Schistosoma mansoni: characterization of the gene encoding Sm23, an integral membrane protein.
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Amino Acid Sequence↗
Biomedical subjects
P T LoVerde
Publications and source records attributed to P T LoVerde.
Schistosoma mansoni: cloning and characterization of a gene encoding cytosolic Cu/Zn superoxide dismutase.
Two forms of Cu/Zn superoxide dismutase (SOD) have been identified in Schistosoma mansoni, signal peptide-containing SOD (SP-SOD, Simurda et al., 1988, Exp. Parasitol. 67, 73-84) and cytosolic SOD (CT-SOD, Hong et al., 1992, Exp. Parasitol. 75, 308-322). The SP-SOD gene has been previously characterized. We report the isolation and characterization of the gene encoding CT-SOD. A cDNA clone of CT-SOD was radiolabeled and used to screen an EMBL3 genomic library. Of 100,000 plaques, 1 hybridizing phage contained an insert of 14.6 kb. The CT-SOD gene spans 8.5 kb of chromosomal DNA and possesses three exons (150, 285, and 234 bp, respectively) and two introns (4.9 and 2.7 kb, respectively) which interrupt the coding region. Separate copies of the gene as determined by restriction fragment patterns on Southern blots are found on two YAC clones. Using YAC clones that contain the CT-SOD gene, fluorescence in situ hybridization on chromosomes identified a copy of the gene located on the middle region of chromosome 1 and another copy localized to the middle region of the long arm of chromosome 3. Primer extension and nuclease protection assay performed on total RNA define the transcription initiation site and allow the identification of potential cis-acting sequences, such as a CAAT box and a hexanucleotide GCCCGG. Comparison of this gene with Cu/Zn SOD genes from Drosophila, humans, and mice identifies conserved exons that represent functional domains in the SOD protein.
Amino Acid Sequence↗
Schistosoma mansoni: cloning the gene encoding glutathione peroxidase.
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Alternative Splicing↗
Yeast artificial chromosome (YAC)-based genome mapping of Schistosoma mansoni.
Schistosoma mansoni has 7 pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm and ZW for a female), of a haploid genome size of 2.7 x 10(8) bp. We initiated the molecular genetic approach for the detailed characterization and understanding of the evolutionary biology of schistosomes. We have constructed a yeast artificial chromosome (YAC)-library with partially digested parasite genomic DNA, and the chromosome location of each insert was detected by fluorescent in situ hybridization (FISH). The library contains > 2283 clones with an average insert size of 358 kb, which represents a 2.6-fold coverage of the genome (> 7.2 x 10(8) bp). 100 randomly selected YAC clones were localized by FISH and found to be distributed widely among all chromosomes. The assembly of 14 YACs distributed almost the whole region of chromosome 3. Generated expressed sequenced tags (ESTs) derived from a unidirectional cDNA library were also used for further characterization of the YAC inserts. These results indicate that an extensive contig assembly of the entire chromosomes and a reasonably detailed gene map should be feasible in the near future.
Animals↗
Cloning of the gene for phosphoglycerate kinase from Schistosoma mansoni and characterization of its gene product.
As molecules on the surface or associated with the outer covering (tegument) of Schistosoma mansoni are a major focus as potential vaccine candidates, affinity purified antibodies which are specific to the tegumental antigens were used to immunoscreen a lambda gt11 S. mansoni cercarial cDNA library. One of the identified clones was found to encode the glycolytic enzyme phosphoglycerate kinase (PGK, EC 2.7.2.3). The 1.5-kb cDNA clone has a single open reading frame encoding 416 amino acids and exhibits over 60% identity to PGKs from a number of eukaryotic species. Recombinant S. mansoni PGK (SmPGK) was overexpressed in Escherichia coli, purified, and shown to have PGK enzyme activity. Native protein affinity purified from S. mansoni adult worms was shown by microsequencing to have the same amino-acid sequence as deduced from the cDNA sequence, thus confirming the cDNA clone we identified encodes S. mansoni phosphoglycerate kinase. Antibodies localize the native SmPGK to various tissues including the tegument of 3-h schistosomula and 42-day adult worms.
