An enzyme study of purine nucleotide biosynthesis in the plerocercoids of cestodes in the fam. Ligulidae]

By means of spectrophotometric method there was determined the activity of three enzymes of biosynthesis of purine nucleotides: amino imidazole ribonucleotide-carboxylase (AIR-carboxylase, EC 4.1.1.21), an enzyme of biosynthesis of purine nucleotides de novo in plerocercoids of Schistocephalus pungitii and Digramma interrupta; inosine monophosphate-dehydrogenase (IMPh-dehydrogenase, EC 1.2.1.14), an enzyme of salvage path, and adenylosuccinate lyase (EC 4.3.2.2), an enzyme taking part both in biosynthesis de novo and salvage in plerocercoids of Schistocephalus pungitii. The activity of AIR-carboxylase was not determined. Specific activities of adenylosuccinate lyase and IMPh-dehydrogenase amount to (1.3 +/- 0.3) x 10(-3) and (1.2 +/- 0.4) x 10(-3) mumole/min.mg protein, respectively. The activity of the three enzymes was determined in the liver of ten-spined stickleback, a host of S. pungitii plerocercoids. The question of metabolic dependence of Ligulidae plerocercoids on hosts to provide for purine bases is discussed.     

Isozyme patterns of Schistosoma japonicum and S. mansoni

Isozyme patterns of six enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, hexokinase, malate dehydrogenase, 6-phosphate dehydrogenase and phosphoglucomutase were examined in electrophoresed homogenates of adult male worms of Schistosoma japonicum and S. mansoni. In general, enzyme patterns obtained from the parasite homogenates differed from that of host (mouse) blood and muscle, indicating that electrophoretic patterns from parasite extracts are most probably of parasite origin. Adult male and female S. mansoni worms yielded identical patterns. However, all six enzyme patterns showed distinct differences between S. japonicum and S. mansoni. These results suggest that S. japonicum is clearly distinguishable from S. mansoni at the molecular level.     

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.     

Crystal structure of fully ligated adenylosuccinate synthetase from Plasmodium falciparum

In the absence of the de novo purine nucleotide biosynthetic pathway in parasitic protozoa, purine salvage is of primary importance for parasite survival. Enzymes of the salvage pathway are, therefore, good targets for anti-parasitic drugs. Adenylosuccinate synthetase (AdSS), catalysing the first committed step in the synthesis of AMP from IMP, is a potential target for anti-protozoal chemotherapy. We report here the crystal structure of adenylosuccinate synthetase from the malaria parasite, Plasmodium falciparum, complexed to 6-phosphoryl IMP, GDP, Mg2+ and the aspartate analogue, hadacidin at 2 A resolution. The overall architecture of P. falciparum AdSS (PfAdSS) is similar to the known structures from Escherichia coli, mouse and plants. Differences in substrate interactions seen in this structure provide a plausible explanation for the kinetic differences between PfAdSS and the enzyme from other species. Additional hydrogen bonding interactions of the protein with GDP may account for the ordered binding of substrates to the enzyme. The dimer interface of PfAdSS is also different, with a pronounced excess of positively charged residues. Differences highlighted here provide a basis for the design of species-specific inhibitors of the enzyme.     

A model to explain the origin of a parasite sex-specific population structure

A discrete time model was built to understand the origin of the sex-specific population structure of the human blood fluke, Schistosoma mansoni. We have estimated both male/female individual ratio and male/female genotype ratio of this parasite taking into account all the experimental published values on differential male and female life-history traits all along the life cycle. We considered in our model male and female life-history traits when both separated and together. The model showed that both male/female individual ratio and male/female genotype ratio of S. mansoni adults are biased toward males in each combination. This bias was more important in male/female genotype ratio than in the male/female individual ratio for the same initial values of cercarial development success. This model could explain the sex specific population structure of this parasite. Firstly, we showed that the male-biased individual ratio finds its origin in the vertebrate host. Secondly, we showed that the male-biased genotype ratio originates prior to any interrelationship between adult worms and could generate by itself a sex-specific genetic structure.     

