Analysis of regulatory protease sequences identified through bioinformatic data mining of the Schistosoma mansoni genome
© Bos et al; licensee BioMed Central Ltd. 2009
Received: 15 April 2009
Accepted: 21 October 2009
Published: 21 October 2009
New chemotherapeutic agents against Schistosoma mansoni, an etiological agent of human schistosomiasis, are a priority due to the emerging drug resistance and the inability of current drug treatments to prevent reinfection. Proteases have been under scrutiny as targets of immunological or chemotherapeutic anti-Schistosoma agents because of their vital role in many stages of the parasitic life cycle. Function has been established for only a handful of identified S. mansoni proteases, and the vast majority of these are the digestive proteases; very few of the conserved classes of regulatory proteases have been identified from Schistosoma species, despite their vital role in numerous cellular processes. To that end, we identified protease protein coding genes from the S. mansoni genome project and EST library.
We identified 255 protease sequences from five catalytic classes using predicted proteins of the S. mansoni genome. The vast majority of these show significant similarity to proteins in KEGG and the Conserved Domain Database. Proteases include calpains, caspases, cytosolic and mitochondrial signal peptidases, proteases that interact with ubiquitin and ubiquitin-like molecules, and proteases that perform regulated intramembrane proteolysis. Comparative analysis of classes of important regulatory proteases find conserved active site domains, and where appropriate, signal peptides and transmembrane helices. Phylogenetic analysis provides support for inferring functional divergence among regulatory aspartic, cysteine, and serine proteases.
Numerous proteases are identified for the first time in S. mansoni. We characterized important regulatory proteases and focus analysis on these proteases to complement the growing knowledge base of digestive proteases. This work provides a foundation for expanding knowledge of proteases in Schistosoma species and examining their diverse function and potential as targets for new chemotherapies.
Schistosomiasis is a common parasitic disease, affecting millions of people, mostly in tropical, developing countries . A causative agent of the disease is a trematode worm, Schistosoma mansoni. Treatment of schistosomiasis is commonly accomplished with praziquantel, for which the mechanism of action is not precisely defined but is thought to affect calcium ion channels  and/or purine nucleotide uptake . Despite the effectiveness of treatment, reinfection is common, and even more troubling, strains of S. mansoni resistant to praziquantel have been found . Thus, additional chemotherapeutic agents and an effective vaccine against this parasite have long been desired .
Ideally, vaccination of at-risk populations against the debilitating effects of schistosomiasis is desired, but no such treatment option is currently available. Surface receptors and other proteins are currently being tested for their potential to act as vaccines, but numerous challenges in the search for an effective vaccine have yet to be overcome . Some candidate vaccines are effective agents but cannot be mass produced. Most other candidate proteins have little potential as a vaccine, providing only 40-50% protection . While the search for an effective vaccine continues, it is critical to continue to identify molecular targets and their potential for chemotherapeutic disruption.
Proteases have been under scrutiny as targets of immunological or chemotherapeutic anti-Schistosoma agents because of their vital role in many stages of the parasitic life cycle [7, 8]. In addition, proteases are known to act as important regulatory elements in a variety of species [9, 10]. They also play a vital role as effectors of virulence in pathogens in general, often serving to alter host signal transduction and modify the immune response [11–14]. By targeting proteases specific to parasitic life style or those with significant dissimilarity to homologous proteases in the host species, investigators hope to find anti-schistosomal chemotherapies with minimal side effects to the host. However, few proteolytic enzymes have been purified in Schistosoma, and even fewer have well characterized functions and interactions.
Proteases of all five catalytic classes have been identified from Schistosoma species through proteomic or genetic analysis. Function has been established for only a handful of identified S. mansoni proteases, and the vast majority of these are the digestive proteases involved in metabolic food processing or host tissue penetration [15–18]. Additional proteases that are involved in reproduction, evasion of host immune system, and development have also been characterized [7, 19]. Very few proteases have been evaluated for the potential to serve as chemotherapeutic targets against schistosomiasis (e.g. ). Fortunately, it is almost certain that additional proteases exist in the S. mansoni genome, as the conserved classes of many regulatory proteases have not been identified from Schistosoma species. Since current therapies for a wide variety of disorders and diseases target regulatory molecules, such proteases may serve as new and effective targets for anti-helminthic treatments. The challenge of developing new therapies involves several steps, the first of which is to identify and characterize potential targets of drug or vaccine treatments. This is currently a task that is increasingly accomplished and streamlined with genomic and bioinformatic tools [9, 21].