Amino Acid Sequence↗
FISH techniques for constructing physical maps on schistosome chromosomes.
In an effort to provide useful information about parasites important in tropical diseases, the WHO has initiated genome mapping projects for a number of parasites. One goal of this effort is to establish physical maps of the genomes of the targeted parasites. Multicellular parasites (helminths) contain various numbers of chromosomes, some large, that condense during the cell cycle. Here Hirohisa Hirai and Phil LoVerde present details of fluorescence in situ hybridization as a means to localize genes and DNA fragments to schistosome chromosomes. Although the techniques presented are for schistosome chromosomes, they are applicable to any system where the chromosomes condense at metaphase.
Journal Article↗
Immune response to Schistosoma mansoni phosphoglycerate kinase during natural and experimental infection: identification of a schistosome-specific B-cell epitope.
A cDNA clone encoding Schistosoma mansoni phosphoglycerate kinase (SmPGK) was previously identified by affinity-purified antibodies which are specific for 3-h-old schistosomula tegumental antigens. Antibodies to the recombinant SmPGK which has enzymatic activity were localized to various tissues including the tegument of the 3-h-old schistosomula and 42-day-old adult worms. In this study, we show that SmPGK is an immunogenic molecule in both natural infection in humans and experimental vaccination in animals. To understand the role that a highly conserved molecule like SmPGK played during schistosome infection, we affinity purified antibodies to SmPGK from patients with chronic schistosomiasis and demonstrated that they did not cross-react with human PGK. However, affinity-purified rabbit anti-SmPGK antibodies did show immunoreactivity to both human PGK and rabbit PGK. Thus, during natural infection antibodies that cross-react with human PGK are not produced; however, as a result of active immunization with an intact conserved molecule, such cross-reacting antibodies are produced. Immunological analysis of cyanogen bromide digests of SmPGK with monoclonal antibodies that recognize SmPGK but not human PGK identifies a B-cell epitope on a 12.2-kDa fragment represented by amino acids 61 to 174.
Animals↗
Invertebrate host-parasite relationships: convergent evolution of a tropomyosin epitope between Schistosoma sp., Fasciola hepatica, and certain pulmonate snails.
Monoclonal antibodies (mAb) directed against Schistosoma mansoni tropomyosin isoform, SMTM (Xu et al. Experimental Parasitology 69, 373-392, 1989), were used to test for cross-reactivity with Biomphalaria glabrata antigens. One mAb (1F10) recognized antigens of 39, 41, and 80 kDa in a snail head/foot antigen preparation but not a hepatopancreas antigen preparation. Another mAb (1C1) cross-reacted with a 39-kDa antigen in the head/foot extract but not in the hepatopancreas extract. Epitope mapping revealed the 1F10 epitope to be between amino acids 135 and 188 of both Bg39 (Dissous et al. Molecular and Biochemical Parasitology 43, 245-256, 1990) and BgTMII (Weston and Kemp, Experimental Parasitology 76, 358-370, 1993), while the 1C1 epitope was located between amino acids 189 and 213 of BgTMII. Various invertebrate species, including members from Trematoda, Pulmonata, Annelida, and Arthropoda, were tested for cross-reactivity with the monoclonal antibodies. While the 1F10 mAb displayed broad invertebrate cross-reactivity, the 1C1 mAb cross-reactivity was restricted to schistosomes, F. hepatica, and the pulmonate snails B. glabrata and Physa sp.
Amino Acid Sequence↗
Schistosoma mansoni: a Cu/Zn superoxide dismutase is glycosylated when expressed in mammalian cells and localizes to a subtegumental region in adult schistosomes.