Dolichol phosphate mannose synthase is differentially expressed in male and female worms of Schistosoma mansoni

The cDNA encoding the Schistosoma mansoni dolichol phosphate mannose synthase was completely sequenced, displaying the highest homology with Cricetulus griseus and Saccharomyces pombe genes. The Schistosome enzyme had a K(m) of 0.127 microM, a value that is within the range of those reported for several other species. Thin-layer chromatography of the radiolabelled schistosome lipid intermediate showed it was identical to dolichol-phosphate (C80-C105). Expression of dolichol phosphate mannose synthase of S. mansoni (SmDPMS) was analysed by Northern blot and quantified by semi-quantitative RT-PCR with cDNA from mature and immature male and female worms. Northern blot analysis revealed a single 1-kb band. Both approaches confirmed a higher level of expression in mature female worms, as compared to immature and male worms.     

Fasciola hepatica: molecular cloning, nucleotide sequence, and expression of a gene encoding a polypeptide homologous to a Schistosoma mansoni fatty acid-binding protein.

Immunization of mice with an antigenic polypeptide from Fasciola hepatica adult worms and having an apparent molecular mass of 12,000 Da (Fh12) has been shown to reduce the worm burden from challenge infection with Schistosoma mansoni by more than 50%. Moreover, mice infected with S. mansoni develop antibodies to Fh12 after 5-6 weeks of infection, indicating that this Fasciola-derived antigen is a cross-reactive, cross-protective protein. A lambda gt11 F. hepatica cDNA library was constructed from poly(A)+ RNA extracted from adult worms. A cDNA encoding a cross-reactive polypeptide (Fh15) was cloned by screening the F. hepatica lambda gt11 library with a monospecific, polyclonal rabbit antiserum against pure, native Fh12. The cDNA was sequenced and the predicted amino acid sequence revealed an open reading frame encoding a 132-amino-acid protein with a predicted molecular weight of 14,700 Da. This protein has significant homology to a 14-kDa S. mansoni fatty acid-binding protein. Comparison of the protective-inducing activity of recombinant Fh15 with that of purified Fh12 against schistosomes and Fasciola is warranted.     

The structure of adenylosuccinate lyase, an enzyme with dual activity in the de novo purine biosynthetic pathway

Background: Adenylosuccinate lyase is an enzyme that plays a critical role in both cellular replication and metabolism via its action in the de novo purine biosynthetic pathway. Adenylosuccinate lyase is the only enzyme in this pathway to catalyze two separate reactions, enabling it to participate in the addition of a nitrogen at two different positions in adenosine monophosphate. Both reactions catalyzed by adenylosuccinate lyase involve the beta-elimination of fumarate. Enzymes that catalyze this type of reaction belong to a superfamily, the members of which are homotetramers. Because adenylosuccinate lyase plays an integral part in maintaining proper cellular metabolism, mutations in the human enzyme can have severe clinical consequences, including mental retardation with autistic features. Results: The 1.8 A crystal structure of adenylosuccinate lyase from Thermotoga maritima has been determined by multiwavelength anomalous dispersion using the selenomethionine-substituted enzyme. The fold of the monomer is reminiscent of other members of the beta-elimination superfamily. However, its active tetrameric form exhibits striking differences in active-site architecture and cleft size. Conclusions: This first structure of an adenylosuccinate lyase reveals that, along with the catalytic base (His141) and the catalytic acid (His68), Gln212 and Asn270 might play a vital role in catalysis by properly orienting the succinyl moiety of the substrates. We propose a model for the dual activity of adenylosuccinate lyase: a single 180 degrees bond rotation must occur in the substrate between the first and second enzymatic reactions. Modeling of the pathogenic human S413P mutation indicates that the mutation destabilizes the enzyme by disrupting the C-terminal extension.     

Cloning of a cDNA encoding adenylosuccinate lyase by functional complementation in Escherichia coli

Adenylosuccinate lyase was cloned by functional complementation of an Escherichia coli purB mutant using an avian liver cDNA expression library. The derived amino acid sequence is homologous to the bacterial purB-encoded adenylosuccinate lyase which catalyzes the same two steps in purine biosynthesis as the enzyme from animals. Avian adenylosuccinate lyase also shows regions of extensive sequence similarity to the urea cycle enzyme, argininosuccinate lyase. This homology suggests a similar mechanism for catalysis. Homology of adenylosuccinate and argininosuccinate lyases is intriguing because chickens do not utilize the urea cycle in nitrogen excretion. This is the first report of the cloning of a eukaryotic cDNA encoding adenylosuccinate lyase, and it affords a route to isolate the corresponding human gene which has been suggested to be defective in autistic children.     

