Cestode VALs – McCrisp proteins

Whilst numerous SCP/TAPS proteins have been identified and characterized in the phylum Nematoda, comparably little is known about SCP/TAPS family members in the other major phylum containing worms of medical importance, the Platyhelminthes. As this review and others have highlighted (Gibbs et al. Reference Gibbs, Roelants and O'Bryan2008; Cantacessi et al. Reference Cantacessi, Campbell, Visser, Geldhof, Nolan, Nisbet, Matthews, Loukas, Hofmann, Otranto, Sternberg and Gasser2009), there is a wide range of naming conventions for SCP/TAPS proteins depending on the species discussed (i.e. PR-1 proteins for plants, ASP proteins for hookworms and CRISP proteins in humans). For this review, and according to our previous naming convention (Chalmers et al. Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008), we have decided to refer to these platyhelminth proteins as the Venom Allergen-Like (VAL) family. The first published report of platyhelminth VAL family members originated from investigations on the cestode Mesocestoides corti – a mouse model for host/cestode relationships (Britos et al. Reference Britos, Lalanne, Castillo, Cota, Senorale and Marin2007). After serendipitously discovering a VAL family member while searching for homeobox containing genes, Britos et al. (Reference Britos, Lalanne, Castillo, Cota, Senorale and Marin2007) amplified 4 different VAL transcripts from the larval parasite life stage (tetrathyridia). Due to strong sequence similarity to human CRISP proteins, the authors named these VAL transcripts McCrisp1–4 (Table 1). Of the 4 M. corti VAL family members, only the full-length sequence of McCrisp2 was determined. Analysing the full-length sequence, the authors were able to determine that McCrisp2 encoded a protein containing a signal peptide with a complete SCP/TAPS domain. Additional in situ hybridization experiments revealed that McCrisp2 expression was focused to the proglottids in adult worms and to the apical region (where the frontal gland develops) in tetrathyridia. This latter observation suggested that cestode VALs could be involved in host/parasite inter-relationships. Indeed, platyhelminth VAL expression in larval secretory glands/secretions has also been discovered in several trematode species (detailed below), further supporting a role for VALs in host interactions.

Table 1. Published findings on platyhelminth venom allergen-like proteins

^a Names of VAL proteins listed in ‘Studies on platyhelminth VALs’ section are as listed in the original publication. For the ‘Platyhelminth VAL proteins identified in global proteomic studies’ section, names are derived from this review's platyhelminth VAL analysis and are listed Supplementary File 1, online version only. Names used in the original publications are present in parentheses.

Trematode VALs – SmVAL, SjVAL and OvVAL proteins

In 2006, a study examining S. mansoni cercarial/schistosomule excretion/secretion (E/S) products by 2-D gel electrophoresis paired with Tandem mass spectrometry (MS/MS) analysis identified 3 VAL proteins (20–25kDA in size) released by in vitro cultured parasites (Curwen et al. Reference Curwen, Ashton, Sundaralingam and Wilson2006). Now named SmVAL4, SmVAL10 and SmVAL18 (previously named SmSCP_a, SmSCP_c and SmSCP_b respectively), these were the first SCP/TAPS family proteins described in a trematode species (Table 1). Further characterization of these VAL family members was hampered at the time of publication due to the incomplete nature of the S. mansoni genome. However, the same research group did discover that SmVAL10 and 18 were glycosylated in a later study (Jang-Lee et al. Reference Jang-Lee, Curwen, Ashton, Tissot, Mathieson, Panico, Dell, Wilson and Haslam2007). In 2008, using the version 4 assembly of the S. mansoni genome as a reference, a comprehensive analysis of the SmVAL family was performed, identifying 28 (SmVAL1–28) genes encoding complete SCP/TAPS domains (Table 1; (Chalmers et al. Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008)). Using a combination of genomic, transcriptomic, phylogenetic and tertiary structure analyses, it was discovered that the SmVAL family contain 2 distinct types of SCP/TAPS proteins. Group 1 SmVALs (SmVAL1, 2, 3, 4, 5, 7, 8, 9, 10, 12, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28) contain signal peptides, 3 conserved disulphide bonds and an extended first loop region, while group 2 SmVALs (SmVAL6, 11, 13, 16 and 17) do not possess these features but do contain other unique elements such as highly conserved histidine and tyrosine residues (i.e. His21-Tyr82 in SmVAL13). It has been postulated that these conserved amino acids help to stabilize the first and third helices of group 2 SCP/TAPS domains by intramolecular hydrogen bond formation (Chalmers, Reference Chalmers2009). Further, multi-species phylogenetic analysis has discovered that group 1 and group 2 proteins were not limited to S. mansoni but are present in all examined species of the Kingdom Animalia (Chalmers, Reference Chalmers2009). Examples of group 2 proteins include Hs-GAPR-1 in humans ((Eberle et al. Reference Eberle, Serrano, Fullekrug, Schlosser, Lehmann, Lottspeich, Kaloyanova, Wieland and Helms2002), CG4270 in Drosophila (Kovalick and Griffin, Reference Kovalick and Griffin2005) and Ss-NIE in nematodes (Ravi et al. Reference Ravi, Ramachandran, Thompson, Andersen and Neva2002). Functionally, several of the group-defining SmVAL characteristics (such as disulphide bonds) suggest different cellular localizations, with group 1 SmVALs likely to be extracellular in nature while group 2 SmVALs are enriched in intracellular compartments (Chalmers et al. Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008). This assertion is now supported by findings derived from several global proteomic studies (see Table 1, (van Balkom et al. Reference van Balkom, van Gestel, Brouwers, Krijgsveld, Tielens, Heck and van Hellemond2005; Curwen et al. Reference Curwen, Ashton, Sundaralingam and Wilson2006; Wu et al. Reference Wu, Sabat, Brown, Zhang, Taft, Peterson, Harms and Yoshino2009)).

