Molecular cloning and expression of human bile acid beta-glucosidase

A novel microsomal beta-glucosidase was recently purified and characterized from human liver that catalyzes the hydrolysis of bile acid 3-O-glucosides as endogenous compounds. The primary structure of this bile acid beta-glucosidase was deduced by cDNA cloning on the basis of the amino acid sequences of peptides obtained from the purified enzyme by proteinase digestion. The isolated cDNA comprises 3639 base pairs containing 524 nucleotides of 5'-untranslated and 334 nucleotides of 3'-untranslated sequences including the poly(A) tail. The open reading frame predicts a 927-amino acid protein with a calculated M(r) of 104,648 containing one putative transmembrane domain. Data base searches revealed no homology with any known glycosyl hydrolase or other functionally identified protein. The cDNA sequence was found with significant identity in the human chromosome 9 clone RP11-112J3 of the human genome project. The recombinant enzyme was expressed in a tagged form in COS-7 cells where it displayed bile acid beta-glucosidase activity. Northern blot analysis of various human tissues revealed high levels of expression of the bile acid beta-glucosidase mRNA (3.6-kilobase message) in brain, heart, skeletal muscle, kidney, and placenta and lower levels of expression in the liver and other organs.     

A coupled assay for bile acid:CoA ligase

A new assay for the enzyme bile acid:CoA ligase is presented. The new assay is designed to supplant the existing radiometric assays which require radiolabeled bile acids. The new assay couples the formation of bile acid-CoA to its glycination in a reaction catalyzed by bile acid-CoA:glycine N-acyltransferase. The coupling reaction utilizes [14C]glycine and the bile acid-CoA is quantitatively converted to [14C]glycobile acid. The [14C]glycobile acid is isolated by solvent extraction and quantitated by liquid scintillation counting. The method is shown to be accurate, highly sensitive, and applicable to a wide variety of bile acids.     

Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase

Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family.     

Molecular cloning of cDNA for rat liver general acyl CoA dehydrogenase and homology between the rat liver and pig kidney enzymes

cDNA clone for general acyl CoA dehydrogenase (GAD) was isolated from a rat liver cDNA expression library in lambda gt11 using anti-pig kidney GAD antibody. Size of the isolated cDNA was estimated to be 1.5-1.6 kb. By immunological analysis of fusion protein and epitope selection, the cDNA clone was identified as that containing the GAD gene. Partial amino acid sequence deduced from nucleotide sequence of the cDNA coincided with that of the pig kidney enzyme. The antibody cross-reacted with rat liver enzyme and molecular weights of these enzyme proteins were shown to be almost the same. All these results indicate that rat liver GAD shares a common structure with pig kidney enzyme.     

Rat liver bile acid CoA:amino acid N-acyltransferase: expression,characterization, and peroxisomal localization

Bile acid CoA:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids taurine and glycine. Rat liver BAT (rBAT) cDNA was isolated from a rat liver lambdaZAP cDNA library and expressed in Sf9 insect cells using a baculoviral vector. rBAT displayed 65% amino acid sequence homology with human BAT (hBAT) and 85% homology with mouse BAT (mBAT). Similar to hBAT, expressed rBAT was capable of forming both taurine and glycine conjugates with cholyl-CoA. mBAT, which is highly homologous to rBAT, forms only taurine conjugated bile acids (Falany, C. N., H. Fortinberry, E. H. Leiter, and S. Barnes. 1997. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase. J. Lipid Res. 38: 86-95). Immunoblot analysis of rat tissues detected rBAT only in rat liver cytosol following homogenization and ultracentrifugation. Subcellular localization of rBAT detected activity and immunoreactive protein in both cytosol and isolated peroxisomes. Rat bile acid CoA ligase (rBAL), the enzyme responsible for the formation of bile acid CoA esters, was detected only in rat liver microsomes. Treatment of rats with clofibrate, a known peroxisomal proliferator, significantly induced rBAT activity, message, and immunoreactive protein in rat liver. Peroxisomal membrane protein-70, a marker for peroxisomes, was also induced by clofibrate, whereas rBAL activity and protein amount were not affected. In summary, rBAT is capable of forming both taurine and glycine bile acid conjugates and the enzyme is localized primarily in peroxisomes in rat liver.     

