Cloning, sequencing, and expression of cDNA for human beta-galactosidase

We cloned and sequenced the full-length cDNA for human placental beta-galactosidase. The 2379-nucleotide sequence contains 2031 nucleotides which encode a protein of 677 amino acids. The amino acid sequence includes a putative signal sequence of 23 amino acids and 7 potential asparagine-linked glycosylation sites. The cDNA in the expression vector pSVL was used to transfect COS cells. Expression of the cDNA in transfected COS cells produced immunoprecipitable proteins and led to an increase in beta-galactosidase activity.     

Rat glutathione S-transferase. Cloning of double-stranded cDNA and induction of its mRNA

Messenger RNA extracted from the livers of normal, phenobarbital-treated, and trans-stilbene oxide-treated rats was translated in a mRNA-dependent protein-synthesizing system. Immunoprecipitation of the translation products by antibodies against the Ya and Yc subunits of glutathione S-transferase detected two polypeptides of molecular weights 23,500 and 25,000. Subsequently, a clone containing glutathione S-transferase sequences was identified from a rat liver double-stranded cDNA library that had been prepared by homopolymeric tailing and cloning into the Pst I site of pBR322. Confirmation of the identity of the clone was obtained by recloning the 550-bp insert DNA into the phage vector M13 and utilizing the single strand recombinant phage DNA in specific hybrid selection of mRNA followed by translation and immunoprecipitation with antibodies to the Ya and Yc subunits. This recombinant phage, M13GST94, was also utilized in a new technique to synthesize 32P-labeled cDNA specific to the glutathione S-transferase insert DNA that was used subsequently in RNA excess solution hybridization to determine the relative concentration of glutathione S-transferase mRNA. Phenobarbital treatment resulted in a 3.2-fold increase in glutathione S-transferase mRNA over levels found in control rats, while trans-stilbene oxide increased glutathione S-transferase mRNA levels 5.7-fold. The DNA sequence of the clone was determined and utilized to propose a partial amino acid sequence.     

Purification and characterization of acidic form of glutathione S-transferase in human fetal livers: high similarity to placental form.

An acidic form of glutathione S-transferase (GST) was purified from human fetal livers by means of affinity chromatography and chromatofocusing. The major peak of the acidic form of GST was focused between pH 4.8 and 4.9. Judging by SDS-PAGE, the purified acidic GST was apparently homogeneous; the subunit molecular weight was estimated to be 23,000. The acidic GST catalyzed the conjugations of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA). The immunochemical properties of the purified acidic GST were indistinguishable from those of human placental GST-pi. The N-terminal amino acid sequence of the acidic GST was identical with that of GST-pi from human placenta. The level of expression of the acidic form of GST was clearly different between human adult and fetal livers as examined on the levels of mRNA and protein.     

Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae).

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.     

Genomic and cDNA cloning, characterization of Delonix regia trypsin inhibitor (DrTI) gene, and expression of DrTI in Escherichia coli

Degenerate primers were designed based on all possible sequences of the N-terminal and C-terminal regions of Delonix regia trypsin inhibitor (DrTI). Five hundred sixty-one bp of polymerase chain reaction (PCR) product was amplified using the above degenerate primers and genomic DNA and cDNA of Delonix regia as a template. The amplified PCR products were cloned and sequenced. DNA sequence analysis of cDNA and genomic clones of DrTI have the same nucleotide sequence in the coding region, and manifested a genomic clone without intervening sequences in the coding region. The amino acid sequence deduced from the DrTI genomic and cDNA clones agreed with that identified via amino acid sequencing analysis, except that two amino acid residues, Ser and Lys, existed between residues Lys141 and Ser142. DrTI open reading frame was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in Escherichia coli to yield a glutathione S-transferase (GST)-fusion protein with a calculated molecular mass of about 45 kDa. The recombinant DrTI (reDrTI) was derived by treating the GST-DrTI fusion protein with thrombin. Both the reDrTI and GST-DrTI fusion protein exhibited a strong identical inhibitory effect on trypsin activity.     

Cloning and partial characterization of a Boophilus microplus (Acari: Ixodidae) glutathione S-transferase

A cDNA of glutathione S-transferase (GST) was isolated from a cDNA library of salivary glands of Boophilus microplus. The recombinant protein was purified by glutathione affinity chromatography and assayed upon the chromogenic substrate CDNB. The 864 bp cloned fragment was sequenced and showed an open reading frame coding for a protein of 220 amino acids. Expression of the GST gene was tested by RT-PCR in tick tissues and larvae mRNA. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian class mu GSTs.     

Nucleotide sequence of a class mu glutathione S-transferase from chicken liver

A clone coding for glutathione S-transferase (GST) CL2 was isolated from a chicken liver cDNA library. This clone (819 bp) encodes a polypeptide comprising 219 amino acids with a molecular weight of 25,717, excluding the initiator methionine. The primary amino acid sequence of the enzyme has 47% identical sequence with other class mu GSTs.     