Expression, purification, and characterization of stable, recombinant human adenylosuccinate lyase

The full length human adenylosuccinate lyase gene was generated by a PCR method using a plasmid encoding a truncated human enzyme as template, and was cloned into a pET-14b vector. Human adenylosuccinate lyase was overexpressed in Escherichia coli Rosetta 2(DE3)pLysS as an N-terminal histidine-tagged protein and was purified to homogeneity by a nickel-nitriloacetic acid column at room temperature. The histidine tag was removed from the human enzyme by thrombin digestion and the adenylosuccinate lyase was purified by Sephadex G-100 gel filtration. The histidine-tagged and non-tagged adenylosuccinate lyases exhibit similar values of Vmax and Km for S-AMP. Analytical ultracentrifugation and circular dichroism revealed, respectively, that the histidine-tagged enzyme is in tetrameric form with a molecular weight of 220 kDa and contains predominantly alpha-helical structure. This is the first purification procedure to yield a stable form of human adenylosuccinate lyase. The enzyme is stable for at least 5 days at 25 degrees C, and upon rapid freezing and thawing. Temperature as well as reducing agent (DTT) play critical roles in determining the stability of the human adenylosuccinate lyase.     

Characterization of Schistosoma mansoni ATPDase2 gene, a novel apyrase family member

Schistosoma mansoni is a major causative agent of schistosomiasis, which constitutes a severe health problem in developing countries. We have previously described the SmATPDase1 gene, encoding a protein from the external surface of the parasites. In this work, we describe the cloning and characterization of SmATPDase2, a novel CD39-like ATP diphosphohydrolase gene in S. mansoni. In silico analysis of the protein encoded by SmATPDase2 predicts a single N-terminal transmembrane domain similar to that described for secreted human apyrase isoforms. Immuno-colocalization experiments detected both SmATPDase proteins at the S. mansoni adult worm tegument basal and apical membranes, but only SmATPDase2 in the tegument syncytium. SmATPDase2 but not SmATPDase1 protein was detected by Western blot in culture medium supernatants following incubation of adult worms in vitro, indicating that SmATPDase2 was secreted by the parasite to the medium. Taken together these data suggest a non-redundant role for SmATPDase2 in the parasite-host interplay.     

The crystal structure of adenylosuccinate lyase from Pyrobaculum aerophilum reveals an intracellular protein with three disulfide bonds

Adenylosuccinate lyase catalyzes two separate reactions in the de novo purine biosynthetic pathway. Through its dual action in this pathway, adenylosuccinate lyase plays an integral part in cellular replication and metabolism. Mutations in the human enzyme can result in severe neurological disorders, including mental retardation with autistic features. The crystal structure of adenylosuccinate lyase from the hyperthermophilic archaebacterium Pyrobaculum aerophilum has been determined to 2.1 A resolution. Although both the fold of the monomer and the architecture of the tetrameric assembly are similar to adenylosuccinate lyase from the thermophilic eubacterium Thermotoga maritima, the archaebacterial lyase contains unique features. Surprisingly, the structure of adenylosuccinate lyase from P. aerophilum reveals that this intracellular protein contains three disulfide bonds that contribute significantly to its stability against thermal and chemical denaturation. The observation of multiple disulfide bonds in the recombinant form of the enzyme suggests the need for further investigations into whether the intracellular environment of P. aerophilum, and possibly other hyperthermophiles, may be compatible with protein disulfide bond formation. In addition, the protein is shorter in P. aerophilum than it is in other organisms. This abbreviation results from an internal excision of a cluster of helices that may be involved in protein-protein interactions in other organisms and may relate to the observed clinical effects of human mutations in that region.     

Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli

Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine. The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238. This paper reports the conditions for hyperexpression of the recombinant protein in E. coli BL21(DE3) and purification of the protein to homogeneity. The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity. Glycerol and EDTA were found to stabilize enzyme activity during storage. The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg. The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively. The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength.     