Group 1 schistosome VALs

As previously noted, 3 group 1 VAL proteins (SmVAL4, 10 and 18) were discovered during analysis of in vitro cultured cercarial/schistosomule E/S products (Curwen et al. Reference Curwen, Ashton, Sundaralingam and Wilson2006). Importantly, SmVAL4 (the most abundantly expressed of the 3, as determined by normalized spot volume (Curwen et al. Reference Curwen, Ashton, Sundaralingam and Wilson2006)) was also found during an ingenious study in which parasite and host proteins were identified by liquid chromatography coupled with tandem MS (LC-MS/MS) in infection tunnels of human skin experimentally exposed to S. mansoni cercariae (Hansell et al. Reference Hansell, Braschi, Medzihradszky, Sajid, Debnath, Ingram, Lim and McKerrow2008). These collective studies, therefore, confirm that SmVAL4, 10 and 18 are all associated with mammalian host invasion. In an intriguing symmetry, proteomic studies of S. mansoni miracidia/sporocyst E/S products show that a different set of group 1 SmVALs are likely to be involved in molluscan parasitism (Wu et al. Reference Wu, Sabat, Brown, Zhang, Taft, Peterson, Harms and Yoshino2009). Employing an in vitro protocol, which mimics the transformation of free-living miracidia to snail-residing sporocyst life-cycle stages, Wu et al. (Reference Wu, Sabat, Brown, Zhang, Taft, Peterson, Harms and Yoshino2009) collected the E/S products and used 1D gel electrophoreses paired with nano LC-MS/MS to identify the released proteins. Of the 99 proteins identified in this study, 5 group 1 SmVALs were conclusively identified – SmVAL2, 9, 15, 27 and the newly identified SmVAL29 (SchistoGeneDB ID, smp_120670) (Table 1; (Wu et al. Reference Wu, Sabat, Brown, Zhang, Taft, Peterson, Harms and Yoshino2009)). At least 2 other SmVAL proteins were identified in the study but due to the high level of sequence similarity between them (e.g. SmVAL3 and 23, SmVAL26 and 28), it is unclear which SmVAL was detected. Interestingly, several of these SmVALs (SmVAL2, 3, 5 and 9) were also detected in a global proteomic study of egg E/S products, indicating that some group 1 SmVAL proteins may be secreted by both egg and miracidial lifestages (Table 1; (Cass et al. Reference Cass, Johnson, Califf, Xu, Hernandez, Stadecker, Yates and Williams2007)). Further research is required to confirm whether SmVALs are actively secreted from the egg. However, as Mathieson and Wilson (Reference Mathieson and Wilson2010) demonstrated, that at least 1 SmVAL (the SmVAL26/28 isoprotein, Table 1) is present in the fluid released during miracidial hatching but could not be detected in the egg E/S products (Mathieson and Wilson, Reference Mathieson and Wilson2009). Irrespective of whether SmVAL proteins are secreted from the egg or are only released after hatching or damage, the evidence above suggests that human hosts are encountering a complex set of group 1 SmVAL proteins during chronic infection (i.e. SmVAL4, 10 and 18 during cercariae invasion and SmVAL2, 3, 5, 9 26/28 during egg embolization or tissue translocation). It, therefore, remains a high priority to characterize if/how these SmVALs modulate/stimulate the mammalian immune system.

In the Asian schistosome (S. japonicum), initial steps have been made to address these immunological questions by studying how mice respond to the group 1 S. japonicum VAL-1 protein (Table 1; (Chen et al. Reference Chen, Hu, He, Wang, Hu, Wang, Zheng, Yang, Liang, Xu and Yu2010)). Amplified from S. japonicum egg cDNA, the Sj-VAL-1 transcript encodes a protein most closely related to SmVAL15 (58% amino acid (AA) identity). Transcript and immunolocalization studies detected Sj-VAL-1 in both cercariae and eggs, although expression was considerably more pronounced in the egg samples (Table 1; (Chen et al. Reference Chen, Hu, He, Wang, Hu, Wang, Zheng, Yang, Liang, Xu and Yu2010)). Analysis of anti-Sj-VAL-1 antibody responses during a chronic murine infection revealed a Th2 bias with anti-Sj-VAL-1 IgG₁ predominating (IgG₁>IgG_2a) (Chen et al. Reference Chen, Hu, He, Wang, Hu, Wang, Zheng, Yang, Liang, Xu and Yu2010). Due to maximal Sj-VAL-1 production being found in the egg stage, increases in murine anti-Sj-VAL-1 IgG₁ were, unsurprisingly, correlated with the onset of schistosome egg production (5–6 weeks post-infection). Unfortunately, examination of anti-Sj-VAL-1 IgE was not performed in this study, so it is currently unknown whether this allergen-like protein is the target of host IgE responses similar to those found for hookworm Na-ASP-2 (Bethony et al. Reference Bethony, Loukas, Smout, Brooker, Mendez, Plieskatt, Goud, Bottazzi, Zhan, Wang, Williamson, Lustigman, Correa-Oliveira, Xiao and Hotez2005). While no other members of the SjVAL family have yet been examined in detail, evidence of 5 additional SjVAL proteins (in addition to Sj-VAL-1) can be found by searching the Liu et al. (Reference Liu, Lu, Hu, Wang, Cui, Chi, Yan, Wang, Song, Xu, Wang, Zhang, Zhang, Wang, Xue, Brindley, McManus, Yang, Feng, Chen and Han2006) proteomic dataset derived from 5 different S. japonicum life-cycle stages (cercariae, 2-week schistosomula, 6-week mixed sex adult worms, eggs and miracidia; see Table 1) (Liu et al. Reference Liu, Lu, Hu, Wang, Cui, Chi, Yan, Wang, Song, Xu, Wang, Zhang, Zhang, Wang, Xue, Brindley, McManus, Yang, Feng, Chen and Han2006). Outside of the Schistosoma genus, VAL proteins have been experimentally detected in only 1 other trematode species – the human liver fluke Opisthorchis viverrini. Notably, a group 1 VAL protein (GeneBank Accession EL619323) was identified in the proteomic study of E/S products released from adult O. viverrini. This datum suggests that O. viverrini group 1 VALs, similar to Schistosoma VALs, are also present at the mammalian host/adult parasite interface (Table 1; (Mulvenna et al. Reference Mulvenna, Sripa, Brindley, Gorman, Jones, Colgrave, Jones, Nawaratna, Laha, Suttiprapa, Smout and Loukas2010)).

