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Developmentally regulated expression, alternative splicing and distinct sub-groupings in members of the Schistosoma mansoni venom allergen-like (SmVAL) gene family.

Chalmers IW, McArdle AJ, Coulson RM, Wagner MA, Schmid R, Hirai H, Hoffmann KF - BMC Genomics (2008)

Bottom Line: The Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain is found across phyla and is a major structural feature of insect allergens, mammalian sperm proteins and parasitic nematode secreted molecules.Analysis of SmVAL6 transcript diversity demonstrated statistically significant, developmentally regulated, alternative splicing.Our results highlight the existence of two distinct SCP/TAPS protein types within the Platyhelminthes and across taxa.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK. iwc21@cam.ac.uk

ABSTRACT

Background: The Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain is found across phyla and is a major structural feature of insect allergens, mammalian sperm proteins and parasitic nematode secreted molecules. Proteins containing this domain are implicated in diverse biological activities and may be important for chronic host/parasite interactions.

Results: We report the first description of an SCP/TAPS gene family (Schistosoma mansoni venom allergen-like (SmVALs)) in the medically important Platyhelminthes (class Trematoda) and describe individual members' phylogenetic relationships, genomic organization and life cycle expression profiles. Twenty-eight SmVALs with complete SCP/TAPS domains were identified and comparison of their predicted protein features and gene structures indicated the presence of two distinct sub-families (group 1 & group 2). Phylogenetic analysis demonstrated that this group 1/group 2 split is zoologically widespread as it exists across the metazoan sub-kingdom. Chromosomal localisation and PCR analysis, coupled to inspection of the current S. mansoni genomic assembly, revealed that many of the SmVAL genes are spatially linked throughout the genome. Quantitative lifecycle expression profiling demonstrated distinct SmVAL expression patterns, including transcripts specifically associated with lifestages involved in definitive host invasion, transcripts restricted to lifestages involved in the invasion of the intermediate host and transcripts ubiquitously expressed. Analysis of SmVAL6 transcript diversity demonstrated statistically significant, developmentally regulated, alternative splicing.

Conclusion: Our results highlight the existence of two distinct SCP/TAPS protein types within the Platyhelminthes and across taxa. The extensive lifecycle expression analysis indicates several SmVAL transcripts are upregulated in infective stages of the parasite, suggesting that these particular protein products may be linked to the establishment of chronic host/parasite interactions.

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SmVAL6 exhibits developmentally-regulated alternative splicing . Sixty-seven individual SmVAL6 cDNA clones (spanning exons 4–38) were isolated and sequenced from parasite material: 32 clones were derived from 7-week mixed-sex adult worm cDNA and 35 clones from mixed-sex cercarial cDNA (see Methods). A) Thirty-five distinct SmVAL6 isoforms were identified from the sixty-seven clones sequenced. Columns represent exon number (as described in Fig. 5) and rows indicate the 35 detected isoforms. Filled, grey boxes represent presence of exon, whereas empty, white boxes represent absence of exon in each of the 35 isoforms. The presence of an asterisk indicates an exon encoding a premature stop codon. The diagonal lines in the exon 35 column indicate that this exon has been detected in previous studies but was not observed in any of the transcripts in this experiment. B) Presence of exons 20 and 26 is associated with adult cDNA clones. Frequency of exon 20 and 26 usage in adult (black bars) and cercariae clones (grey bars) is shown with the χ2 test p values indicated. The amino acids encoded by each exon are shown below, with amino acids spanning splice acceptor/donator sites indicated in parentheses.
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Figure 8: SmVAL6 exhibits developmentally-regulated alternative splicing . Sixty-seven individual SmVAL6 cDNA clones (spanning exons 4–38) were isolated and sequenced from parasite material: 32 clones were derived from 7-week mixed-sex adult worm cDNA and 35 clones from mixed-sex cercarial cDNA (see Methods). A) Thirty-five distinct SmVAL6 isoforms were identified from the sixty-seven clones sequenced. Columns represent exon number (as described in Fig. 5) and rows indicate the 35 detected isoforms. Filled, grey boxes represent presence of exon, whereas empty, white boxes represent absence of exon in each of the 35 isoforms. The presence of an asterisk indicates an exon encoding a premature stop codon. The diagonal lines in the exon 35 column indicate that this exon has been detected in previous studies but was not observed in any of the transcripts in this experiment. B) Presence of exons 20 and 26 is associated with adult cDNA clones. Frequency of exon 20 and 26 usage in adult (black bars) and cercariae clones (grey bars) is shown with the χ2 test p values indicated. The amino acids encoded by each exon are shown below, with amino acids spanning splice acceptor/donator sites indicated in parentheses.

