Purification and characterization of 3-hydroxymethylglutaryl-coenzyme A reductase of Schistosoma mansoni: regulation of parasite enzyme activity differs from mammalian host

The enzyme 3-hydroxymethylglutaryl-CoA (HMG-CoA) reductase plays a critical role in regulating the production of cholesterol, dolichols, and ubiquinones in mammals. The inhibition of this enzyme in Schistosoma mansoni is accompanied by a cessation of egg production by the female parasite and a reduced ability of the parasite to properly glycoslyate their proteins. Furthermore, we recently demonstrated that mevinolin, if given continuously over a period of 10-14 days, is a potent antischistosomal drug. In this paper, we describe the properties of purified HMG-CoA reductase from S. mansoni. Using affinity chromatography, we were able to obtain a 417-fold purification of the enzyme which had Km values similar to the rat enzyme for HMG-CoA and NADPH. The Ki value for mevinolin, a potent and selective inhibitor of the rat reductase (Ki = 0.6 nM), was significantly higher (Ki = 46 nM) for the schistosome enzyme. SDS-PAGE and HPLC of the purified enzyme resulted in the appearance of a single protein, which had a molecular weight (66,000) in the range reported for the rat enzyme. Parasite reductase activity, unlike that of its host, did not display a circadian rhythm. Furthermore, agents which elevate (cholestyramine) or decrease (cholesterol) mammalian reductase activity had no effect on the parasite enzyme. Our results suggest that the mechanism which regulates production of the parasite's enzyme may differ from its mammalian host.     

An association between Schistosoma mansoni worms and an enzymatically-active protease/peptidase in mouse blood

An enzyme found previously in extracts of adult Schistosoma mansoni worms, that hydrolysed the chromogenic substrate N-acetyl-DL-phenylalanine beta-naphthyl-ester, has here been further investigated and characterized. Evidence that the molecule found in the parasite was antigenically and enzymatically homologous with a constituent of normal mouse plasma has been consolidated using a monospecific serum in immunoelectrophoresis and Western immunoblotting. The molecular size of the enzyme was found to be approximately 70 kDa and it was inhibited by a serine protease inhibitor, but not by inhibitors of other classes of protease. The enzymatic activity found in normal mouse serum was also found in normal rat serum, but not in sera from several other mammalian species.     

The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli

Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.     

A regulatory domain in the N terminus of tryptophan hydroxylase 2 controls enzyme expression

Serotonin is involved in a variety of physiological processes in the central nervous system and the periphery. As the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase plays an important role in modulating these processes. Of the two variants of tryptophan hydroxylase, tryptophan hydroxylase 2 (TPH2) is expressed predominantly in the central nervous system, whereas tryptophan hydroxylase 1 (TPH1) is expressed mostly in peripheral tissues. Although the two enzymes share considerable sequence homology, the regulatory domain of TPH2 contains an additional 41 amino acids at the N terminus that TPH1 lacks. Here we show that the extended TPH2 N-terminal domain contains a unique sequence involved in the regulation of enzyme expression. When expressed in cultured mammalian cells, TPH2 is synthesized less efficiently and is also less stable than TPH1. Removal of the unique portion of the N terminus of TPH2 results in expression of the enzyme at a level similar to that of TPH1, whereas protein chimeras containing this fragment are expressed at lower levels than their wild-type counterparts. We identify a region centered on amino acids 10-20 that mediates the bulk of this effect. We also demonstrate that phosphorylation of serine 19, a protein kinase A consensus site located in this N-terminal domain, results in increased TPH2 stability and consequent increases in enzyme output in cell culture systems. Because this domain is unique to TPH2, these data provide evidence for selective regulation of brain serotonin synthesis.     

