressed in a baculovirus/Sf21 cell system, the recombinant enzyme was localized in the endoplasmic reticulum. Hydrolase B and C both have putative N-linked glycosylation sites at Asn1 and Asn61. The active site of hydrolase B and C appears to be composed of a nucleophile, Ser203, a basic residue, His448, and an acidic residue, either Asp97 or Glu228. Based on cloning experiments, restriction endonuclease mapping and Northern blotting, hydrolase B is expressed in both rat liver and kidney, whereas hydrolase C is expressed predominantly, perhaps exclusively, in liver. When expressed in Escherichia coli, hydrolase C was catalytically inactive and unstable, but when expressed in the baculovirus/Sf21 cell system hydrolase C it was stable and catalytically active toward 1-naphthylacetate and esters of para-nitrophenol. Hydrolase C is the fourth member of the rat carboxylesterase family to be cloned and sequenced. In terms of nucleotide and deduced amino acid sequence, hydrolase C is highly similar to hydrolase B, but differs from hydrolase B in terms of its catalytic activity and tissue distribution. Recombinant hydrolase C has properties similar to those described for esterase RL2, which was purified from rat liver microsomes by Hosokawa et al. (Arch. Biochem. Biophys. 277, 219-227, 1990), although additional studies will be required to establish conclusively the identity of this enzyme. The high degree of sequence identity (96%) between hydrolase B and C, particularly in the 3' untranslated region, suggests that the genes encoding these two carboxylesterases evolved by duplication and divergence of a common ancestral gene.

We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase B, that catalyzes the hydrolysis of para-nitrophenylacetate with low affinity (Km approximately 400 microM) and is relatively insensitive to the inhibitory effects of phenylmethylsulfonyl fluoride. A carboxylesterase with identical properties is also present in rat kidney microsomes, at levels comparable to those in liver microsomes. The kidney enzyme is immunochemically indistinguishable from hydrolase B by Western immunoblotting and Ouchterlony double diffusion analysis. This study describes the cloning and sequencing of hydrolase B. A 1809-base pair (bp) cDNA was isolated from a rat kidney cDNA library screened with antibody against hydrolase B. Screening the same cDNA library by two-step polymerase chain reaction with external and internal primers based on the sequence of the 1809-bp cDNA and a primer based on the sequence of the adjoining lambda gt11 arm yielded a 279-bp cDNA that overlapped by 179 bp with the 1809-bp-sequence. Together these two cDNAs spanned a 1909-bp sequence with an opening reading frame encoding 561 amino acids, which includes all 543 amino acid residues in the mature protein plus an 18-amino acid signal peptide at the N terminus. The mature protein encoded by this kidney cDNA matches perfectly the N-terminal amino acid sequence of purified hydrolase B for 30 amino acid residues, as determined by automated Edman degradation. The mature protein contains 5 cysteine residues, two potential N-linked glycosylation sites, and a C-terminal tetrapeptide (His-Asn-Glu-Leu) that matches the HXEL consensus sequence for retaining proteins in the lumen of the endoplasmic reticulum. Based on alignment of conserved amino acid sequences in several mammalian carboxylesterases, and based on the mechanism of catalysis of serine proteases, the catalytic triad in hydrolase B is apparently composed of the nucleophile Ser203, the basic amino acid His448, and the acidic amino acid Asp97 or Glu228. Northern blots probed with the 1809-bp cDNA identified high levels of a approximately 2-kilobase mRNA for hydrolase B in liver and kidney. Little or no mRNA for hydrolase B was detected in testis, lung, prostate, brain, and heart, which confirms the tissue distribution of hydrolase B based on catalytic activity and Western immunoblotting. Immunocytochemical studies established that hydrolase B is localized in the centrilobular region of the liver and in the proximal tubules of the kidney, where it presumably plays a role in the metabolism of xenobiotics and possibly endogenous lipids, although a precise physiological role for hydrolase B remains to be determined.