We previously isolated a gene from Schistosoma mansoni with the capacity to encode a 20-kDa polypeptide that had homology to Cu/Zn superoxide dismutase from 19 other species. The predicted protein sequence contained a hydrophobic N-terminus as well as a site for N-linked glycosylation, suggesting that the protein was a secreted or membrane-associated form of the enzyme. To study this protein further, we first expressed it in a prokaryotic system and used the gene product to make both monoclonal and polyclonal antibodies. We then expressed the protein in CMT3 cells, a monkey kidney cell line, to investigate possible post-translational modifications. Our results demonstrated that the schistosomal protein expressed in CMT3 cells migrated on an SDS-polyacrylamide gel as multiple glycosylated species with molecular masses of 20, 22, and 23 kDa. Glycosylation was inhibited by tunicamycin, resulting in the expression of an unglycosylated product which migrated with a molecular mass of 18 kDa. The CMT3-cell expressed protein eluted from a gel filtration column with a molecular mass larger than 200 kDa, suggesting that it was membrane-associated or bound to a high-molecular-weight component. The product could not be detected in the medium of the CMT3 cell culture and apparently was not secreted. Comparison between the protein expressed in CMT3 cells and that found in adult schistosomes showed that the parasite-derived gene product was also heterogeneous but had different molecular masses (20, 23, and 25 kDa). The protein was localized in frozen sections of adult worms to the subtegumental area as detected by indirect immunofluorescence using monoclonal antibodies. Since the protein was glycosylated but not secreted we suggest it be called signal peptide-containing superoxide dismutase.
Animals↗
Schistosoma mansoni: chromosomal localization of female-specific genes and a female-specific DNA element.
Two genes, p14 and p48, that are each expressed in a female-specific manner in vitelline cells in response to male pairing were each localized by in situ hybridization to chromosome 2. Biotinylated DNA was hybridized to target chromosomes and hybridization was detected using either alkaline phosphatase-labeled avidin or fluorescein-labeled avidin. In addition Southern blots of genomic DNA separated by pulsed-field gradient gel electrophoresis was used to localize the genes to large chromosome fragments. p14 was localized to two EagI fragments (150 and 240 kb) found in the distal portion of the lower (q) arm of chromosome 2 in 79 of 109 metaphase preparations examined. p48 was localized to two EagI fragments (50 and 700 kb) found in the proximal portion of the lower arm of chromosome 2 in 52 of 63 metaphase preparations examined. A DNA repeat element, pW1, that hybridizes to genomic DNA from female but not male Schistosoma mansoni was also used as a hybridization probe. In all cells examined the biotinylated pW1 only hybridizes to a euchromatic gap region (eg3) within the large heterochromatin block of the long arm of the W chromosome. This specific hybridization coupled with the lack of chiasma formation in this region of the ZW bivalent (presumably due to heterochromatization) is offered as an explanation for the female-specific hybridization of pW1.
Animals↗
Characteristics of phenol oxidase of Schistosoma mansoni and its functional implications in eggshell synthesis.
It is proposed that the phenol oxidase enzyme of schistosomes and other trematodes has a crucial role in the cross-linking of precursor proteins in the formation of the eggshell. However, to date there is no direct evidence to show that the enzyme catalyzes the reactions necessary for the posttranslational modification of eggshell precursor proteins. In this report we demonstrate that an eggshell precursor protein acts as a substrate for a schistosome fraction that catalyzed the 2 steps in the oxidation of tyrosine. This action of the phenol oxidase-containing worm fraction resulted in the tyrosine-dependent insolubilization and aggregation of the protein, suggesting a role for the enzyme in the posttranslational modification and cross-linking of schistosome eggshell proteins. The enzyme-rich fraction from Schistosoma mansoni catalyzed both steps of the reactions necessary for the conversion of tyrosine residues on putative eggshell precursor protein (p48) to quinones. The parasite fraction also catalyzed the hydroxylation of free L-tyrosine to DOPA (monophenol oxidase activity) and the oxidation of L-DOPA to dopaquinone (diphenol oxidase activity). Both activities of the enzyme are avidly bound to membranous structures, are susceptible to agents known to inhibit a functionally related enzyme, tyrosinase, and may reside on the same protein.
Animals↗
Molecular mimicry of hepatitis B surface antigen by an anti-idiotype-derived synthetic peptide.