Serotonin synthesis by two distinct enzymes in Drosophila melanogaster

Annotation of the sequenced Drosophila genome suggested the presence of an additional enzyme with extensive homology to mammalian tryptophan hydroxylase, which we have termed DTRH. In this work, we show that enzymatic analyses of the putative DTRH enzyme expressed in Escherichia coli confirm that it acts as a tryptophan hydroxylase but can also hydroxylate phenylalanine, in vitro. Building upon the knowledge gained from the work in mice and zebrafish, it is possible to hypothesize that DTRH may be primarily neuronal in function and expression, and DTPH, which has been previously shown to have phenylalanine hydroxylation as its primary role, may be the peripheral tryptophan hydroxylase in Drosophila. The experiments presented in this report also show that DTRH is similar to DTPH in that it exhibits differential hydroxylase activity based on substrate. When DTRH uses tryptophan as a substrate, substrate inhibition, catecholamine inhibition, and decreased tryptophan hydroxylase activity in the presence of serotonin synthesis inhibitors are observed. When DTRH uses phenylalanine as a substrate, end product inhibition, increased phenylalanine hydroxylase activity after phosphorylation by cAMP-dependent protein kinase, and a decrease in phenylalanine hydroxylase activity in the presence of the serotonin synthesis inhibitor, alpha-methyl-(DL)-tryptophan are observed. These experiments suggest that the presence of distinct tryptophan hydroxylase enzymes may be evolutionarily conserved and serve as an ancient mechanism to appropriately regulate the production of serotonin in its target tissues.     

Characterisation of carbonic anhydrase in Plasmodium falciparum

Here we report the existence, purification and characterisation of carbonic anhydrase in Plasmodium falciparum. The infected red cells contained carbonic anhydrase approximately 2 times higher than those of normal red cells. The three developmental forms of the asexual stages, ring, trophozoite and schizont were isolated from their host red cells and found to have stage-dependent activity of the carbonic anhydrase. The enzyme was purified to homogeneity from the crude extract of P. falciparum using multiple steps of fast liquid chromatographic techniques. It had a Mr of 32 kDa and was active in a monomeric form. The human red cell enzyme was also purified for comparison with the parasite enzyme. The parasite enzyme activity was sensitive to well-known sulfonamide-based inhibitors of both bacterial and mammalian enzymes, sulfanilamide and acetazolamide. The kinetic properties and the amino terminal sequences of the purified enzymes from the parasite and host red cell were found to be different, indicating that the purified protein most likely exhibited the P. falciparum carbonic anhydrase activity. In addition, the enzyme inhibitors had antimalarial effect against in vitro growth of P. falciparum. Moreover, the vital contribution of the carbonic anhydrase to the parasite survival makes the enzyme an attractive target for therapeutic evaluation.     

Increased Hippocampal 5-Lipoxygenase mRNA Content in Melatonin-Deficient, Pinealectomized Rats

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is activated by protein kinase A and calcium/calmodulin-dependent protein kinase. One important aspect of the regulation of any enzyme by a phosphorylation-dephosphorylation cascade, and one that is lacking for tryptophan hydroxylase, lies in the identification of its site of phosphorylation by protein kinases. Recombinant forms of brain tryptophan hydroxylase were expressed as glutathione S -transferase fusion proteins and exposed to protein kinase A. This protein kinase phosphorylates and activates full-length tryptophan hydroxylase. The inactive regulatory domain of the enzyme (corresponding to amino acids 1–98) was also phosphorylated by protein kinase A. The catalytic core of the hydroxylase (amino acids 99–444), which expresses high levels of enzyme activity, was neither phosphorylated nor activated by protein kinase A. Conversion of serine-58 to arginine resulted in the expression of a full-length tryptophan hydroxylase mutant that, although remaining catalytically active, was neither phosphorylated nor activated by protein kinase A. These results indicate that the activation of tryptophan hydroxylase by protein kinase A is mediated by the phosphorylation of serine-58 within the regulatory domain of the enzyme.     