The sequencing and annotation of the S. mansoni genome , combined with large EST libraries, provide a wealth of data from which to identify new vaccine or therapy targets [23–25]. These data, combined with bioinformatics tools and specialized databases, can fast-track the identification of potential anti-trematode agents by complementing and supplementing traditional genetic and proteomic identification techniques. Therefore, as an initial step in characterizing some of these potential targets, we survey the S. mansoni genome and EST library for protease genes. In doing so, we identify for the first time, numerous potentially important proteases in trematodes, many of which will have essential functions and may serve as targets of effective chemotherapeutic or immunological treatments.
Results and Discussion
Summary of characteristics from putative protease sequences from the Schistosoma mansoni genome.
Num. protease sequences
Num. protease families
Proteases with predicted transmembrane helices
Proteases with signal sequence
The recently published genome sequence of S. mansoni estimates the presence of 335 protease sequences in 60 families . For the protease families represented in both publications, estimated numbers are identical in 19 families, and 27 families are estimated by Berriman et al.  to have more members. In cases where estimates differ, most are very similar, the exception being families A01, C01, and S09, which we estimate to have a little more than half the previously published numbers. Such differences are easily attributed to differences in data mining methodologies. For instance, Berriman et al. used a Markov method to identify divergent homologues and culled sequences less than 80 residues or those that overlapped less than 50% of the most similar homologue. In comparison, our methods utilized no specialized methods to identify divergent homologues and culled sequences less than 100 residues or that overlapped less than 80% of the most similar homologue.
Below, we review the findings and highlight several important regulatory proteases which are identified for the first time in this species. We focus on regulatory proteases because numerous digestive proteases are already known and being studied in S. mansoni, but relatively little is known about the regulatory proteases despite their potential as targets for novel inhibitive chemotherapies.
Eleven loci encoding aspartic proteases from just three families were identified. Four of the aspartic proteases, all in the A1 family, were found to contain a signal peptide. Transmembrane (TM) predictions differed between methods: TMHMM found three proteases with a TM domain, all in family A22; TUPS detected a total of 5 TM domains among the A1 and A22 families (Table 1; see Additional file information 1). Two loci were significantly similar to family A2 proteases, but were subsequently found to lack similarity to any conserved domain, protease or not, in the CD database. We found five loci encoding proteins with high similarity to Pepsin/Cathepsin type aspartic proteases, which are broadly distributed among taxa and are most active at an acidic pH. Many of these sequences have already been identified in S. mansoni, and are known to be involved in digestion of host proteins . Other functions of this class of proteases are also known, and it is possible that specific sub-functions may be portioned among loci. However, only two of these loci show similarity to sequences in the S. mansoni EST library, so this possibility should be investigated further.
We also identify three A22 protease family members from S. mansoni for the first time. This class of proteases serves as important regulatory elements through their exceptional ability to lyse peptide bonds within a cell membrane . The capacity to lyse peptide bonds that lie embedded within a plasma membrane (also termed regulated intramembrane proteolysis, or RIP) is shared only among A22, M50 and S54 family proteases . These proteases associate with latent, membrane bound cell signaling molecules; upon intramembrane proteolysis, bioactive domains of the signaling molecules are freed to initiate a signaling cascade or act as an enzyme .
We detected 68 loci with significant similarity to known cysteine proteases; of these, 19% had an identifiable signal sequence and thus are likely to enter the secretory pathway. There was a large discrepancy between the two TM domain prediction algorithms: TMHMM found a TM domain in just one cysteine protease, whereas TUPs detected such a domain in 60% of the members of this catalytic class (Table 1). We detected 15 members of the C1 family of proteases, which contain the well-known cysteine-type cathepsin activity involved in digestion of host proteins . Eight of these loci had a conserved cathepsin B domain and other cathepsin family members were detected as well. Cathepsin proteases have already shown promise as targets of new anti-schistosomal therapies and further investigation is warranted . In addition to the digestive enzymes characterized by the cathepsins, we also detected numerous regulatory cysteine proteases.
