Schistosome Enzyme Research Articles

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni.

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

The schistosome glutathione S-transferase P28GST, a unique helminth protein, prevents intestinal inflammation in experimental colitis through a Th2-type response with mucosal eosinophils.

Intestinal helminth parasites are potent inducers of T helper type 2 (Th2) response and have a regulatory role, notably on intestinal inflammation. As infection with schistosomes is unlikely to provide a reliable treatment of inflammatory bowel diseases, we have investigated the beneficial effect of a schistosome enzymatic protein, the 28-kDa glutathione S-transferase (P28GST), on the modulation of disease activity and immune responses in experimental colitis. Our results showed that immunization with recombinant P28GST is at least as efficient as established schistosome infection to reduce colitis lesions and expression of pro-inflammatory cytokines. Considering underlying mechanisms, the decrease of inflammatory parameters was associated with the polarization of the immune system toward a Th2 profile, with local and systemic increases of interleukin (IL)-13 and IL-5. Dense eosinophil infiltration was observed in the colons of P28GST-immunized rats and mice. Depletion of eosinophils by treatment with an anti-Siglec-F monoclonal antibody and use of IL-5-deficient mice led to the loss of therapeutic effect, suggesting the crucial role for eosinophils in colitis prevention by P28GST. These findings reveal that immunization with P28GST, a unique recombinant schistosome enzyme, ameliorates intestinal inflammation through eosinophil-dependent modulation of harmful type 1 responses, representing a new immuno-regulatory strategy against inflammatory bowel diseases.

The schistosome enzyme that activates oxamniquine has the characteristics of a sulfotransferase

Available evidence suggests that the antischistosomal drug oxamniquine is converted to a reactive ester by a schistosome enzyme that is missing in drug-resistant parasites. This study presents data supporting the idea that the active ester is a sulfate and the activating enzyme is a sulfotransferase. Evidence comes from the fact that the parasite extract loses its activating capability upon dialysis, implying the requirement of some dialyzable cofactor. The addition of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) restored activity of the dialyzate, a strong indication that a sulfotransferase is probably involved. Classical sulfotransferase substrates like beta-estradiol and quercetin competitively inhibited the activation of oxamniquine. Furthermore, these substrates could be sulfonated in vitro using an extract of sensitive (but not resistant) schistosomes. Gel filtration analysis showed that the activating factor eluted in a fraction corresponding to a molecular mass of about 32 kDa, which is the average size of typical sulfotransferase subunits. Ion exchange and affinity chromatography confirmed the sulfotransferase nature of the enzyme. Putative sulfotransferases present in schistosome databases are being examined for their possible role as oxamniquine activators.

Cellular responses to Schistosoma japonicum cathepsin D aspartic protease.

Lymphocyte proliferation and cytokine production were measured in groups of mice vaccinated (but not subsequently challenge infected) with recombinant forms of Schistosoma japonicum cathepsin D aspartic protease, rSjASP1 (expressed in bacteria; enzymatically inactive) and rSjASP2 (expressed in insect cells; enzymatically active). Both forms of the schistosome enzyme induced significant proliferation of splenocytes recovered from vaccinated mice, and expression of interferon (IFN)-gamma, interleukin (IL)-4 and IL-10 mRNA in these cells was detected using reverse transcriptase-polymerase chain reaction. Secretion of IFN-gamma, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using enzyme-linked immunosorbent assay. IFN-gamma was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order. These findings indicated that vaccination of mice with the schistosome protease induces a mixed Th1/Th2 cytokine response, which may explain the modest level of protection after challenge infection in cathepsin D-vaccinated mice, reported previously.

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 μM, 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 (C 80–C 105). 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.

Enzymatic basis for the lack of oxamniquine activity in Schistosoma haematobium infections.

The notion that oxamniquine is active against Schistosoma mansoni but inactive against S. haematobium was confirmed using in vitro cultures of adult worms. Since oxamniquine and hycanthone have been shown to become effective upon activation by a schistosome enzyme, enzymatic tests were carried out to detect possible differences between the enzyme of S. mansoni and that of S. haematobium. It was found that the S. mansoni enzyme could activate hycanthone and, to a lesser extent, oxamniquine. The S. haematobium enzyme, on the other hand, was capable of activating hycanthone but virtually incapable of activating oxamniquine. It is concluded that the different activity of oxamniquine in the two species is due to differences in the drug-activating enzyme.

Identification of the active sites of human and schistosomal hypoxanthine-guanine phosphoribosyltransferases by GMP-2',3'-dialdehyde affinity labeling.