Group 2 schistosome VALs

Whilst there is growing evidence that many group 1 VAL proteins are associated with parasite secretions in trematode species (e.g. S. mansoni, S. japonicum and O. viverrini), information related to group 2 VAL proteins is sparse. The one exception is the highly unusual SmVAL6 – a group 2 SmVAL expressed throughout the mammalian S. mansoni lifestages (cercariae through adult, (Chalmers et al. Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008)). While other group 1 and group 2 SmVAL family members possess very few amino acids outside of the SCP/TAPS domain, SmVAL6 contains a C-terminal region of variable length and sequence (40–295AA) with no similarity to any characterized protein. Examination of the SmVAL6 gene revealed a complex structure of 34 exons (ranging from 6 to 294 bp in size) encoding the C-terminal region, which provided a template for extensive alternative splicing detected in the SmVAL6 transcripts (Chalmers et al. Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008). Intriguingly, the presence of 17 exons less than 20 bp in length, allied with the high level of alternative splicing over this region, suggests that the C-terminal region of SmVAL6 is related to the recently discovered Micro-Exon Gene (MEG) families (Berriman et al. Reference Berriman, Haas, LoVerde, Wilson, Dillon, Cerqueira, Mashiyama, Al-Lazikani, Andrade, Ashton, Aslett, Bartholomeu, Blandin, Caffrey, Coghlan, Coulson, Day, Delcher, DeMarco, Djikeng, Eyre, Gamble, Ghedin, Gu, Hertz-Fowler, Hirai, Hirai, Houston, Ivens, Johnston, Lacerda, Macedo, McVeigh, Ning, Oliveira, Overington, Parkhill, Pertea, Pierce, Protasio, Quail, Rajandream, Rogers, Sajid, Salzberg, Stanke, Tivey, White, Williams, Wortman, Wu, Zamanian, Zerlotini, Fraser-Liggett, Barrell and El-Sayed2009; DeMarco et al. Reference DeMarco, Mathieson, Manuel, Dillon, Curwen, Ashton, Ivens, Berriman, Verjovski-Almeida and Wilson2010; Verjovski-Almeida and DeMarco, Reference Verjovski-Almeida and DeMarco2011).

Defined by their gene structure, which is comprised of several micro-exons (<36 bp) flanked by conventional exons (>36 bp) at the 5′ and 3′ ends, MEGs are exclusive to the Schistosoma genus with 18 separate families identified to date (DeMarco et al. Reference DeMarco, Mathieson, Manuel, Dillon, Curwen, Ashton, Ivens, Berriman, Verjovski-Almeida and Wilson2010). While the function of these proteins is unknown, recent proteomic analysis has detected members of the MEG-3 family in E/S products derived from in vitro-transformed schistosomula and mature eggs, while members of the MEG-2 family were identified in mature egg E/S products only (DeMarco et al. Reference DeMarco, Mathieson, Manuel, Dillon, Curwen, Ashton, Ivens, Berriman, Verjovski-Almeida and Wilson2010). The secretion of MEG proteins during mammalian host lifestages has led to the hypothesis that the high levels of alternative splicing in MEG transcripts is an attempt to evade the host immune response. While SmVAL6 cannot truly be classified as a MEG (due to the presence of conventional exons and a non-schistosome-specific SCP/TAPS domain), the proposal by Verjovski-Almeida and DeMarco (Reference Verjovski-Almeida and DeMarco2011) that an SmVAL6 ancestor was formed by the combination of a MEG gene and a group 2 VAL gene is highly plausible.

As the only known MEG-like protein with a characterized domain, study of the SmVAL6 protein may well provide insight into both the function of group 2 VALs and MEG proteins. Interestingly, proteomic evidence by van Balkom et al. (Reference van Balkom, van Gestel, Brouwers, Krijgsveld, Tielens, Heck and van Hellemond2005) shows that SmVAL6 (referred to as TC10634 and TC10635 by the authors in the study) is found in adult worm tegumental preparations (van Balkom et al. Reference van Balkom, van Gestel, Brouwers, Krijgsveld, Tielens, Heck and van Hellemond2005). However, the absence of SmVAL6 in proteomic studies examining surface tegumental membrane preparations suggests it is, like the human group 2 member Hs-GAPR-1, an intracellular protein (Braschi and Wilson, Reference Braschi and Wilson2006; Castro-Borges et al. Reference Castro-Borges, Dowle, Curwen, Thomas-Oates and Wilson2011). Recently, microarray analysis of different parasite tissues/regions has provided further localization data for SmVAL6, identifying the transcript to be 31-fold enriched in the female head region when compared to the whole female worm (Nawaratna et al. Reference Nawaratna, McManus, Moertel, Gobert and Jones2011). In contrast to SmVAL6, the SmVAL13 transcript, which is also a group 2 member, was found to be 14-fold enriched in the male head. Additional studies are required to shed light on the role of these different group 2 members at these locations, and to investigate whether these roles are conserved across platyhelminth species.

Monogenean and Tubellarian VALs

Currently there are no experimental studies of VAL proteins from either monogenean or turbellarian species, which limits our understanding of this family in either of these platyhelminth classes. However, a recent large-scale proteomic study of the turbellarian Schmidtea mediterranea provides evidence that at least 19 S. mediterranea VALs (SmdVALs) are present in the adult worm (Table 1; (Adamidi et al. Reference Adamidi, Wang, Gruen, Mastrobuoni, You, Tolle, Dodt, Mackowiak, Gogol-Doering, Oenal, Rybak, Ross, Sanchez Alvarado, Kempa, Dieterich, Rajewsky and Chen2011)). These data suggest that VAL proteins are also participating in aspects of non-parasitic platyhelminth biology. With 19 SmdVALs identified in the adult worm, potential issues of functional redundancy (especially when using RNA interference) may hamper ascertaining functions for these proteins.