Cloning and expression of isocitrate lyase from human round worm Strongyloides stercoralis

A full length cDNA (1463 bp) encoding isocitrate lyase (EC 4.1.3.1) of Strongyloides stercoralis is described. The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase. The conceptually translated amino acid sequence of the open reading frame for S. stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39. The sequence is 69% A/T, reflecting a characteristic A/T codon bias of S. stercoralis. The amino acid sequence of S. stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus. The full length cDNA of S. stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system. S. stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels. The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S. stercoralis is also discussed.     

Enzymes of Purine Metabolism in Mycoplasma mycoides subsp. mycoides

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides were proposed previously from studies of its usage of radioactive purines and pyrimidines. To interpret more fully the pattern of purine usage, we have assayed cell-free extracts of this organism for several enzymes associated with the salvage synthesis of purine nucleotides. M. mycoides possessed phosphoribosyltransferases for adenine, guanine, and hypoxanthine, purine nucleoside phosphorylase, GMP reductase, GMP kinase, adenylosuccinate synthetase, and adenylosuccinate lyase. Purine nucleoside kinase and adenosine deaminase were not detected. Examination of kinetic properties and regulation of some of the above enzymes revealed differences between M. mycoides and Escherichia coli. Most notable of these were the greater susceptibility of the enzymes from M. mycoides to inhibition by nucleotides and the more widespread involvement of GMP as an inhibitor. Observations on enzyme activities in vitro allow an adequate explanation of the capacity of guanine to provide M. mycoides with its full requirement for purine nucleotides.     

Cloning and gene expression of Schistosoma mansoni protease

Schistosomes utilize proteases (termed hemoglobinases) for degradation of host globin. cDNA clones encoding Schistosoma mansoni protease were isolated by immunologically screening an expression cDNA library with antisera raised against purified hemoglobinase. Confirmation of the identities of the clones was obtained immunologically and biochemically. The bacterially produced fusion protein encoded by one clone, lambda Hb2, degraded hemoglobin in vitro. The sequence of this clone suggested that this S. mansoni protease is synthesized in a precursor form in vivo. Gene titrations indicated that S. mansoni contains multiple genes corresponding to this cDNA. The expression of these genes may be regulated during the organism's life cycle since adult, female worms contained the highest abundances of homologous mRNA and protein compared to other stages.     

Schistosoma mansoni tropomyosin: cDNA characterization, sequence, expression, and gene product localization

We have defined the polypeptide pattern of 3-hr Schistosoma mansoni schistosomula on nonequilibrium two-dimensional gels (NEPHGE). An acidic group of polypeptides with a molecular weight of about 40 kDa and a pI value of around 5.0 (numbered 48/59/53) were identified as antigens on Western blots probed with chronic human infection sera or vaccinated mouse sera. Polypeptides 48/49/53 from silver-stained NEPHGE gels produced antisera that were specific as demonstrated by Western blot analysis and immunoprecipitations of in vitro translation products. A cDNA clone (clone 1) from a S. mansoni adult worm pBR322 library was isolated by using cDNA probes made from size-fractionated mRNA and defined as encoding polypeptide 49 by hybridization selection of the mRNA which was in vitro translated and immunoprecipitated with specific mouse antiserum. A lambda gt 11 expression clone which contained an insert close to the full length mRNA was isolated from a S. mansoni cercariae library. The complete sequence of the mRNA was determined by sequencing the insert of this clone as well as primer extension of total RNA. The only open reading frame coding for 284 amino acids in the 1316 nucleotide sequence showed a 44.76 to 55.44% homology with the amino acid sequences of 18 different tropomyosins from various species. Computer-predicted secondary structure of schistosome tropomyosin was mainly alpha-helix which was very similar to other tropomyosins. Northern analysis showed the mRNA to be about 1.5 kb in size and detectable at much higher levels in the adult worm stage as compared to the cercariae and the egg stages. Western blot analysis likewise showed that greater amounts of tropomyosin were detected in extracts from adult worm stage as compared to extracts from cercariae and egg stages. Immunocytochemical analysis shows that tropomyosin is strongly associated with the tegument of adult worms. The restriction digestion pattern given by genomic Southern analysis suggests the existence of introns and/or multiple gene copies. Thus polypeptide 49, an immunodominant antigen, represents schistosome tropomyosin.