Characterization of antibodies to a rabbit hepatic UDP-glucuronosyltransferase and the identification of an immunologically similar enzyme in human liver

An antibody to a UDP-glucuronosyltransferase (UDPGT) isoenzyme which catalyzes the glucuronidation of p-nitrophenol (PNP) in rabbit liver was raised in sheep and used to identify immunologically similar UDPGTs in rabbit and human livers. Immunoblotting experiments showed that the antisera specifically recognized PNP UDPGT but not estrone UDPGT purified from rabbit liver. Sheep anti-rabbit liver PNP UDPGT IgG immunoprecipitated PNP, 1-naphthol, and 4-methylumbelliferone glucuronidation activities in rabbit and human liver microsomal preparations. In rabbit liver microsomes the antibody did not immunoprecipitate estrone or estradiol glucuronidation activities. In human liver microsomes, 4-aminobiphenyl but not estriol glucuronidation activities were immunoprecipitated, suggesting that the antibody recognizes a specific UDPGT (pI 6.2) in human liver microsomes.     

The full length coding sequence of rat liver androsterone UDP-glucuronyltransferase cDNA and comparison with other members of this gene family.

Cloned cDNAs coding for hepatic UDP-glucuronyltransferase (UDPGT) have been isolated from a rat liver cDNA library in the expression vector bacteriophage lambda gt11 using anti-UDPGT antibodies. Four different mRNAs have been identified by sequencing of 15 UDPGT cDNA clones. The sequences of the four classes of cDNA were determined to be 85-95% homologous. Restriction fragments were isolated from the cDNA in each class and used as class specific probes. Hybridisation of these probes to northern blots of total RNA prepared from the livers of normal and genetically deficient Wistar rats identified the cDNA in class 4 with androsterone UDPGT. Translation of the cDNA sequence of clone rlug 23, the longest member of class 4, allowed determination of the complete amino acid sequence of androsterone UDPGT.     

Cloning and expression of a cDNA coding for a rat liver plasma membrane ecto-ATPase. The primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I

The amino acid sequence of the ecto-ATPase from rat liver was deduced from analysis of cDNA clones and a genomic clone. Immunoblots with antibodies raised against a peptide sequence deduced from the cDNA sequence indicated that the determined amino acid sequence is that of the ecto-ATPase. The deduced sequence predicts a 519-amino acid protein with a calculated molecular mass of 57,388 daltons. There are 16 potential asparagine-linked glycosylation sites in the protein. Hydropathy analysis of the deduced amino acid sequence indicates that the protein has two hydrophobic stretches. One is located at the N-terminal and the other is near the C-terminal end. A full-length clone encoding the ecto-ATPase was expressed transiently in mouse L cells and human HeLa cells. The cell lysate from the transfected cells contained immunoreactive ecto-ATPase and Ca2+-stimulated ATPase activities. The expressed protein is glycosylated and has an apparent molecular weight (100,000) similar to that of the rat liver plasma membrane ecto-ATPase.     

Rat liver carboxylesterase: cDNA cloning, sequencing, and evidence for a multigene family

A cDNA clone was isolated from a rat liver lambda gt11 expression library by screening with polyclonal antibodies raised against a rat liver microsomal carboxylesterase. This clone of 1.8 kb contained an open reading frame encoding a mature protein of 531 amino acids with a predicted molecular weight of 58,084. The 5' portion of the clone coded for 9 amino acids of a putative signal peptide. The 3' end of the clone included an untranslated region and a poly (A) tail. Carboxylesterase active site regions, five potential N-linked glycosylation sites, and 2 postulated cystine disulfide bridges were found in the cDNA-deduced amino acid sequence. Sequences obtained from tryptic peptides and the NH2-terminus of the purified native carboxylesterase were aligned with the deduced amino acid sequence, and the overall identity was 84%. Southern blot analysis suggested the presence of multiple genes. Thus it is concluded that we have cloned a rat liver carboxylesterase, and that this enzyme is a member of a multigene family.     

Isolation and sequence analysis of a cDNA encoding rat liver alpha-L-fucosidase.

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].     

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.     

Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA.

A cDNA clone (HLUG 25) encoding the complete sequence of a human liver UDP-glucuronosyltransferase was isolated from a lambda gt11 human liver cDNA library. The library was screened by hybridization to a partial-length human UDP-glucuronosyltransferase cDNA (pHUDPGT1) identified from a human liver pEX cDNA expression library by using anti-UDP-glucuronosyltransferase antibodies. The authenticity of the cDNA clone was confirmed by hybrid-select translation and extensive sequence homology to rat liver UDP-glucuronosyltransferase cDNAs. The sequence of HLUG 25 cDNA was determined to be 2104 base-pairs long, including a poly(A) tail, and contains a long open reading frame. The possible site of translation initiation of this sequence is discussed with reference to a rat UDP-glucuronosyltransferase cDNA clone (RLUG 38).     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Expression of a human liver cDNA encoding a UDP-glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture

A cDNA encoding a human liver UDPGT (HLUG 25) transcribed and translated in vitro showed that the encoded protein was synthesized as a precursor and was cleaved and glycosylated when dog pancreatic microsomes were present during translation. The UDPGT cDNA was transiently expressed in mammalian cell culture (COS-7 cells) resulting in the biosynthesis of a polypeptide of 52 kDa. This expressed UDPGT glycoprotein catalysed the glucuronidation of hyodeoxycholic acid forming an ether glucuronide. These results suggest that this UDPGT isoenzyme may be responsible for the glucuronidation of 6 alpha-hydroxy bile acids in human liver.     

Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-gamma-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis

L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.     

Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor

A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.     

The structure of phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4. Production and characterization of site-specific antibodies

Fifteen peptides corresponding in sequence to segments of the major phenobarbital-inducible forms of rat hepatic cytochrome P-450 (termed P-450 PB-4 and P-450 PB-5) were chemically synthesized, conjugated to carrier proteins, and used to prepare site-specific rabbit and/or mouse antipeptide antibodies. Four of the synthetic peptides were recognized by rabbit heterosera raised against purified P-450 PB-4. The titer of these heterosera measured against P-450 PB-4 was only partially reduced upon complete adsorption of antipeptide activity suggesting that these peptides represent minor antigenic determinants. Each of the antipeptide antibodies recognized purified P-450 PB-4 and the highly homologous P-450 PB-5 as demonstrated by a solid-phase enzyme-linked immunosorbent assay. Although each antipeptide immunoprecipitated both purified 125I-labeled P-450 PB-4 and also in vitro-synthesized apo-P-450 PB-4, the yields of immunoprecipitation were low relative to that obtained using anti-P-450 heterosera. Only one of the antipeptide antibodies gave a good signal in an immunoblot analysis of either microsomal or purified P-450s PB-4 and PB-5. Three antipeptide antibodies raised against hydrophilic segments located in the amino-terminal one-third of P-450 PB-4 markedly inhibited the P-450 PB-4-catalyzed O-deethylation of the model substrate 7-ethoxycoumarin. Four of the antipeptide antibodies were found to cross-react with P-450 beta NF-B, the major aromatic hydrocarbon-inducible rat hepatic P-450, suggesting that certain amino acid sequences or regions of secondary structure are conserved between the major phenobarbital-induced and polycyclic-induced rat liver P-450 isoenzymes. These studies demonstrate the utility of antipeptide antibodies for evaluation of antigenic sites exposed in native P-450 PB-4, for identification of specific amino acid sequences important for the interaction of P-450 PB-4 with its substrate and/or with cytochrome P-450 reductase in a reconstituted system and for elucidation of structural and immunochemical homologies between P-450 PB-4 and other P-450 isoenzymes present in rat liver endoplasmic reticulum.     

Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme.

6-Pyruvoyl-tetrahydropterin synthase (PTPS) is involved in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes such as the hepatic phenylalanine hydroxylase. BH4 deficiency causes malignant hyperphenylalaninemia. We cloned the human liver cDNA encoding PTPS. The coding region for PTPS contains 145 amino acids and predicts a polypeptide of 16'387 Da. The human amino acid sequence showed a 82% identity with the rat liver sequence. Expression of the cDNA in E. coli yielded the active enzyme and showed immunoreactivity with antibodies against the rat liver PTPS. This is the basis for the molecular understanding of BH4 deficiency in patients suffering from a defect in PTPS activity.     

Cloning of the human glucocorticoid receptor cDNA.

We show that the human glucocorticoid receptor (GR), isolated from the breast cancer cell line MCF-7, has an apparent molecular weight identical to that of rat liver GR (94 kDa) and reacts with antibodies raised against the latter. These antibodies were used to clone cDNA sequences corresponding to the human GR from a lambda gt11 expression library constructed using MCF-7 poly(A)+ RNA. Three non-homologous cDNA clones with inserts of 125, 220 and 350 bp, which express epitopes recognised by the rat liver GR antibodies, were isolated. Rat liver GR antibodies, immunopurified using the immobilised purified beta-galactosidase fusion proteins, detect partially purified rat liver and human GRs on Western blots. In addition, these antibodies immuno-adsorb rat liver and human GRs affinity-labelled with [3H] triamcinolone acetonide. Northern blot analysis, using all three cDNA probes, reveals the presence of a major MCF-7 poly(A)+ RNA species of approximately 7 kb.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.     

cDNA encoding type B subunit of rat phosphoglycerate mutase: its isolation and nucleotide sequence.

A cDNA encoding the nonmuscle-specific (type B) subunit of phosphoglycerate mutase (PGAM-B) was isolated and characterized. A cDNA probe, synthesized by the polymerase chain reaction (PCR) from rat liver cell mRNA using mixed primers specific to the amino acid sequence of human PGAM-B, was used to screen a rat liver cell cDNA library. The identity of the cDNA was confirmed by amino acid sequence data for 24 peptides obtained by digesting the purified protein with three different endopeptidases. The coding region encoded a polypeptide composed of 253 amino acid (plus the initiator Met). RNA blot analysis showed a single mRNA species of 1.7 kilobases in rat liver cell. The deduced amino acid sequence of rat PGAM-B was identical to that of human PGAM-B except for only one substitution at position 251 near the carboxyl terminus (valine for the rat and alanine for the human).