Characterization of a variant rat glutathione S-transferase by cDNA expression in Escherichia coli

We have isolated a glutathione S-transferase Yb1 subunit cDNA from a lambda gt11 cDNA collection constructed from rat testis poly(A) RNA enriched for glutathione S-transferase mRNA activities. This Yb1 cDNA, designated pGTR201, is identical to our liver Yb1 cDNA clone pGTR200 except for a shorter 5'-untranslated sequence. Active glutathione S-transferase is expressed from this Yb1 cDNA driven by the tac promoter on the plasmid construct pGTR201-KK. The expressed glutathione S-transferase protein begins with the third codon (Met) of the cDNA, and is missing the N-terminal proline of rat liver glutathione S-transferase 3-3. Therefore, our Escherichia coli expressed glutathione S-transferase protein represents a variant form of glutathione S-transferase 3-3 (Yb1Yb1), designated GST 3-3(-1). The expressed Yb1 subunits are assembled into a dimer as purified from sonicated E. coli crude extracts. In the absence of dithiothreitol three active isomers can be resolved by ion-exchange chromatography. The pure protein has an extinction coefficient of 9.21 x 10(4) M-1 cm-1 at 280 nm or E0.1% 280 = 1.78 and a pI at 8.65. It has a substrate specificity pattern similar to that of the authentic glutathione S-transferase 3-3. The GST 3-3(-1) has a KM of 202 microM for reduced GSH and of 36 microM for 1-chloro-2,4-dinitrobenzene. The turnover number for this conjugation reaction is 57 s-1. Results of kinetic studies of this reaction with GST 3-3(-1) are consistent with a sequential substrate binding mechanism. We conclude that the first amino acid proline of glutathione S-transferase 3-3 is not essential for enzyme activities.     

Molecular cloning and expression of mouse brain sialidase.

Sialidase (EC 3.2.1.18) catalyzes the release of sialic acid from sialo-oligosaccharides, gangliosides, or sialo-glycoproteins. In this investigation, we cloned a novel cDNA for mouse brain sialidase and expressed the cDNA in COS-7 cells. This 1,699 bp cDNA codes for a 41.6 kDa protein consisting of 372 deduced amino acid residues. In COS-7 cells transiently transfected with the cDNA, a 250-fold increase was observed in specific activity toward 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Similarity searches of the nonredundant GenBank peptide sequence database by the PSI-BLAST program identified rat, hamster, human, and bacterial sialidases homologous to this mouse brain sialidase. Amino acid sequence identities to rat and hamster sialidases (84% and 77%, respectively) suggest that this form of sialidase is conserved in rodents. Sequence identities to human and mouse lysosomal sialidases (30% and 28%, respectively) indicate that the mouse brain sialidase is distinct from the lysosomal enzyme. Mouse brain sialidase has two amino acid sequence motifs common to bacterial sialidases: the 'F/YRIP' motif and the 'Asp-box' motif. The 'F/YRIP' motif is present near the N terminus while two 'Asp-box' motifs are present downstream.     

Structural studies on human glutathione S-transferase pi. Substitution mutations to determine amino acids necessary for binding glutathione.

In order to identify amino acids involved in binding the co-substrate glutathione to the human glutathione S-transferase (GST) pi enzyme, we assembled three criteria to implicate amino acids whose role in binding and catalysis could be tested. Presence of a residue in the highly conserved exon 4 of the GST gene, positional conservation of a residue in 12 glutathione S-transferase amino acid sequences, and results from published chemical modification studies were used to implicate 14 residues. A bacterial expression vector (pUC120 pi), which enabled abundant production (2-26% of soluble Escherichia coli protein) of wild-type or mutant GST pi, was constructed, and, following nonconservative substitution mutation of the 14 implicated residues, five mutants (R13S, D57K, Q64R, I68Y, L72F) showed a greater than 95% decrease in specific activity. A quantitative assay was developed which rapidly measured the ability of wild-type or mutant glutathione S-transferase to bind to glutathione-agarose. Using this assay, each of the five loss of function mutants showed a greater than 20-fold decrease in binding glutathione, an observation consistent with a recent crystal structure analysis showing that several of these residues help to form the glutathione-binding cleft.     

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.     

Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells.

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.     

Clonorchis sinensis: molecular cloning and characterization of 28-kDa glutathione S-transferase

A 28-kDa glutathione S-transferase (Cs28GST) was purified from a Clonorchis sinensis cytosolic fraction through anion-exchange and glutathione-affinity column chromatographies. A monoclonal antibody raised against Cs28GST reacted specifically to the C. sinensis antigen among trematode proteins. A putative peptide of 212 amino residues deduced from a cDNA clone appeared homologous with 28-kDa GST of trematodes, and its secondary structural elements predicted a GSH-binding site. Recombinant Cs28GST showed GST enzyme activity with CDNB substrate and was sensitive to the model inhibitors. The recombinant Cs28GST was antigenically indistinguishable from the native form and was recognized specifically by C. sinensis-infected human sera. The Cs28GST was localized in the tegument and underlying mesenchymal tissues. It is suggested that Cs28GST may play significant physiological roles against bioreactive molecules and be a useful reagent for serodiagnosis of clonorchiasis.     

The human Hb (mu) class glutathione S-transferases are encoded by a dispersed gene family

The human glutathione S-transferases are products of a gene superfamily which consists of at least four gene families. The various glutathione S-transferase genes are located on different human chromosomes, and new gene(s) are still being added to the gene superfamily. We have characterized a cDNA in pGTH4 encoding human glutathione S-transferase subunit 4 (GST mu) and mapped its gene (or a homologous family member) on chromosome 1 at p31 by in situ hybridization. Genomic Southern analysis with the 3' noncoding region of the cDNA revealed at least four human DNA fragments with highly homologous sequences. Using a panel of DNAs from mouse-human somatic cell hybrids in genomic DNA hybridization we show that the Hb (or B) genes of human glutathione S-transferases are on three separate chromosomes: 1, 6, and 13. Therefore, the glutathione S-transferase B gene family, which encodes the Hb (mu) class subunits, is a dispersed gene family. The GST mu (psi) gene, whose expression is polymorphic in the human population, is probably located on chromosome 13. We propose that the GST mu (psi) gene was created by a transposition or recombination event during evolution. The null phenotype may have resulted from a lack of DNA transposition just as much as from the deletion of an inserted gene.     

Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage

We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths. cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST. The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids. The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K(m) value of a recombinant enzyme was 0.78 mM. Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST.     

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.