Regulatory cysteine proteases include the calcium-dependent proteases typified by the calpain and caspase proteases. Calpain proteases belong to the C2 family, of which we found eight members encoded in the S. mansoni genome. Calpains are regulatory proteases with a broad array of functions and are involved in cell mobility, cell cycle progression, and the regulation of clotting factors. Calpains are one class of proteases already under investigation as vaccine candidates in S. japonicum, with results indicating a reduction in worm burden and egg production in immunized mice .
Identity matrix of selected caspase proteases
Another group of important regulatory elements include the cysteine proteases that interact with ubiquitin, SUMO and NEDD molecules (hereafter collectively referred to as ub-like). Ub-like molecules are known to function in the targeting of proteins for destruction by the 26S proteasome, but they also play a prominent regulatory role in the cell through post-translational modification, and protein turn-over and half life [40, 41]. We have putatively identified more than 30 members from the C12, C19, and C48 proteases families, which interact with ub-like protein molecules. These proteases are responsible for the activation (through cleavage of C-terminal di-glycine motifs) and recycling (by removing ub-like molecules from targeted proteins) of ubiquitin and SUMO molecules, controlling the regulatory roles these molecules play . For instance, ubiquitin is known in part to regulate apoptosis by antagonizing the reaction of caspases with the apoptosis complex; cleavage by ubiquitin proteases is central to this process .
It is interesting to note that some of the molecules that effect pathogenesis in bacteria use cysteine proteases that operate on host ub-like molecules to interfere with the regulation of proteins during infection . In fact, numerous effector proteins causing virulence in plant and animal pathogens such as Yersia and Pseudomonas are cysteine proteases, which target SUMO and other regulatory and signaling molecules such as NF-κB .
Metalloproteases make up a large fraction of proteolytic enzymes in the S. mansoni genome; one hundred loci were detected, and 12% of these contained signal sequence. Nineteen percent of metalloproteases have one or more trans-membrane alpha helices, indicating that a substantial portion of these proteases may be membrane bound (Table 1). The bioinformatic approach found a locus that may correspond to the previously identified LAP protease of family M17 . Additionally, we found a putative locus for a previously isolated protein thought to be the SmDPPIII, in family M28 . Many other metalloproteases, previously unidentified, were also detected.
There are hundreds of proteins that operate in the mitochondria, and the vast majority of them are encoded in the nucleus. The mitochondrial processing protease (MPP) and the mitochondrial intermediate protease (MIP) often function in concert to cleave signal peptides from immature mitochondrial proteins that are synthesized in the cytoplasm . Failure to do so obstructs further protein sorting, assembly, and function. We detected both the α- and β- (which should be catalytically active) subunits of the S. mansoni MPP. The β-MPP of S. mansoni has the conserved HxxEHx(76)E motif, indicating that it is probably catalytically active (Additional file 3). The S. mansoni α-MPP and the β-MPP share 20% identity, which is within the normal range of this comparison for several taxa. Interestingly, S. mansoni β-MPP shares 50% sequence identity with its human homolog, which is a higher similarity than the S. mansoni - C. elegans β-MPP homologs (40%).
A predicted protein for MIP was also detected, and contains a HExxH zinc binding domain, critical to catalysis. The putative S. mansoni MIP sequence contains 18 cysteine residues, consistent with cysteine enrichment in the MIP proteins of other species (human and rat MIPs have 18 and 16 cysteine residues respectively) . However, the C terminus of the protein truncates part of the larger catalytic domain conserved in this family of proteases, indicating that this sequence may either be artificially abbreviated by the gene prediction algorithms, or represent a pseudogene.
ATP-dependent mitochondrial proteases are also known, but their function is less well defined. Generally however, these play an essential role in quality control, turnover, and assembly of the respiratory chain complex proteins . Nine ATP-dependent proteases of the M41 family are detected in S. mansoni, and all contain an ATP binding motif, but only one sequence (Smp_165550) contains the active site HExxH motif. Treatment of S. mansoni with metalloprotease inhibitors results in unexplained paralysis of adult worms . This could be related to mitochondrial ATP-dependent protease inhibition, as defects or loss of this protease results in spastic paraplegia in humans and mice .
