Genetic complementation to identify DNA elements that influence complement resistance in Leishmania chagasi.

Past studies showed that Leishmania spp. promastigotes exhibit differential sensitivity to complement mediated lysis (CML) during development in vitro and in vivo. Leishmania chagasi promastigotes in cultures during logarithmic and stationary growth phases are CML-sensitive or CML-resistant when exposed to human serum, respectively, but only in cultures recently initiated with parasites from infected animals; serially passaged cultures become constitutively CML-sensitive regardless of growth phase. Building on these observations, a genetic screen was conducted to identify novel complement resistance factors of L. chagasi. A cosmid library containing genomic DNA was transfected into a promastigote line previously subjected to >50 serial passages. Selection with human serum for CML resistance yielded 12 transfectant clones. Cosmids isolated from 7 of these clones conferred CML resistance when transfected into an independent, high-passage promastigote culture; at 12% human serum, the mean survival of transfectants was 37% (+/- 11.6%), and that of control transfectants was about 1%. Inserts within the 7 cosmids were unique. Determination of the complete DNA sequence for 1 cosmid indicated that its 32-kilobase insert was 89% identical (overall) to a 31-kilobase region of Leishmania major chromosome 36, which is predicted to encode 6 genes, all of which encode hypothetical proteins.

Cress and potato soluble epoxide hydrolases: purification, biochemical characterization, and comparison to mammalian enzymes.

Affinity chromatographic methods were developed for the one-step purification to homogeneity of recombinant soluble epoxide hydrolases (sEHs) from cress and potato. The enzymes are monomeric, with masses of 36 and 39 kDa and pI values of 4.5 and 5.0, respectively. In spite of a large difference in sequence, the two plant enzymes have properties of inhibition and substrate selectivity which differ only slightly from mammalian sEHs. Whereas mammalian sEHs are highly selective for trans- versus cis-substituted stilbene oxide and 1,3-diphenylpropene oxide (DPPO), plant sEHs exhibit far greater selectivity for trans- versus cis-stilbene oxide, but little to no selectivity for DPPO isomers. The isolation of a covalently linked plant sEH-substrate complex indicated that the plant and mammalian sEHs have a similar mechanism of action. We hypothesize an in vivo role for plant sEH in cutin biosynthesis, based on relatively high plant sEH activity on epoxystearate to form a cutin precursor, 9,10-dihydroxystearate. Plant sEHs display a high thermal stability relative to mammalian sEHs. This stability and their high enantioselectivity for a single substrate suggest that their potential as biocatalysts for the preparation of enantiopure epoxides should be evaluated.

Regulatory sequences and a novel gene in the msp (GP63) gene cluster of Leishmania chagasi.

The surface protease GP63 of Leishmania chagasi is encoded by a cluster of more than 18 tandem major surface protease (msp) genes belonging to three classes (mspL, mspS, mspC). mspL and mspS transcripts are differentially expressed during parasite growth. RNAs from mspS genes predominate during stationary phase, the time when parasite virulence and GP63 expression are maximal. We hypothesized that the unique regions downstream of mspS genes contain signals important for gene expression. The 2.8 kb region between tandem mspS genes was found to contain an 882 bp open reading frame designated mag. Copies of mag were found downstream of all mspS genes in the cluster. mag hybridized faintly to bands on Northern blots and a fully processed mag cDNA was identified in a promastigote cDNA library, providing evidence that mag genes are expressed at low levels. Similar to mspS RNAs, the abundance of mag RNAs was greater in stationary phase than logarithmic phase organisms, although mag RNAs were less abundant than mspS RNAs throughout growth. Northern blots and enzyme assays of promastigotes containing plasmid constructs in which the beta-galactosidase gene was followed by sequences between mspS coding regions, either with or without mag and its downstream sequences, suggest these regions have several regulatory effects accounting for the growth-associated changes in mspS expression.

Glycoprotein 46 mRNA abundance is post-transcriptionally regulated during development of Leishmania chagasi promastigotes to an infectious form.