As this overview suggests, research into platyhelminth VAL family members has not progressed as quickly as that performed on the nematode VAL homologues (reviewed by Cantacessi et al. (Reference Cantacessi, Campbell, Visser, Geldhof, Nolan, Nisbet, Matthews, Loukas, Hofmann, Otranto, Sternberg and Gasser2009)). One of the main reasons for this has been the paucity of characterized platyhelminth genomic and transcriptomic datasets in comparison to those elucidated for the nematodes. In the last 5–10 years, however, a number of small-, medium- and large-scale platyhelminth transcriptomes (Verjovski-Almeida et al. Reference Verjovski-Almeida, DeMarco, Martins, Guimaraes, Ojopi, Paquola, Piazza, Nishiyama, Kitajima, Adamson, Ashton, Bonaldo, Coulson, Dillon, Farias, Gregorio, Ho, Leite, Malaquias, Marques, Miyasato, Nascimento, Ohlweiler, Reis, Ribeiro, Sa, Stukart, Soares, Gargioni, Kawano, Rodrigues, Madeira, Wilson, Menck, Setubal, Leite and Dias-Neto2003; Zayas et al. Reference Zayas, Hernandez, Habermann, Wang, Stary and Newmark2005; Liu et al. Reference Liu, Lu, Hu, Wang, Cui, Chi, Yan, Wang, Song, Xu, Wang, Zhang, Zhang, Wang, Xue, Brindley, McManus, Yang, Feng, Chen and Han2006; Morris et al. Reference Morris, Ladurner, Rieger, Pfister, Del Mar De Miguel-Bonet, Jacobs and Hartenstein2006; Young et al. Reference Young, Campbell, Hall, Jex, Cantacessi, Laha, Sohn, Sripa, Loukas, Brindley and Gasser2010a , Reference Young, Campbell, Hall, Jex, Cantacessi, Laha, Sohn, Sripa, Loukas, Brindley and Gasser b , Reference Young, Jex, Cantacessi, Hall, Campbell, Spithill, Tangkawattana, Tangkawattana, Laha and Gasser2011) have been made publicly available in addition to the genomes of S. mansoni (Berriman et al. Reference Berriman, Haas, LoVerde, Wilson, Dillon, Cerqueira, Mashiyama, Al-Lazikani, Andrade, Ashton, Aslett, Bartholomeu, Blandin, Caffrey, Coghlan, Coulson, Day, Delcher, DeMarco, Djikeng, Eyre, Gamble, Ghedin, Gu, Hertz-Fowler, Hirai, Hirai, Houston, Ivens, Johnston, Lacerda, Macedo, McVeigh, Ning, Oliveira, Overington, Parkhill, Pertea, Pierce, Protasio, Quail, Rajandream, Rogers, Sajid, Salzberg, Stanke, Tivey, White, Williams, Wortman, Wu, Zamanian, Zerlotini, Fraser-Liggett, Barrell and El-Sayed2009), S. japonicum, (2009) and S. mediterranea, (Robb et al. Reference Robb, Ross and Sanchez Alvarado2008). Interrogating these datasets in a systematic fashion has facilitated the first large-scale comparative genomic/transcriptomics/phylogenetic analysis of VAL diversity across the Platyhelminthes.

VAL proteins are present in all classes of platyhelminth species

To identify VAL homologues from these newly-available nucleotide datasets, BLAST searches and protein domain interrogation were combined (see Table 2 legend for full description of methods), resulting in the identification of 228 complete VAL family members from 18 different platyhelminth species (Table 2; sequences excluded due to incomplete SCP/TAPS domains are listed in Supplementary File 1, online version only). Of the 59 published VAL proteins (Table 1), 56 were reassuringly found in this dataset with only McCrisp3, McCrisp4 and OvEL619323 excluded due to the incomplete nature of their respective SCP/TAPS domains. At the time this analysis was performed (11/11/2011), the S. haematobium genome predictions were not publicly available. Since that date, the S. haematobium genome was published (Young et al. Reference Young, Jex, Li, Liu, Yang, Xiong, Li, Cantacessi, Hall, Xu, Chen, Wu, Zerlotini, Oliveira, Hofmann, Zhang, Fang, Kang, Campbell, Loukas, Ranganathan, Rollinson, Rinaldi, Brindley, Yang, Wang and Gasser2012), allowing a preliminary examination of VAL diversity within this species. Here, a total of 21 ShVAL genes were found using a Pfam domain search (see Supplementary file 1, online version only). However, a comprehensive analysis is required to identify the full repertoire of ShVAL diversity. Of the 21 ShVALs present in the genome Pfam list, only SHA_103186 is represented in this analysis (ShVAL6).

Table 2. Venom allergen-like family distribution across the phylum Platyhelminthes

VAL members from platyhelminth species were identified by tBLASTn searches of NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi), Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/) and Gasser Laboratory (http://gasser-research.vet.unimelb.edu.au/) EST databases and genome gene predictions for S. mansoni, (http://www.genedb.org/Homepage/Smansoni), S. japonicum (http://www.genedb.org/Homepage/Sjaponicum) and S. mediterranea (http://smedgd.neuro.utah.edu) using SmVAL1-29 protein sequences. All sequences with a tBLASTn e-value of <1 e-04 were then clustered to create a non-redundant dataset using a CAP3 clustering and additional pair-wise alignment interrogation (98% match over 150 bp minimum). Database searches were preformed on the 11 November 2011 (a) Number of VAL members refers to the number of unique sequences encoding a protein sequence containing at least 90% of a SCP/TAPS domain as defined by Pfam (PF00188). (b) Number of Group 1 and Group 2 members were defined by phylogenetic clustering (Fig. 2) with known SmVAL group 1 and group 2 members.