Monoclonal antibody 2F10 is an "internal-image" anti-idiotype (anti-id) antibody capable of mimicking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg). By mRNA sequencing and computer-assisted molecular modeling of monoclonal antibody 2F10, we identified a 15-amino acid region of the heavy-chain hypervariable region that has partial residue homology with sequences of the "a" determinant epitopes of HBsAg. We have established that a linear 15-mer peptide from a contiguous region on the anti-id antibody can (i) generate anti-HBsAg-specific antibodies when injected into mice, (ii) prime murine lymph node cells for in vitro HBsAg-specific T-cell proliferative responses, and (iii) stimulate in vitro human CD4+ T cells that were primed in vivo to HBsAg by natural infection with hepatitis B virus or vaccination with a commercially available HBsAg vaccine. Significantly, this peptide could also stimulate CD4+ T cells of human hepatitis B virus carriers. We conclude that a 15-mer peptide derived from the anti-id sequence can duplicate the B- and T-cell stimulatory activity of the intact anti-id antibody and the antigen that is mimicked, HBsAg.
Amino Acid Sequence↗
Schistosoma mansoni: cloning of a complementary DNA encoding a cytosolic Cu/Zn superoxide dismutase and high-yield expression of the enzymatically active gene product in Escherichia coli.
We recently purified a 16-kDa cytosolic Cu/Zn superoxide dismutase (CT Cu/Zn-SOD) from Schistosoma mansoni, a human parasite. Three peptide sequences were obtained, one from the unblocked N-terminal and two from internal peptides which were generated by digestions with trypsin and cyanogen bromide. These sequences were aligned to the corresponding sequences of 19 cytosolic Cu/Zn-SODs from various species. Degenerate oligonucleotides were then designed according to the sequence and the position of each peptide. The oligonucleotides were used to amplify a complete cDNA using the polymerase chain reaction with either adult schistosome total RNA or a cercariae lambda gt11 phage cDNA library as the template. The protein encoded by the cDNA has 153 amino acids with a calculated molecular weight of 15,693. It also has 60-65% homology to 19 cytosolic Cu/Zn-SOD from various species. All of the copper/zinc binding sites and SOD activity sites are conserved. Computer analysis predicts that the Cu/Zn-SOD has a pI value of 6.6, which is very close to the experimental results of IEF analysis (6.0 and 6.3). The entire coding sequence from the cDNA was cloned into a bacterial alkaline phosphatase cytosolic expression vector and a large amount of soluble product was expressed and purified to homogeneity. We compared the bacterially expressed Cu/Zn-SOD with the native enzyme derived from schistosomes and found that they are identical by the following criteria: (1) They focus at the same positions on IEF gels; (2) they form dimers in solution as measured by gel filtration; (3) they have the same unblocked N-terminal sequence; (4) they both are enzymatically active with comparable specific activities. The specific activity of the bacterially derived enzyme was increased somewhat (approximately 10%) by incubation with copper and zinc ions.
Amino Acid Sequence↗
Schistosoma mansoni p48 eggshell protein gene: characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene.
Egg production by worm pairs is a major cause of pathogenesis in schistosomiasis. To further the understanding of female reproductive development, we have isolated and characterized a complete copy of an eggshell protein precursor gene, p48. Sequence analysis reveals that the gene has 3 open reading frames and does not contain an intron. One of the open reading frames, ORF1, encodes a polypeptide of 50 kDa which shows strong homology to insect chorion proteins. Determination of the position of the mRNA cap-site facilitated identification of putative regulatory elements in the 5' upstream region of the gene. Some of these elements (e.g., TCACGT) have been shown to play a role in the regulation of chorion gene expression in insects. p48 mRNA is detectable only in mature female worms and the ability to detect the mRNA coincides temporally with worm pairing. Quantitative comparisons, during female reproductive development, of p48 transcripts to those from another eggshell protein precursor gene, p14, show that the p48 mRNA is significantly less abundant than p14 mRNA. In mature female worms, p48 mRNA can only be detected in vitelline cells. Antibodies made against the polypeptide sequence deduced from ORF1 of the p48 gene recognize a 50-kDa molecule in extracts from mature female worms, but not in extracts from immature females or males.
Amino Acid Sequence↗
Identification and purification of a second form of Cu/Zn superoxide dismutase from Schistosoma mansoni.