Molecular cloning and expression of rat liver N-heparan sulfate sulfotransferase.

N-Heparan sulfate sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the nitrogen of glucosamine in heparan sulfate. The enzyme has been previously purified to apparent homogeneity from rat liver (Brandan, E., and Hirschberg, C. B. (1988) J. Biol. Chem. 263, 2417-2422). We have now cloned the rat liver enzyme using the following strategy: (a) the amino acid sequence was obtained from tryptic peptides of the purified protein, (b) mixed oligonucleotides were generated based on the sequence of the tryptic peptides, (c) a polymerase chain reaction fragment was obtained using mixed oligonucleotide interprimer amplification of cDNA, and (d) this fragment was used to screen rat liver lambda gt 10 and lambda ZAP libraries. Three clones were obtained, one of which seems to contain the complete coding sequence of the N-heparan sulfate sulfotransferase (N-HSST). Evidence that the cDNA clone corresponds to the previously purified and characterized N-HSST was the following: (a) the predicted sequence of the N-HSST contains all of the 11 tryptic peptides obtained from the purified protein, (b) when a cDNA containing the sequence coding for the N-HSST was introduced in a eukaryotic expression vector and transfected in COS-1 cells, the enzyme activity was expressed 9-fold over controls, and (c) the characteristic of the predicted protein fits with the purified protein in terms of molecular weight, membrane localization, and its being an N-linked glycoprotein. The size of the longest cDNA isolated is 4.1 kilobases, which is in close agreement with the 4.2-kilobase size of one of the mRNA observed in Northern analyses. In addition, messages of 7.0 and 8.5 kilobases were also observed, suggesting that a large portion is untranslated. The latter messages were the major mRNA species detected.     

Molecular cloning and functional expression of rat liver cytosolic acetyl-CoA hydrolase.

A cytosolic acetyl-CoA hydrolase (CACH) was purified from rat liver to homogeneity by a new method using Triton X-100 as a stabilizer. We digested the purified enzyme with an endopeptidase and determined the N-terminal amino-acid sequences of the two proteolytic fragments. From the sequence data, we designed probes for RT-PCR, and amplified CACH cDNA from rat liver mRNA. The CACH cDNA contains a 1668-bp ORF encoding a protein of 556 amino-acid residues (62 017 Da). Recombinant expression of the cDNA in insect cells resulted in overproduction of functional acetyl-CoA hydrolase with comparable acyl-CoA chain-length specificity and Michaelis constant for acetyl-CoA to those of the native CACH. Database searching shows no homology to other known proteins, but reveals high similarities to two mouse expressed sequence tags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.     

Molecular cloning and sequencing of human palmitoyl-CoA ligase and its tissue specific expression

A complimentary DNA clone encoding the entire human palmitoyl-CoA ligase has been isolated from a liver cDNA library and sequenced in it's entirety. The predicted product is a 699 amino acid protein. Southern analysis utilizing the human palmitoyl-CoA ligase gene as a probe revealed varying degrees of similarity amongst various mammalian species. The palmitoyl-CoA ligase gene is highly expressed in liver, heart, skeletal muscle and kidney, and to a lesser extent in brain, lung, placenta and pancreas. The expression of palmitoyl-CoA ligase in various tissue parallels the function of this enzyme in the metabolism of fatty acids in these tissues.     

Molecular cloning and expression of rat interferon-γ

The nueclotide sequence data reported in this paper have been submitted to the EMBL nucleotide databse and have been assigned the accession number X86972     

Molecular cloning and expression of rat betacellulin cDNA

The cDNA encoding an entire open reading frame of rat betacellulin has been cloned from rat kidney. Expression of this cDNA in COS7 cells showed a significant amount of mitogenic activity in the culture media. Western blotting of the cell lysates suggested that the membrane-anchored precursor was cleaved to release its ectodomain very efficiently.     

Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis

Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.     

Molecular cloning of cDNA for rat liver catalase

For the studies on the induction of peroxisomal enzymes by hypolipidemic agents, we have tried to isolate a cDNA clone for rat liver catalase. A recombinant clone, pMJ501, was isolated, of which cDNA insert specifically hybridized to catalase mRNA in hybridization-selected translation. On RNA blot hybridization, it hybridized to 2.4-kilobases RNA which was increased about 1.5-fold by the administration of di-(2-ethylhexyl)phthalate to the rats. The nucleotide sequence of the cDNA contains a reading frame for 109 amino acid residues which match the reported amino acid sequence of bovine liver catalase at the carboxyl end with 82% homology. It is concluded that pMJ501 contains a cDNA sequence for rat liver catalase.     

Molecular cloning and amino acid sequence of rat enkephalinase

cDNA clones encoding rat enkephalinase (neutral endopeptidase, EC 3.4.24.11) have been isolated in lambda gt10 libraries from both brain and kidney mRNAs and the complete 742 amino acid sequence of rat enkephalinase is presented. The enzyme possesses a single transmembrane spanning domain near the N-terminal of the molecule but lacks a signal sequence. Because enkephalinase has it active site located extracellularly and is thus an ectopeptidase, we suggest that the N-terminal transmembrane region of the enzyme anchors the protein in membranes and that the majority of the protein, including the carboxy terminus, is extracellular. Enkephalinase, a zinc-containing metallo enzyme, displays homology with other zinc metallo enzymes such as carboxypeptidase A, B and E, suggesting enzymatic similarities in these enzymes.     

Molecular weights of subunits of acetyl CoA carboxylase in rat liver cytoplasm

Monomeric [14C] methyl avidin was shown to bind to sodium dodecyl sulfate-denatured biotinyl proteins and remain bound through polyacrylamide gel electrophoresis which allowed their detection by fluorography. This method was used to show that purified rat liver acetyl CoA carboxylase contained two high molecular weight forms of the enzyme (MR = 241,000 and 252,000) while rapidly prepared, crude rat liver cytoplasm contained two larger molecular weight (MR = 257,000 and 270,000) forms. Thus, the enzyme had undergone substantial proteolysis during purification. The crude enzyme preparation also contained a smaller biotinyl protein (MR = 141,000) which is likely a proteolytic product of the larger forms of acetyl CoA carboxylase.     

Characterization of rat liver bile acid acyl glucuronosyltransferase

Recent studies have suggested that bile acid acyl glucuronides form covalently bound protein adducts which may cause hypersensitivity reactions and increased morbidity in patients. Although the preferential biosynthesis of the acyl glucuronides has been known, the characterization of hepatic bile acid acyl glucuronosyltransferase has not yet been clearly elucidated. We have investigated the substrate specificity of the hepatic bile acid acyl glucuronosyltransferase using five common bile acids as substrates. The glucuronidation rate was dependent on the number of the hydroxy group on the steroid nucleus and mono-hydroxylated lithocholic acid, the more lipophilic common bile acid, was most effectively metabolized into its acyl glucuronide. The tri-hydroxylated cholic acid, the more water-soluble common bile acid, barely transformed into its glucuronide. Results showed decreasing of the initial velocity of the acyl glucuronidation with increasing of the concentration of substrate, lithocholic acid, owing to the substrate inhibition of the hepatic bile acid acyl glucuronosyltransferase. The substrate analogues, glycine and taurine conjugated bile acids, which exist in the body fluids in high concentrations, also inhibited the enzyme's activity. In addition, enzymatic reaction products, bile acid acyl glucuronides, also inhibited the activity. These inhibitory mechanisms may be responsible for the low concentration of bile acid acyl glucuronides in urine and may be an important detoxification system in the body.     

Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.     

Molecular cloning and expression of rat interleukin-1 alpha cDNA

A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.