GP46 is an abundant glycoprotein of 46 kDa on the surface of the promastigote form of most Leishmania species. We show that the steady state level of GP46 mRNA increases >>30-fold as Leishmania chagasi promastigotes develop in vitro from a less infectious form during logarithmic growth to a highly infectious form in the stationary phase of cultivation. Nuclear run-on experiments demonstrate that this increase in GP46 mRNA abundance is regulated post-transcriptionally. Plasmids containing the 3'-untranslated regions (UTRs) and downstream intergenic regions (IRs) of two different GP46 genes fused immediately downstream of the beta-galactosidase coding region were transfected into L. chagasi, and beta-galactosidase activity and mRNA levels were examined. The presence of the 3'-UTR + IR of one GP46 gene (gp46A) resulted in a steady increase in beta-galactosidase activity and mRNA level as the transfected promastigotes developed from logarithmic to stationary phase. This differential effect parallels that of the 3'-UTRs + IRs of a family of genes for an unrelated Leishmania surface glycoprotein, GP63. Thus, post-transcriptional regulation of the genes for two different surface glycoproteins of Leishmania occurs via a similar mechanism.

Visualization of a covalent intermediate between microsomal epoxide hydrolase, but not cholesterol epoxide hydrolase, and their substrates.

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of alpha/beta-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with [1-(14)C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1,1,1-trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluorochalcone-oxide-sensitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-(14)C]cholesterol-5alpha,6alpha-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.

Molecular and biochemical evidence for the involvement of the Asp-333-His-523 pair in the catalytic mechanism of soluble epoxide hydrolase.

In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.

Gene evolution of epoxide hydrolases and recommended nomenclature.

We have analyzed amino acid sequence relationships among soluble and microsomal epoxide hydrolases, haloacid dehalogenases, and a haloalkane dehalogenase. The amino-terminal residues (1-229) of mammalian soluble epoxide hydrolase are homologous to a haloacid dehalogenase. The carboxy-terminal residues (230-554) of mammalian soluble epoxide hydrolase are homologous to haloalkane dehalogenase, to plant soluble epoxide hydrolase, and to microsomal epoxide hydrolase. The shared identity between the haloacid and haloalkane dehalogenases does not indicate relatedness between these two types of dehalogenases. The amino-terminal and carboxy-terminal homologies of mammalian soluble epoxide hydrolase to the respective dehalogenases suggests that this epoxide hydrolase, but not the soluble epoxide hydrolase of plant or the microsomal epoxide hydrolase, derives from a gene fusion. The homology of microsomal to soluble epoxide hydrolase suggests they derive from a gene duplication, probably of an ancestral bacterial (epoxide) hydrolase gene. Based on homology to haloalkane dehalogenase, the catalytic residues for the soluble and microsomal epoxide hydrolases are predicted. A nomenclature system based on divergent molecular evolution is proposed for these epoxide hydrolases.

Cloning and expression of soluble epoxide hydrolase from potato.

Five cDNAs encoding a putative soluble epoxide hydrolase (sEH) from potato were isolated and characterized. The cDNAs contained open reading frames encoding 36 kDa polypeptides which were highly homologous to the carboxy terminal region of mammalian sEH. When one of the cDNAs was expressed in a baculovirus system a soluble 38 kDa protein with epoxide hydrolase activity was produced. The recombinant enzyme hydrolyzed a commonly used diagnostic substrate for the soluble form of mammalian EH. Inhibitor profiles of the recombinant potato and mammalian sEH were also similar. The expression of sEH in potato was found to be regulated by both developmental and environmental signals. Levels of mRNA for sEH were higher in meristematic tissue than in mature leaves. This mRNA was also observed to accumulate on wounding and application of exogenous methyl jasmonate.

Characterization of an Arabidopsis cDNA for a soluble epoxide hydrolase gene that is inducible by auxin and water stress.

A cDNA (1122 bp) was isolated from a cDNA library prepared from Arabidopsis thaliana L. that had been subjected to drought stress for 1 h. The sequencing of a genomic clone corresponding to the cDNA and S1 mapping analysis revealed that the cDNA lacked the first 6 bp from its translational start (ATG). The resulting open reading frame encodes a polypeptide of 321 amino acids, and the calculated molecular weight of this polypeptide is 36,423 Da. The deduced amino acid sequence shows a high degree of similarity to C terminal halves of those of soluble epoxide hydrolases (sEHs) of human, mouse and rat, 35.5%, 34.1% and 33.1%, respectively. The cDNA was expressed in Escherichia coli cells, and the expressed protein migrates at 40 kDa when analyzed by SDS-PAGE. The recombinant protein at 40 kDa is much smaller than the mammalian sEH (58 kDa) but has characteristics of activity and inhibition similar to the mammalian sEHs when assayed with the substrate trans-stilbene oxide and the inhibitors 4-fluorochalcone oxide (4FCO), (2R,3R)-3-(4-nitrophenyl) glycidol (RRNPG), and (2S,3S)-3-(4-nitrophenyl)glycidol (SSNPG), which indicates that the cDNA did encode a soluble epoxide hydrolase of A. thaliana (AtsEH). Drought stress, but not heat or cold stress, slightly increased the accumulation of the mRNAs for AtsEH. The level of AtsEH transcripts increased strongly after treatment with a plant hormone, auxin (2,4-dichlorophenoxyacetic acid, 2,4-D; naphthalene-acetic acid, NAA; and indole-3-acetic acid, IAA) in young, pre-bolting plants. Treatment with cytokinin (6-benzylaminopurine, BA), abscisic acid (ABA) or gibberellin (GA3) had no detectable effect on AtsEH transcript levels. The transcripts for AtsEH gene were detected in the aerial vegetative organs of bolting plants (i.e. stems and leaves), but not in roots, flowers and seeds. The possible function of AtsEH is discussed. A similar sEH cDNA has recently been characterized in potato (Stapleton et al., 1994).