Our laboratories previously isolated a putative extracellular or membrane-associated Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene, designated a signal peptide-containing (SP) Cu/Zn-SOD, from Schistosoma mansoni. SOD activity was thus investigated throughout the life cycle of S. mansoni and found in all stages: eggs, miracidia, cercariae, schistosomula, lung-stage worms, and adult worms. The adult worms had the highest SOD activity (53 +/- 9 nitrite units), which was five times higher than that of eggs or miracidia and twice as high as that of 3-h-old mechanically transformed schistosomula. Cu/Zn-SOD constituted over 95% of the total SOD activity found in S. mansoni, compared with that of Mn-SOD. Most of Cu/Zn-SOD specific activity was associated with a detergent-extractable fraction of the parasite. Isoelectric focusing gel electrophoresis analysis revealed that there were four major pI variants of Cu/Zn-SOD present in the adult worms. Only two of these Cu/Zn-SOD pI variants were present in the 3-h-old mechanically transformed schistosomula. Fast protein liquid chromatography gel filtration fractionation of adult parasite extract was carried out to correlate the SP Cu/Zn-SOD with the SOD activity by using anti-SP Cu/Zn-SOD monoclonal antibodies, which separated the immunoreactive gene product and the SOD activity into different fractions. Quantitative tissue fractionation also revealed a discordant distribution of the gene product compared with that of Cu/Zn-SOD activity. These results indicated the existence of another Cu/Zn-SOD(s) in the parasite. Purification of the Cu/Zn-SOD activity from the adult worms showed that it represented the two lower-pI variants found in both adult worms and 3-h-old schistosomula. Peptide sequence analysis of the purified Cu/Zn-SOD confirmed that there is a second form of Cu/Zn-SOD in the parasite.
Amino Acid Sequence↗
Schistosoma haematobium and S. japonicum: analysis of the ribosomal RNA genes and determination of the "gap" boundaries and sequences.
We have determined the intragenic organization of the rRNA genes of Schistosoma haematobium and S. japonicum and found them to be similar to that of S. mansoni and other eukaryotes. An entire ribosomal repeat approximately 10 kbp in size from each species was isolated as a SalI fragment from a genomic library constructed in bacteriophage lambda. The segments encoding both the small and large rRNAs have been identified using three cloned EcoRI fragments of S. mansoni as probes. There were three EcoRI fragments (4.2, 3.0, 1.6 kbp) from S. haematobium and four EcoRI fragments (4.6, 2.3, 1.7, 1.0 kbp) from S. japonicum. As in a wide variety of organisms within the protostome phyla, the 28S rRNA in schistosomes contains a "gap" which separates it into two fragments. The length of the gap sequence in S. haematobium is 54 bases and it is identical to that in S. mansoni in both length and sequence. However, in S. japonicum the sequence is between 64-67 bases long. In each case, irrespective of the species, the gap is located at the same position within the 28S rRNA. Secondary structures of the gap sequence derived by computer analysis predict a conformation with the minimum free energy that has an UAAU tract in a hairpin loop for S. haematobium and an UAUU tract for S. japonicum.
Animals↗
Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes.
As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum.
Amino Acid Sequence↗
Characterization of a 54-nucleotide gap region in the 28S rRNA gene of Schistosoma mansoni.
We have analyzed 572 bp in the 28S rDNA of the human blood fluke Schistosoma mansoni which correspond to expansion segment 5 of domain IV as defined by Clark et al. for the Xenopus laevis 28S rRNA. S1 nuclease mapping and primer extension analysis comparing this region with the mature 28S rRNA indicate that there are 54 nucleotides present in the 28S rDNA which are absent from the mature rRNA. This defines a gap that creates two 28S rRNA subunits (28S alpha and 28S beta). Comparison of the S. mansoni sequence with rDNAs of other organisms which contain gaps in their 28S rRNA shows that the overall features are conserved except that the S. mansoni gap is less A + T-rich. The conserved features include: (1) the location of the gap within the 28S rRNA; (2) the predicted secondary structure of the gap, containing a stem-loop with a UAAU sequence within the loop; and (3) a conserved CGAAAGGG on the 3' side of the gap.
Animals↗