Examination of the platyhelminth VALs by species distribution reveals this protein family to be present across all 4 classes within the phylum (Table 2). Notably, this is the first published description of VAL family members in several of these species – S. haematobium, Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis (Class: Trematoda), Moniezia expansa, Echinococcus multilocularis, Taenia asiatica, Taenia solium, Taenia saginata (Class: Cestoda), Neobenedenia melleni (Class: Monogenea), Macrostomum lignano, Dugesia japonica, Dugesia ryukyuensis and S. mediterranea (Class: Turbellaria). Interestingly, while the experimental data on platyhelminth VAL proteins (described above) have found a strong association with early events in parasite infection, the largest VAL family is present in the free-living planarian S. mediterranea (51 members – including the 19 identified proteomically in Adamidi et al. (Reference Adamidi, Wang, Gruen, Mastrobuoni, You, Tolle, Dodt, Mackowiak, Gogol-Doering, Oenal, Rybak, Ross, Sanchez Alvarado, Kempa, Dieterich, Rajewsky and Chen2011)). Whereas the final number of S. mediterranea VALs (SmdVALs) may be amended as newer versions of the S. mediterranea genome are assembled and annotated, our analysis finds transcriptomic support (EST coverage over gene prediction; 98% match over 150 bp minimum) for 32 of the 51 SmdVALs, confirming that a larger protein family exists in this species than S. mansoni (Supplementary File 1, online version only). It is interesting to note that a recent bioinformatic study of G protein-coupled receptors (GPCRs) found that the S. mediterranea genome contained 4 times the number of GPCRs in comparison to the S. mansoni genome (Zamanian et al. Reference Zamanian, Kimber, McVeigh, Carlson, Maule and Day2011). Whether this reflects a general trend for larger gene families in free-living compared to parasitic platyhelminths needs to be further investigated by comparative genomics.

Cestodes provided the fewest numbers of VAL proteins with only 8 members identified across the 6 analysed species. This general under-representation of cestode VALs implies that fewer family members are required in these species. Caution must be made when drawing this conclusion, however, as the cestode EST databases currently available are represented by small-scale studies using few lifestages, while many VALs are known to have expression profiles tightly restricted to particular developmental forms (Chalmers et al. Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008). A clearer view of the cestode VAL family will undoubtedly arrive when cestode genome projects (such as T. solium, E. multilocularis, E. granulosus and Hymenolepis microstoma; reviewed by (Olson et al. Reference Olson, Zarowiecki, Kiss and Brehm2011)) are published. Although the publicly available E. multilocularis genomic assembly (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/Echinococcus) is not annotated with gene predictions or fully assembled, a preliminary, non-exhaustive search for VAL genes identifies 5 scaffolds containing at least 5 different group 1 VAL genes (pathogen_EMU_scaffold_006139, _62143, _007768, _47586 and _007761; data not shown) and 2 different scaffolds containing at least 4 different group 2 VAL genes (pathogen_EMU_scaffold_007285, _007768 and _008000; data not shown). One example of a probable E. multilocularis VAL gene is present on EMU_scaffold_008000 (1226851–1235374 bp). This gene (named EmVAL11 in this study) possesses the same structure as SmVAL11 over the SCP/TAPS regions with a 50% identity at the amino acid level (Fig. 1). The detection of group 2 VAL genes in the draft E. multilocularis genome is especially important as our analysis (using cestode ESTs) failed to identify a group 2 cestode VAL (Table 2). Further research is required to confirm whether EmVAL11, or any other E. multilocularis group 2 gene is transcribed.

Fig. 1. Comparison of Schistosoma mansoni and Echinococcus multilocularis VAL11 gene structure. (A) SmVAL11 gene structure over the SCP/TAPS domain-encoding exons. Structure obtained from S. mansoni genome v. 4 (http://www.genedb.org/Homepage/Smansoni). (B) EmVAL11 gene structure over the SCP/TAPS domain-encoding exons. The genomic region (scaffold_008000) was identified by a tBLASTn search of the E. multilocularis genome (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/Echinococcus) using SmVAL11. EmVAL11 gene structure was manually predicted with all exon/intron junctions conforming to the consensus (GT/AG) splice donor/acceptor sequences for eukaryotes. Exons are represented by boxes with the length shown in base pairs above. Introns are represented by lines with the length shown in base pairs below. Exon regions coloured red represent regions encoding the SCP/TAPS domain.

Overall, the presence of large VAL families in both parasitic (e.g. N. melleni) and non-parasitic (e.g. S. mediterranea) species most likely is explained by these proteins participating in functions critical to platyhelminth life cycles, regardless of trophic strategy. Whether these functions are the same in all platyhelminth organisms is currently unknown. However, detailed interrogation of phylogenetic relationships (described below and illustrated in Fig. 2 and Supplementary File 2, online version only) indicates that conservation of function across species may differ between group 1 and group 2 VAL proteins.

Fig. 2. Phylogenetic analysis of platyhelminth VAL proteins. In total, 237 platyhelminth SCP/TAPS domain amino acid sequences were aligned using ClustalW (Larkin et al. Reference Larkin, Blackshields, Brown, Chenna, McGettigan, McWilliam, Valentin, Wallace, Wilm, Lopez, Thompson, Gibson and Higgins2007) with Bayesian inference phylogenetic analysis performed using MrBayes software (version 3.1.2, WAG protein substitution model used, 3×10⁶ generations run). The resulting unrooted consensus phylogenetic tree was visualized using Mesquite software. Branches are coloured to indicate the taxonomic class each sequence derives from: Trematoda (red), Cestoda (yellow), Turbellaria (blue) or Monogenea (green). Group 1 (dashed black line) and Group 2 proteins (solid black line) are indicated, as are the 2 major group 2 clades – Clade 2a (light grey line) and 2b (dark grey line). Examples of class-specific group 1 clades are highlighted red (trematode-specific), yellow (cestode-specific), green (monogenean-specific) or blue (turbellarian-specific) depending on the taxonomic class. Bayesian posterior probability support values greater than 0·6 are indicated. Species identifiers are as follows; Schistosoma mansoni (Sm), Schistosoma japonicum (Sj), Schistosoma haematobium (Sh), Opisthorchis viverrini (Ov), Fasciola hepatica (Fh), Fasciola gigantica (Fg), Clonorchis sinensis (Cs), Mesocestoides corti (Mc), Taenia asiatica (Ta), Taenia solium (Ts), Taenia saginata (Tsg), Moniezia expansa (Me), Echinococcus multilocularis (Em), Neobenedenia melleni (Nm), Dugesia japonica (Dj), Dugesia ryukyuensis (Dr), Schmidtea mediterranea (Smd) and Macrostomum lignano (Ml).