Mentions: In the process of cloning SmVAL6, and likely due to its genomic complexity (Fig. 5), many different individual full-length ORFs were identified. Interestingly, all sequence variability was limited to the region C-terminal to the SCP/TAPS domain. We used primers (indicated in Fig. 5) designed to amplify this specific region, both to further examine the level of SmVAL6 variation and to compare the diversity of SmVAL6 products expressed in different developmental stages. Analysis of restriction digests from SmVAL6 PCR amplicons throughout the life cycle showed that the greatest qualitative differences in RFLP (restriction fragment length polymorphism) patterns existed between cercarial and 7-week adult worms (data not shown). Therefore, clones derived from cercarial and adult 7-week mixed-sex cDNA were subsequently isolated and sequenced. Analysis of the randomly-selected clones, 32 from 7-week adult worm samples and 35 from cercarial samples, showed that the SmVAL6 transcript is highly variable in both developmental stages (Fig. 8). From the 67 clones sequenced, 35 separate isoforms were observed (Fig. 8A). The variation observed was found to be due either to the absence/presence of exons or alternative 3' splicing within an exon with none of the single nucleotide polymorphisms detected shared by more than one clone. No isoform was predominant; the most abundant isoform accounting for only 7 clones (data not shown). To represent the different isoforms identified in this study, each exon found in the SmVAL6 gene was numbered (according to Fig. 5) and each isoform was scored for the presence or absence of each exon (Fig. 8A). Using the χ2 test, statistically significant developmental regulation of exon expression was found for two exons – 20 (p < 0.001) and 26 (p < 0.001) (Fig. 8B). Interestingly, these two exons were found specifically associated with SmVAL6 transcripts expressed by the adult 7-week life stage and encode very similar amino acid pentamers (KDDQY & KDEQY, respectively) (Fig 8B).


Developmentally regulated expression, alternative splicing and distinct sub-groupings in members of the Schistosoma mansoni venom allergen-like (SmVAL) gene family.

Chalmers IW, McArdle AJ, Coulson RM, Wagner MA, Schmid R, Hirai H, Hoffmann KF - BMC Genomics (2008)