Labeling of human and schistosomal hypoxanthine-guanine phosphoribosyltransferases (HGPRTases) with GMP-2',3'-dialdehyde (ox-GMP) results in nearly complete inactivation of the enzymes. Digestion of the [3H]ox-GMP-modified HGPRTases with trypsin followed by high-performance liquid chromatographic fractionation, partial amino acid sequencing, and mass spectral analysis of the labeled peptides revealed that four peptides from each of the two HGPRTases were labeled with ox-GMP. The conclusion from these studies indicates that two segments of the human enzyme protein, Ser 4-Arg 47 and Ser 91-Arg 100, and one region in the schistosomal enzyme, Gly 95-Lys 133, were labeled by ox-GMP. Since the ox-GMP labeling of human HGPRTase was effectively blocked by either GMP or PRibPP, whereas that of schistosomal HGPRTase was inhibited only by GMP [Kanaaneh, J., Craig, S. P., III, & Wang, C. C. (1994) Eur. J. Biochem. 223, 595-601], the two labeled peptides in human enzyme may be involved in binding to both GMP and PRibPP while the one peptide in schistosomal enzyme may be implicated only in GMP binding. We have also confirmed a previous observation [Keough, D. T., Emmerson, B. T., & de Jersey, J. (1991) Biochim. Biophys. Acta 1096, 95-100] that carboxymethylation of Cys 22 in the human HGPRTase by iodoacetate was inhibited by PRibPP. We also demonstrated that the carboxymethylation of Cys 25 in schistosomal HGPRTase by iodoacetate was specifically blocked by PRibPP. Apparently, the N-terminal regions in both enzymes are involved in PRibPP binding. The fact that ox-GMP labels the N-terminal region in human enzyme but not in schistosomal enzyme and that PRibPP protects against ox-GMP labeling in human enzyme but not in schistosomal enzyme both suggest that the amino-terminal PRibPP-binding site may be in close proximity to the GMP-binding site in human HGPRTase but not in schistosomal HGPRTase. This clear distinction between the active sites of human and schistosomal HGPRTases could be further exploited for potential opportunities for antischistosomal chemotherapy.

Differential inhibitory effects of GMP-2',3'-dialdehyde on human and schistosomal hypoxanthine-guanine phosphoribosyltransferases.

The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of human and the parasitic trematode, Schistosoma mansoni, were expressed at high levels in transformed Escherichia coli in their native forms. Guanosine 2',3'-dialdehyde 5'-phosphate (ox-GMP) was shown to bind irreversibly to both enzymes in a time-dependent manner. This binding was stabilized by sodium borohydride reduction, suggesting that a Schiff's base is formed between the dialdehyde groups of ox-GMP and the amino group of a lysine residue in the enzymes. This linkage formation applies also to inosine 2',3'-dialdehyde 5'-phosphate but not to adenosine 2',3'-dialdehyde 5'-phosphate. GMP was found to be protective against ox-GMP inactivation and [3H]ox-GMP labeling of both HGPRTases. 5-Phosphoribosyl-1-diphosphate (PRibPP) also protects human HGPRTase against the ox-GMP inactivation and [3H]ox-GMP labeling but provides virtually no protection against the ox-GMP inactivation and labeling of the schistosomal enzyme, even though PRibPP binds to the latter with a threefold higher affinity. These results imply that PRibPP and ox-GMP compete with each other for binding to the human HGPRTase but not for binding to the schistosomal enzyme. This discrepancy could be exploited for the purpose of designing selective inhibitors of the schistosomal HGPRTase. Guanosine 2',3'-dialdehyde (ox-guanosine) is nearly as active as ox-GMP in inhibiting schistosomal HGPRTase but much less potent in inhibiting human HGPRTase, suggesting that ox-guanosine and ox-GMP may bind equally well to the parasite enzyme. PRibPP can protect human but not schistosomal HGPRTase against the inactivation by ox-guanosine. Therefore, ox-GMP and ox-guanosine must be forming Schiff's bases with the same amino acid residues in each of the two HGPRTases.

Comparing the human and schistosomal hypoxanthine-guanine phosphoribosyltransferases by circular dichroism

The hypoxanthine-guanine phosphoribosyltransferases (HGPRTases) of human and the parasitic trematode, Schistosoma mansoni, are of biomedical importance. The conformations of these two enzymes were studied by circular dichroism (CD). The schistosomal HGPRTase is estimated to contain 27% α-helix and 30% β-structure. This result is consistent with what is predicted from a tertiary model (Craig, S.P., Cohen, F.E., Yuan, L., McKerrow, J.H. and Wang, C.C. (1991) in Molecular & Immunological Aspects of Parasitism (Wang, C.C., ed.), pp. 122–138, Am. Assoc. Adv. Sci., Washington DC, USA), which proposes that the enzyme is an alpha/beta barrel protein. The human enzyme is estimated to contain 21% α-helix and 53% β-form. The two enzymes are different in their thermostability. The human enzyme remains active after being heated to 85°C for 15 min, while the schistosomal enzyme only retains its activity at temperature below 65°C. The transition temperature (T1/2) of the schistosomal HGPRTase was determined by CD measurement to be 57.5°C. One of the enzyme substrates, phosphoribose pyrophosphate (PRPP), stabilizes the HGPRTases by preventing the human enzyme from unfolding at 85°C and partially protecting the schistosomal enzyme from unfolding at 65°C. It is suggested that the amino-acid substitutions in the human enzyme improve the spatial structure and stability of its α-helices, which may lead to an enhanced thermostability.