Group 1/Group 2 VAL division is maintained across platyhelminth species

As first identified in the D. melanogaster and S. mansoni VAL family studies (Kovalick and Griffin, Reference Kovalick and Griffin2005; Chalmers et al. Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008), our phylogenetic reconstruction confirms that the major division within the platyhelminth VAL members is between group 1 and group 2 proteins (Fig. 2, Bayesian inference 100% support; Supplementary File 2, online version only; Maximum Likelihood 90% support). This division of platyhelminth group 1 or group 2 VALs is also supported by evidence from multiple sequence alignment and signal peptide analysis (summarized in Supplementary File 2, online version only).

Examination of the platyhelminth VALs showed that the vast majority of group 1 members (87%; 150/171 SCP/TAPS domains) contain all 6 disulphide bond-forming cysteines characteristic of group 1 SmVALs (C1-C6) (indicated in Supplementary File 2, online version only). These 6 cysteines were absent in all group 2 proteins analysed (Supplementary File 2, online version only), as previously found for group 2 SmVALs. Signal peptide analysis confirmed that the presence of signal peptides was, as found by Chalmers et al. (Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008) in the SmVAL family, to be characteristic of group 1 VALs with a majority (74%) of the platyhelminth group 1 proteins encoding a signal peptide (as defined by SignalP 3.0 Neural Network analysis, using default Dscore threshold). The prevalence of this feature was similar across all 4 taxonomic classes (Trematoda – 78%, Cestoda – 87%, Monogenea – 68% and Turbellaria – 71%; Supplementary File 1, online version only). In contrast to this result, not one group 2 VAL encoded a signal peptide, indicating that these members are likely to be found as intracellular proteins.

Group 1 VALs are restricted to class-specific clades

One of the most notable findings from phylogenetic inspection is the strong evidence for multiple group 1 class-specific VAL clades (Fig. 2). For example, 7 of the 8 group 1 cestode VALs (McCrisp3, McCrisp2, TsVAL1, TsVAL2, TsgVAL1, TaVAL1 and MeVAL1) form a single, cestode-specific clade (Fig. 2; cestode VALs highlighted yellow). Further interrogation of all group 1 VALs demonstrates that this observation is ubiquitous across the phylum with 92% of family members (157/171 SCP/TAPS domains) contained within class-specific clades (Fig. 2), thus having no clear orthologue outside of that taxonomic class. Within the turbellarian group 1 VALs, taxonomic subdivisions are also reflected, with 43 of the 55 VALs from Dugesidea species (D. japonica, D. ryukyuensis and S. mediterranea) present in a single clade (highlighted blue in Fig. 2), while the distantly related Macrostomum lignano VALs are present in additional species-specific clades. Within the trematodes (Fig. 2; coloured red), all group 1 schistosome VALs are present in class-specific clades with the exception of SmVAL20, which does not cluster within any clade. Interestingly, the 3 cercarial/schistosomal E/S SmVALs (4, 10 and 18) form a distinct clade (along with SmVAL19) lacking orthologues from other species (Fig. 2, posterior probability support 0·82; Maximum Likelihood 53% support). This finding provides molecular evidence for potential species specificity in these mammalian-associated, invasion proteins. Monogenean group 1 VALs also showed clear class specificity with 79% of the N. melleni VALs clustering into class-specific clades. Of the 171 group 1 SCP/TAPS domains examined, only 3 clustered in a non-class-specific clade – NmVAL4 (N. melleni; Monogenea), SmdVAL4 (S. mediterranea; Turbellaria) and DrVAL12 (D. ryukyuensis; Turbellaria) (posterior probability score 0·79; Fig. 2). This clade, however, was not observed by Maximum Likelihood analysis (Supplementary File 2, online version only), casting doubt on the relationship of these 3 proteins.

In contrast to the divergent relationship amongst group 1 proteins (i.e. class-specific members), the platyhelminth group 2 proteins are more highly conserved across the phylum. Phylogenetic analysis of the 65 group 2 SCP/TAPS domains provides strong support (Fig. 2, posterior probability score 0·99; Maximum Likelihood, 81% support) for at least 2 major clades within the Platyhelminthes – Clade 2a and 2b (Fig. 2, highlighted in grey (2a) and black (2b)). The presence of turbellarian, cestode and trematode members in both clades provides evidence that these two group 2 clades diverged early in platyhelminth evolution and have both been maintained across taxa. Published genomic structure analysis of the group 2 SmVALs (Chalmers et al. Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008) supports this early divergence, finding different intron boundary positions over exons encoding the N-terminal and C-terminal SCP/TAPS domain. Of the two clades, Clade 2b contains the vast majority of the group 2 SCP/TAPS domains, while Clade 2a contains only 11 members – 9 from trematode species, 1 from the E. multilocularis genome and 1 from the turbellarian S. mediterranea. Interestingly, all of the double SCP/TAPS domain group 2 proteins identified (SmVAL11, SjVAL11 CsVAL11, OvVAL11, FgVAL2, EmVAL11 and SmdVAL46) possess a Clade 2a N-terminal SCP/TAPS domain and a Clade 2b C-terminal SCP/TAPS domain. These 7 double-domain VALs from 7 different species are highly likely to represent orthologous proteins. Given the early divergence of these two group 2 domain types, it is likely that each domain type possesses a different function. Double SCP/TAPS domain VALs such as SmVAL11 (Fig. 1), therefore, would possess 2 different functions mediated through the different SCP/TAPS domains.