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Schistosoma mansoni. A potential target for chemotherapy

Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities are essential for the supply of guanine nucleotides in Schistosoma mansoni schistosomules. In crude extracts of adult S. mansoni, these two activities co-elute in size exclusion, ion exchange, and chromatofocusing chromatography and exhibit similar stabilities to heat treatment, suggesting that they are associated in one enzyme protein hypoxanthine-guanine phosphoribosyltransferase. This enzyme has been purified by a combination of heat treatment at 85 degrees C and chromatofocusing chromatography with elution at an apparent pI of 5.27 +/- 0.15. Pore gradient electrophoresis of the native enzyme followed by subsequent activity staining demonstrate an enzyme molecular weight of 105,000. The activity staining pattern remains the same whether hypoxanthine or guanine is used as the substrate, further supporting the existence of a single protein, hypoxanthine-guanine phosphoribosyltransferase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein results in a single protein band with a subunit molecular weight estimate of 64,000, suggesting that the native enzyme is a dimer. Preliminary kinetic studies showed that the purified hypoxanthine-guanine phosphoribosyltransferase reacted with guanine at a rate twice as fast as it did with hypoxanthine, but it did not act on xanthine at all. A full-length mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase cDNA clone pHPT5 and a plasmid pSV2-gpt containing the xanthine-guanine phosphoribosyltransferase gene for Escherichia coli were utilized as probes on Southern blots of S. mansoni DNA digests, and no significant hybridization was found under relatively relaxed conditions. Polyclonal antibodies made against human erythrocyte hypoxanthine-guanine phosphoribosyltransferase and E. coli xanthine-guanine phosphoribosyltransferase were tested in enzyme-linked immunosorbent assays of S. mansoni protein extracts, and no detectable cross-reacting protein was found. S. mansoni hypoxanthine-guanine phosphoribosyltransferase thus may bear rather limited homology to mammalian hypoxanthine-guanine phosphoribosyltransferase or bacterial xanthine-guanine phosphoribosyltransferase and could be an attractive target for antischistosomal chemotherapeutic drug design.     

The glutathione transferase activity and tissue distribution of a cloned Mr28K protective antigen of Schistosoma mansoni.

A protective Mr28K antigen of Schistosoma mansoni, expressed from its cDNA, has been purified in a single step and shown to possess glutathione (GSH) transferase activity as predicted from sequence homologies with two mammalian GSH transferase multigene families. It is notable for its high 1-chloro-2,4-dinitrobenzene GSH transferase and linoleic acid hydroperoxide GSH peroxidase activities. The major GSH transferase of S. mansoni has been purified and its subunit is identical to this Mr28K antigen by criteria of Mr, immunochemistry, substrate specificity and peptide sequence analysis. In the parasite, the antigen is present in the tegument, protonephridial cells and subtegumental parenchymal cells. No significant immunological cross-reactivity between the S.mansoni and mammalian (human and rat) GSH transferases was observed.     

Compartmentalization of neuronal and peripheral serotonin synthesis in Drosophila melanogaster

In Drosophila, one enzyme (Drosophila tryptophan-phenylalanine hydroxylase, DTPHu) hydroxylates both tryptophan to yield 5-hydroxytryptophan, the first step in serotonin synthesis, and phenylalanine, to generate tyrosine. Analysis of the sequenced Drosophila genome identified an additional enzyme with extensive homology to mammalian tryptophan hydroxylase (TPH), which we have termed DTRHn. We have shown that DTRHn can hydroxylate tryptophan in vitro but displays differential activity relative to DTPHu when using tryptophan as a substrate. Recent studies in mice identified the presence of two TPH genes, Tph1 and Tph2, from distinct genetic loci. Tph1 represents the non-neuronal TPH gene, and Tph2 is expressed exclusively in the brain. In this article, we show that DTRHn is neuronal in expression and function and thus represents the Drosophila homologue of Tph2. Using a DTRHn-null mutation, we show that diminished neuronal serotonin affects locomotor, olfactory and feeding behaviors, as well as heart rate. We also show that DTPHu functions in vivo as a phenylalanine hydroxylase in addition to its role as the peripheral TPH in Drosophila, and is critical for non-neuronal developmental events.     

Identification and characterization of Paragonimus westermani leucine aminopeptidase

Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.     