A protective monoclonal antibody specifically recognizes and alters the catalytic activity of schistosome triose-phosphate isomerase.

mAb M.1 was previously shown to recognize a 28-kDa Ag in all stages of the human helminth parasite, Schistosoma mansoni, and to bind to the surface membranes of newly transformed schistosomula in a transient manner. Here we demonstrate that M.1 passively transfers partial resistance (41-49%) to cercarial challenge in naive mice. Thus, the 28-kDa Ag recognized by M.1 is a putative vaccine candidate. After immunoaffinity purification, tryptic digests of the 28-kDa Ag were prepared and individual peptides were sequenced. Amino terminus sequences of tryptic peptides of the 28-kDa Ag had high (79-87%) sequence homology with the mammalian glycolytic/gluconeogenic enzyme triosephosphate isomerase (TPI). Purified, native 28-kDa Ag from adult parasites was shown to function enzymatically in an analogous manner to yeast and mammalian TPI in the reverse reaction. Addition of M.1 antibody to the enzyme reaction altered the catalytic activity of schistosome TPI. To determine the immunologic cross-reactivity of this vaccine candidate with mammalian TPI, Western blot analysis was performed and demonstrated that M.1 was immunologically specific for the schistosome enzyme.

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 K m values similar to the rat enzyme for HMG-CoA and NADPH. The K i value for mevinolin, a potent and selective inhibitor of the rat reductase ( K i = 0.6 n M), was significantly higher ( K i = 46 n M) 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.

Acetylcholinesterase from Schistosoma mansoni: immunological characterization

The enzyme acetylcholinesterase (AChE) is present in the trematode Schistosoma mansoni, which infects humans and causes a severe disease called schistosomiasis or Bilharzia. We have purified this enzyme and raised polyclonal antibodies against it. The specificity of these antibodies against the schistosome enzyme was demonstrated by their capacity to precipitate exclusively AChE activity from cercariae extract and to recognize the 8S molecular form of the parasite's AChE. On the other hand, they did not cross-react at all with AChE from human erythrocytes. By employing immunogold electron microscopy, AChE was located on the surface, in the membranal bodies of the tegument and in the muscles of schistosomula. The antibodies raised against the purified AChE of S. mansoni are of protective value, as they led to efficient complement-mediated killing of schistosomula in vitro. It was also demonstrated that antibodies specific towards S. mansoni AChE are present in the sera of mice and of human patients infected with the parasite, suggesting that this enzyme partakes in the immune response towards the parasite during infection. These cumulative data, particularly the schistosomicidal activity of the antibodies and their lack of cross-reactivity with human AChE, are of significance in the consideration of the S. mansoni AChE for vaccination purposes.

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.

Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni

This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG 1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complement-dependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule.

Hemoglobinolytic activity of serum in mice infected with Schistosoma mansoni.

Sera from mice infected with Schistosoma mansoni were found to contain substantial amounts of an acid-active hemoglobinolytic enzyme. Recovery of this enzyme from aliquots of pooled 12-week infected serum, using a phenylalanine-agarose affinity column, showed that a portion of the enzyme binds tightly to the column at pH 4.0, and can be eluted with 0.01 M formic acid. Another larger portion of hemoglobin-digesting activity is not bound to the column and emerges with the buffer front. Sera from rats which were exposed to cercariae, but in whom worms were stunted and did not develop to maturity, were found not to contain hemoglobin-active protease. At the present time, the source of the enzyme has not been unequivocably proven. The enzyme found in the serum binds to and releases from the affinity column in the same manner as does hemoglobinase recovered from freeze-dried S. mansoni worms. Maximal activity of both enzymes against the substrate occurs at pH 4.5-5.0. Present evidence suggests that the protease present in the serum is of worm origin. It is postulated that this protein may be excreted by the parasite during the process of regurgitation of gut contents. Presence of worm enzyme circulating in the host plasma would correlate with the known sensitization of the host to schistosomal hemoglobin-digesting enzyme.

Species specificity of the immediate hypersensitivity to schistosomal proteolytic enzyme

An acidic proteolytic enzyme which digests host haemoglobin can be isolated and purified from schistosomes. This small glycoprotein is an allergen which sensitizes the host, as shown by immediate hypersensitivity reactions. These are specific for either Schistosoma haematobium or S. mansoni and can be demonstrated by mast cell degranulation in mice or by intradermal skin tests in monkeys. Although high levels of total IgE may be found in acute and chronic schistosomiasis, there was no evident relationship between the worm burden in monkeys and immediate hypersensitivity reactions to either purified enzyme or crude schistosomal extracts. It is suggested that an in vivo correlation between worm burden and manifestations of the allergic response may be perturbed by high titres of non-specific IgE or other homocytotropic antibodies, thus accounting for false negative skin test reactions. Alternatively, a return to low or subnormal IgE levels may allow the restoration of the allergic response, giving rise to false positive reactions. Purified schistosomal antigens offer certain advantages over crude skin test preparations in terms of uniformity of antigen content, dosage and specificity. In addition, the enzyme may represent a species-specific tool for new immunochemical analyses of schistosomiasis.