In addition to an SmVAL11 orthologue (SjVAL11; 89% amino acid identity over N-terminal SCP/TAPS domain, 82% ID for C-terminal SCP/TAPS domain), the S. japonicum genome also contains orthologues for all group 2 SmVALs – SmVAL6 (SjVAL6; 90% ID), SmVAL13 (SjVAL13; 76% ID), SmVAL16 (SjVAL16; 93% ID) and SmVAL17 (SjVAL17; 85% ID). Surprisingly, one group 2 SjVAL (SjVAL18) does not appear to have an S. mansoni orthologue. Derived from 2 S. japonicum ESTs (AY811609 and BU780182), the SjVAL18 transcript has no gene prediction in the current S. japonicum genome and must therefore be viewed with caution (see Supplementary File 1, online version only). In contrast to the group 2 VALs, no clear orthologues can be ascertained for a number of group 1 SmVALs (i.e. SmVAL4, 10, 18, 19 and 20). Further, where group 1 orthologues are identified, the percentage amino acid identities between the Sj and SmVAL members is also consistently lower than those observed in the group 2 analysis, with only SjVAL5′s similarity to SmVAL28 above 80% (summarized in Supplementary File 1, online version only).

The class- and species- specificity of group 1 platyhelminth VALs (in comparison to the group 2 proteins) indicates that these particular SCP/TAPS members undergo rapid evolutionary changes. High levels of divergence between SCP/TAPS families from related species is not unprecedented. For example, only 1 potential orthologue was detected in a phylogenetic comparison of the Arabidopsis thaliana (22 family members) and rice (Oryza sativa; 32 members) PR-1 family (van Loon et al. Reference van Loon, Rep and Pieterse2006). This conservation level is very low in comparison to other gene families such as the serine protease proteins, where nearly 40% of A. thaliana members have identifiable orthologues in rice (Tripathi and Sowdhamini, Reference Tripathi and Sowdhamini2006). The authors explained the near-complete, non-overlap of SCP/TAPS members as being due to gene duplication/gene loss and sequence evolution after the divergence of these 2 species. As with Arabidopsis PR-1 family, the S. mansoni genome contains evidence of local gene duplication events expanding the gene repertoire, with clusters of group 1 SmVAL genes present in particular chromosomal regions (Chalmers et al. Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008). Detailed evolutionary studies are required to address whether gene duplication/loss or sequence divergence are driving the differences observed in this study. If the group 1 platyhelminth VALs are indeed rapidly changing in amino acid sequence, this may support the view that a key role of the SCP/TAPS domain is providing a structural scaffold for functions performed by residues on the loop regions, glycans and/or additional domains N-terminal or C-terminal to the SCP/TAPS domain (Gibbs et al. Reference Gibbs, Roelants and O'Bryan2008). If VAL functional residues are not present in the core SCP/TAPS fold, considerable sequence variation found here would not affect function. Alternatively, as many group 1 VALs are likely to function after excretion/secretion into the environment, the protein differences between species could reflect the co-evolution of these proteins with specific environmental interacting partners (e.g. host proteins for parasitic platyhelminths).

Distinct protein domains are found within Group 1 VAL C-terminal regions

While the phylogenetic analysis focused only on the SCP/TAPS domain regions, comparison of the platyhelminth VALs outside of the SCP/TAPS domain identified further differences in protein structure amongst taxonomic classes. Similar to SCP/TAPS proteins from other phyla (e.g. PR-1 proteins and Hs-GAPR-1), the majority (98%; 223/228) of the platyhelminth VALs encode no protein domains other than an SCP/TAPS domain (as determined by Pfam searches). Only 5 transcripts were found to encode other protein domains; 4 group 1 turbellarian VALs (DrVAL12, DrVAL9, SmdVAL8 and SmdVAL4) encoded a fibronectin type 2 domain (FN2; PF00040) and one group 1 monogenean VAL (NmVAL27) encoded 3 low-density lipoprotein receptor domains (LDL; PF00057) C-terminal to the SCP/TAPS domain (Fig. 3). The identification of FN2 domains in 4 tubellarian VALs is unusual as proteins containing FN2 domains are thought to only be present in vertebrate species (Ozhogina et al. Reference Ozhogina, Trexler, Banyai, Llinas and Patthy2001). From the published literature, invertebrates should only contain the ancestor of the FN2 domain, the Kringle domain (PF00015) (Ozhogina and Bominaar, Reference Ozhogina and Bominaar2009). However, the 4 FN2 regions found within the turbellarian VALs conform in both size and composition (i.e. conserved residues) to the FN2 domain (data not shown). If these turbellarian VALs do contain functional FN2 domains, then this would indicate a role for these proteins in collagen and/or gelatin binding (Banyai et al. Reference Banyai, Tordai and Patthy1994). Protein interaction studies are essential to address whether this represents a novel function for an SCP/TAPS protein.

Fig. 3. Diversity of domain architectures across platyhelminth VALs. (A) Cartoon representation of different domain architectures within platyhelminth group 1 VALs across different taxonomic classes. Signal peptides (represented by a yellow box) were identified by SignalP searches. A question mark indicates when the incomplete nature of the sequences did not allow for presence/absence of a signal peptide to be determined. Protein domains were identified by Pfam searches (red boxes represent SCP/TAPS domains (PF00188), white boxes represent low-density lipoprotein receptor domains (PF00057) and blue boxes represent fibronectin 2 domains (PF00040)). The M sequence subdomain (represented by green boxes) was identified by manual inspection of the alignment using the following amino acid convention derived from Gibbs et al. (Reference Gibbs, Roelants and O'Bryan2008) – C-X(2)-C-X(5-10)-C-X(5-15)-C (where C indicates a cysteine residue and X indicates any amino acid). SCP/TAPS domains containing the additional disulphide bond are represented by 2 circled ‘C’ letters. (B) Homology model of M. corti Crisp2 protein. The McCrisp2 M sequence subdomain is coloured white. Potential disulphide bonds are coloured yellow, with the cysteines involved in the formation of each disulphide bond labelled C1-C6. (C) Homology model of S. mansoni VAL4 (SmVAL4) protein. The SmVAL4 C-terminal region is coloured white, potential disulfide bonds are coloured yellow and the additional disulphide bond between Cysteine 26 and Cysteine 195 (where the starting Methionine is the first amino acid) indicated by an arrow. Homology models were produced, optimized and verified as described by Chalmers et al. (Reference Chalmers, McArdle, Coulson, Wagner, Schmid, Hirai and Hoffmann2008) using MODELLER version 9.1 (Eswar, Reference Eswar, Webb, Marti-Renom, Madhusudhan, Eramian, Shen, Pieper, Sali, Baxevanis, Pearson, Stein, Stormo and Yates III2006). Specific constraints employed to model the SmVAL4 Cys26-Cys195 disulphide bond did not adversely affect model quality by PROSA-web analysis (Wiederstein and Sippl, Reference Wiederstein and Sippl2007). Models were visualized using MacPyMOL (DeLano Scientific LLC).