SmVAL6 exhibits developmentally-regulated alternative splicing . Sixty-seven individual SmVAL6 cDNA clones (spanning exons 4–38) were isolated and sequenced from parasite material: 32 clones were derived from 7-week mixed-sex adult worm cDNA and 35 clones from mixed-sex cercarial cDNA (see Methods). A) Thirty-five distinct SmVAL6 isoforms were identified from the sixty-seven clones sequenced. Columns represent exon number (as described in Fig. 5) and rows indicate the 35 detected isoforms. Filled, grey boxes represent presence of exon, whereas empty, white boxes represent absence of exon in each of the 35 isoforms. The presence of an asterisk indicates an exon encoding a premature stop codon. The diagonal lines in the exon 35 column indicate that this exon has been detected in previous studies but was not observed in any of the transcripts in this experiment. B) Presence of exons 20 and 26 is associated with adult cDNA clones. Frequency of exon 20 and 26 usage in adult (black bars) and cercariae clones (grey bars) is shown with the χ2 test p values indicated. The amino acids encoded by each exon are shown below, with amino acids spanning splice acceptor/donator sites indicated in parentheses.
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Figure 8: SmVAL6 exhibits developmentally-regulated alternative splicing . Sixty-seven individual SmVAL6 cDNA clones (spanning exons 4–38) were isolated and sequenced from parasite material: 32 clones were derived from 7-week mixed-sex adult worm cDNA and 35 clones from mixed-sex cercarial cDNA (see Methods). A) Thirty-five distinct SmVAL6 isoforms were identified from the sixty-seven clones sequenced. Columns represent exon number (as described in Fig. 5) and rows indicate the 35 detected isoforms. Filled, grey boxes represent presence of exon, whereas empty, white boxes represent absence of exon in each of the 35 isoforms. The presence of an asterisk indicates an exon encoding a premature stop codon. The diagonal lines in the exon 35 column indicate that this exon has been detected in previous studies but was not observed in any of the transcripts in this experiment. B) Presence of exons 20 and 26 is associated with adult cDNA clones. Frequency of exon 20 and 26 usage in adult (black bars) and cercariae clones (grey bars) is shown with the χ2 test p values indicated. The amino acids encoded by each exon are shown below, with amino acids spanning splice acceptor/donator sites indicated in parentheses.
Mentions: In the process of cloning SmVAL6, and likely due to its genomic complexity (Fig. 5), many different individual full-length ORFs were identified. Interestingly, all sequence variability was limited to the region C-terminal to the SCP/TAPS domain. We used primers (indicated in Fig. 5) designed to amplify this specific region, both to further examine the level of SmVAL6 variation and to compare the diversity of SmVAL6 products expressed in different developmental stages. Analysis of restriction digests from SmVAL6 PCR amplicons throughout the life cycle showed that the greatest qualitative differences in RFLP (restriction fragment length polymorphism) patterns existed between cercarial and 7-week adult worms (data not shown). Therefore, clones derived from cercarial and adult 7-week mixed-sex cDNA were subsequently isolated and sequenced. Analysis of the randomly-selected clones, 32 from 7-week adult worm samples and 35 from cercarial samples, showed that the SmVAL6 transcript is highly variable in both developmental stages (Fig. 8). From the 67 clones sequenced, 35 separate isoforms were observed (Fig. 8A). The variation observed was found to be due either to the absence/presence of exons or alternative 3' splicing within an exon with none of the single nucleotide polymorphisms detected shared by more than one clone. No isoform was predominant; the most abundant isoform accounting for only 7 clones (data not shown). To represent the different isoforms identified in this study, each exon found in the SmVAL6 gene was numbered (according to Fig. 5) and each isoform was scored for the presence or absence of each exon (Fig. 8A). Using the χ2 test, statistically significant developmental regulation of exon expression was found for two exons – 20 (p < 0.001) and 26 (p < 0.001) (Fig. 8B). Interestingly, these two exons were found specifically associated with SmVAL6 transcripts expressed by the adult 7-week life stage and encode very similar amino acid pentamers (KDDQY & KDEQY, respectively) (Fig 8B).

Bottom Line: The Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain is found across phyla and is a major structural feature of insect allergens, mammalian sperm proteins and parasitic nematode secreted molecules.Analysis of SmVAL6 transcript diversity demonstrated statistically significant, developmentally regulated, alternative splicing.Our results highlight the existence of two distinct SCP/TAPS protein types within the Platyhelminthes and across taxa.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK. iwc21@cam.ac.uk

ABSTRACT

Background: The Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain is found across phyla and is a major structural feature of insect allergens, mammalian sperm proteins and parasitic nematode secreted molecules. Proteins containing this domain are implicated in diverse biological activities and may be important for chronic host/parasite interactions.

Results: We report the first description of an SCP/TAPS gene family (Schistosoma mansoni venom allergen-like (SmVALs)) in the medically important Platyhelminthes (class Trematoda) and describe individual members' phylogenetic relationships, genomic organization and life cycle expression profiles. Twenty-eight SmVALs with complete SCP/TAPS domains were identified and comparison of their predicted protein features and gene structures indicated the presence of two distinct sub-families (group 1 & group 2). Phylogenetic analysis demonstrated that this group 1/group 2 split is zoologically widespread as it exists across the metazoan sub-kingdom. Chromosomal localisation and PCR analysis, coupled to inspection of the current S. mansoni genomic assembly, revealed that many of the SmVAL genes are spatially linked throughout the genome. Quantitative lifecycle expression profiling demonstrated distinct SmVAL expression patterns, including transcripts specifically associated with lifestages involved in definitive host invasion, transcripts restricted to lifestages involved in the invasion of the intermediate host and transcripts ubiquitously expressed. Analysis of SmVAL6 transcript diversity demonstrated statistically significant, developmentally regulated, alternative splicing.

Conclusion: Our results highlight the existence of two distinct SCP/TAPS protein types within the Platyhelminthes and across taxa. The extensive lifecycle expression analysis indicates several SmVAL transcripts are upregulated in infective stages of the parasite, suggesting that these particular protein products may be linked to the establishment of chronic host/parasite interactions.

Show MeSH
Related in: MedlinePlus