Identification of Schistosoma mansoni candidate antigens for diagnosis of schistosomiasis

The development of a more sensitive diagnostic test for schistosomiasis is needed to overcome the limitations of the use of stool examination in low endemic areas. Using parasite antigens in enzyme linked immunosorbent assay is a promising strategy, however a more rational selection of parasite antigens is necessary. In this study we performed in silico analysis of the Schistosoma mansoni genome, using SchistoDB database and bioinformatic tools for screening immunogenic antigens. Based on evidence of expression in all parasite life stage within the definitive host, extracellular or plasmatic membrane localization, low similarity to human and other helminthic proteins and presence of predicted B cell epitopes, six candidates were selected: a glycosylphosphatidylinositol-anchored 200 kDa protein, two putative cytochrome oxidase subunits, two expressed proteins and one hypothetical protein. The recognition in unidimensional and bidimensional Western blot of protein with similar molecular weight and isoelectric point to the selected antigens by sera from S. mansoni infected mice indicate a good correlation between these two approaches in selecting immunogenic proteins.     

Involvement of striatum serotonergic system in the expression of genetically defined catalepsy

The activity of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, and specific binding of [3H]ketanserin to 5-HT2A receptors and [3H]8-OH-DPAT to 5-HT1A receptors in the striatum of genetically predisposed to catalepsy rats and mice have been studied. The activity of tryptophan hydroxylase in the striatum of rats bred for many generations for predisposition to catalepsy was higher than in nonselected rats. Mice of highly susceptible to pinch-induced catalepsy CBA strain also differed from noncataleptic AKR and C57BL mouse strains by higher activity of tryptophan hydroxylase in striatum. Inhibition of tryptophan hydroxylase with p-chlorophenylalanine or p-chloromethamphetamine significantly decreased immobility time in genetically predisposed to catalepsy rats and mice. A decrease in the [3H]ketanserin specific binding in the striatum of cataleptic rats and CBA mice was found indicating a decrease in 5-HT2A receptor density. A decrease in [3H]8-OH-DPAT binding in striatum of cataleptic rats but not in CBA mice was shown. These results indicate that serotonergic system of striatum is involved in the expression of hereditary catalepsy and suggest that hereditary catalepsy may result from genetic changes in the regulation of serotonin metabolism and reception in striatum.     

Uridine phosphorylase from Schistosoma mansoni

Uridine phosphorylase is the only pyrimidine nucleoside cleaving activity that can be detected in extracts of Schistosoma mansoni. The enzyme is distinct from the two purine nucleoside phosphorylases contained in this parasite. Although Urd is the preferred substrate, uridine phosphorylase can also catalyze the reversible phosphorolysis of dUrd and dThd, but not Cyd, dCyd, or orotidine. The enzyme was purified 170-fold to a specific activity of 2.76 nmol/min/mg of protein with a 16% yield. It has a Mr of 56,000 as determined by molecular sieving on Sephadex G-100. The mechanism of uridine phosphorylase is sequential. When Urd was the substrate, the KUrd = 13 microM and the KPi = 533 +/- 78 microM. When dThd was used as a substrate, the KdThd = 54 microM and the KPi = 762 +/- 297 microM. The Vmax with dThd was 53 +/- 9.8% that of Urd. dThd was a competitive inhibitor when Urd was used as a substrate. The enzyme showed substrate inhibition by Urd, dThd (greater than 0.125 mM) and phosphate (greater than 10 mM). 5-(Benzyloxybenzyloxybenzyl)acyclouridine was identified as a potent and specific inhibitor of parasite (Ki = 0.98 microM) but not host uridine phosphorylase. Structure-activity relationship studies suggest that uridine phosphorylase from S. mansoni has a hydrophobic pocket adjacent to the 5-position of the pyrimidine ring and indicate differences between the binding sites of the mammalian and parasite enzymes. These differences may be useful in designing specific inhibitors for schistosomal uridine phosphorylase which will interfere selectively with nucleic acids synthesis in this parasite.