One subdomain not included in the Pfam database is the M (metazoan) sequence (also known as the Hinge region; (Gibbs et al. Reference Gibbs, Roelants and O'Bryan2008)). First identified in the snake venom SteCRISP crystal structure C-terminal to the SCP/TAPS domain (Guo et al. Reference Guo, Teng, Niu, Liu, Huang and Hao2005), the M sequence is a small (~25AA) subdomain present in multiple group 1 metazoan VAL structures such as Na-ASP-2 and mCRISP2 (Asojo et al. Reference Asojo, Goud, Dhar, Loukas, Zhan, Deumic, Liu, Borgstahl and Hotez2005; Gibbs et al. Reference Gibbs, Scanlon, Swarbrick, Curtis, Gallant, Dulhunty and O'Bryan2006). The M sequence comprises 2 anti-parallel beta-strands containing 4 disulphide bond-forming cysteines (Fig. 3B) with the following pattern: C-X(2)-C-X(5-10)-C-X(5-15)-C (where C indicates a cysteine residue and X indicates any amino acid). Crucially, the M sequence is known to be essential in mCRISP2 binding to MAP3KII and gametogenetin 1 (Gibbs et al. Reference Gibbs, Bianco, Jamsai, Herlihy, Ristevski, Aitken, Kretser and O'Bryan2007; Jamsai et al. Reference Jamsai, Bianco, Smith, Merriner, Ly-Huynh, Herlihy, Niranjan, Gibbs and O'Bryan2008), suggesting that this is a critical region for certain protein-protein interactions. In mammalian and reptile CRISP proteins, the M sequence is paired with the vertebrate-specific ion channel regulator subdomain (ICR). However, in other SCP/TAPS proteins, such as those found in Drosophila and the Nematoda, it is the only identifiable C-terminal subdomain. Importantly, the presence/absence of the M sequence appears to be a major area of divergence between the trematode VALs and other platyhelminth VALs (Fig. 3). Visual inspection of alignments finds that all trematode group 1 proteins have lost the M sequence, whereas at least one group 1 VAL from the turbellarians, cestodes and monogeneans contains it. For example, greater than 90% of turbellarian group 1 proteins (57/63) contain the M sequence (summarized in Supplementary File 1, online version only). This number is likely to be 100% as the 6 turbellarian VALs not possessing an M sequence are S. mediterranea gene predictions without any EST support. Thus, these sequences may represent incorrect gene models. Support for this assertion is found in the phylogenetic analysis where these 6 SmdVALs cluster with M sequence-containing VALs (Fig. 2). In cestodes, 63% (5/8) group 1 VALs contain the M sequence (including the published McCrisp2 and 3; McCrisp2 homology model in Fig. 3B). The 3 cestode VALs missing the M sequence originate from EST sequences encoding no 3′ stop codon, thus likely only missing the M sequence due to incomplete sequence. Finally, approximately 50% (20/38) of the monogenean group 1 VALs encode a C-terminal M sequence. The lack of M sequences in some monogenean VALs does not appear to be due to incomplete EST coverage as the majority of these sequences (15/18) encode a 3′ stop codon. Overall, our sequence analyses suggest that this subdomain is differentially found amongst the Platyhelminthes.

Given the near ubiquity of the M sequence in metazoan group 1 VALs (Chordata; (Gibbs et al. Reference Gibbs, Roelants and O'Bryan2008), Arthropoda; (Kovalick and Griffin, Reference Kovalick and Griffin2005), Nematoda; (Asojo et al. Reference Asojo, Goud, Dhar, Loukas, Zhan, Deumic, Liu, Borgstahl and Hotez2005), Gastropoda; (Milne et al. Reference Milne, Abbenante, Tyndall, Halliday and Lewis2003)), the complete loss of this subdomain in the trematode VAL family is highly unusual but not unique. For example, the Ag5 wasp venoms do not possess an M sequence (Henriksen et al. Reference Henriksen, King, Mirza, Monsalve, Meno, Ipsen, Larsen, Gajhede and Spangfort2001). However, these SCP/TAPS domain containing proteins differ from the trematode VALs in that they possess an insect-specific N-terminal subdomain named the I (insect) domain. Oddly, due to the lack of an M sequence, it could be argued that the trematode group 1 VALs most closely resemble the plant PR-1 proteins (Fernandez et al. Reference Fernandez, Szyperski, Bruyere, Ramage, Mosinger and Wuthrich1997). However, a subset of the trematode group 1 VALs (e.g. SmVAL4) appear to contain a trematode-specific structural feature (Fig. 3). Identified by multiple sequence alignment, 2 cysteine residues are co-conserved in 36 trematode VALs originating from all 7 species used in this analysis (Fig. 3). With 1 cysteine present after the first helix of the SCP/TAPS domain and the other C-terminal to the SCP/TAPS domain, this conserved pair of cysteines is unique to these trematode VALs (Fig. 3C). Crucially, homology modelling of SmVAL4 confirms that these 2 cysteines (Cys26-Cys195) could create a disulphide bond within a monomer (Fig. 3C), forming a distinct C-terminal region (Fig. 3C; coloured white). Phylogenetic analysis shows that this fourth disulphide bond is not always maintained, as SmVAL7, SjVAL7 and SmVAL10 do not contain either of the cysteines despite being located in clades containing VALs with the additional disulphide bond (Fig. 2). Further research must be performed to address whether this trematode-specific disulphide bond leads to immunological and/or functional differences in these proteins.