Cloning, characterization, and genetics of the juvenile hormone esterase gene from Heliothis virescens.

The gene for juvenile hormone esterase (JHE) was cloned from Heliothis virescens (Lepidoptera: Noctuidae). A genomic library was constructed from embryonic DNA and screened with a homologous N-terminal probe from the JHE cDNA. Five genomic clones were isolated and analyzed by dot blot hybridization using regions of the JHE cDNA as probes. Clone C hybridized to both 5' and 3' probes from the JHE cDNA, suggesting that clone C contains both ends of JHE gene. This was verified by sequencing the ends of the JHE gene from clone C using primers from both the 5' and 3' ends of the JHE cDNA. Additional sequencing and restriction mapping were used to characterize the gene. The gene is c. 8 kb long and contains four introns with consensus intron-exon junctions. One of the introns is relatively large (4 kb) and is situated near the extreme 5' end of the gene. Genetic analysis of RFLP variation in interspecific and intraspecific crosses shows that the JHE locus is single-copy with no closely related paralogs and is autosomally encoded in Heliothis. Therefore the developmental pattern of expression of this gene and the previously documented sequence variation in cDNA clones is not explainable by reference to a JHE gene family with distinct structural loci for the different forms.

Isolation of a putative hydroxyacyl enzyme intermediate of an epoxide hydrolase.

A putative covalent, alpha-hydroxyacyl intermediate was isolated by the brief exposure of murine soluble epoxide hydrolase to its substrate. The reaction was reversed by time and blocked by competitive inhibitors. The formation of the intermediate was dependent upon the concentration of the enzyme and was increased by incubation under acidic conditions. The structure of the intermediate was supported by microchemical methods.

Sequence similarity of mammalian epoxide hydrolases to the bacterial haloalkane dehalogenase and other related proteins. Implication for the potential catalytic mechanism of enzymatic epoxide hydrolysis.

Direct comparison of the amino acid sequences of microsomal and soluble epoxide hydrolase superficially indicates that these enzymes are unrelated. Both proteins, however, share significant sequence similarity to a bacterial haloalkane dehalogenase that has earlier been shown to belong to the alpha/beta hydrolase fold family of enzymes. The catalytic mechanism for the dehalogenase has been elucidated in detail [Verschueren et al. (1993) Nature 363, 693-698] and proceeds via an ester intermediate where the substrate is covalently bound to the enzyme. From these observations we conclude (i) that microsomal and soluble epoxide hydrolase are distantly related enzymes that have evolved from a common ancestral protein together with the haloalkane dehalogenase and a variety of other proteins specified in the present paper, (ii) that these enzymes most likely belong to the alpha/beta hydrolase fold family of enzymes and (iii) that the enzymatic epoxide hydrolysis proceeds via a hydroxy ester intermediate, in contrast to the presently favoured base-catalyzed direct attack of the epoxide by an activated water.

cDNA cloning and expression of a soluble epoxide hydrolase from human liver.

We report the cloning and expression of a cDNA that encodes a soluble epoxide hydrolase from human liver. The 2101-base clone predicts a 554-residue protein (M(r) 62,640) with an apparently imperfect peroxisomal targeting signal of Ser-Lys-Met at the carboxy terminus. The cDNA was expressed in the baculovirus system in the Spodoptera frugiperda 21 cell line. The recombinant protein was similar to soluble epoxide hydrolase isolated from human liver in terms of molecular weight, hydrolytic activity, inhibition, and immunoreactivity.