M50 family proteases are one of the three families of proteases that can perform RIP, and include mammalian S2P proteases, and bacterial SpoIVFB. One protease of the M50 family with an active site motif (HExxH) was detected in our analysis (Smp_054310). This protease is predicted to have multiple transmembrane helices, and the putative active site is found within the third transmembrane helix (approximately residues 175-195; see Additional file 3), consistent with predicted function . As with other proteases that perform RIP, bioactivation of the substrate is a multi-step process, involving multiple proteases. In mammals, S2P cannot perform RIP unless the substrate protein is first cleaved by S1P, a S08 family protease. A homolog of mammalian S1P is also found in S. mansoni, providing evidence of an active and at least partially conserved functional mechanism .
Sixty serine proteases were detected in our screening of the S. mansoni predicted proteins, and these are divided among 15 families. Sixteen loci were found to have a predicted signal sequence--many of these are in the S1 family, which is consistent with the majority of known S1 family proteases entering the secretory pathway and having a signal sequence. Twelve of the serine proteases have at least one transmembrane alpha helix, as predicted by TMHMM, whereas TUPs identified 35 proteases with a TM alpha helix (Table 1). We identified a locus for the well known cercarial elastase , and note that several loci also identified as having the highest similarity to cercarial elastase were detected, but were excluded from the analysis due to the likelihood of being pseudogenes. Numerous regulatory serine proteases are identified for the first time in S. mansoni with this report.
We also found members of the S08 family that have a catalytic mechanism that is distinct from the typical chymotrypsin activity of S01 family proteases. This family is mostly comprised of endoproteases with broad function, many of which enter the secretory pathway . S. mansoni also has S26 family members, which contain proteases that are responsible for processing numerous precursor proteins to active, mature forms . This occurs when signal peptides of newly synthesized proteins are cleaved upon arrival at a functional site. This cleavage event also acts as a post-translational regulatory event, controlling the activation of these proteins until the signal peptide is removed. Other AAA mitochondrial proteases of the S16 family, which bind DNA and RNA and may participate directly in the metabolism of mtDNA, are also found. Downregulation of this protease causes a general activation of caspases and leads to apoptosis .
We also found members of the S54 family of proteases in the S. mansoni genome. This family of proteases functions to perform RIP, resulting in the liberation of bioactive signaling peptides from anchoring TM domains [13, 59]. Consistent with the expected function, these S54 proteases have several TM helices predicted by both TMHMM and TUPs, indicating a likely conserved structure among taxa (Appendix 1).
Threonine proteases are most closely associated with the elements of the 20S proteasome, and the majority of threonine proteases discovered in our analysis appear to be subunits of that proteasome. At least 5 alpha subunits and 4 beta subunits of the proteasome were detected, all of which show evidence of being expressed. S. mansoni proteasome elements have already been identified and characterized through proteomic analysis, which detected all seven alpha and beta subunits and displayed morphological diversity due mainly to post-translational modifications . Thus, the bioinformatic data mining done here failed to identify all threonine protease genes present in the genome, or the genome annotation and gene detection methods used have failed to detect some genes known to be present. This difference highlights the need for collaborative work and the complementary nature of bioinformatic, and proteomic discovery of genetic elements.
In addition to the proteasome subunits, a taspase-like protease was identified in the S. mansoni genome. This protease cleaves the general transcription factor TFIIA, regulating transcription of numerous gene products.
We used in silico methods to explore the catalog of predicted proteins of S. mansoni for sequences homologous to known proteases. The search resulted in the detection of 255 putative proteases, over 90% of which are recognized for the first time in this species. Along with the proteases already described as having a function primarily in digestion and host invasion, we identify numerous previously unidentified regulatory proteases involved in a variety of biological processes. We focused on regulatory proteases because they generally serve essential functions in the maintenance of homeostasis and the developmental progression of the life cycle of a species. The vital nature of protease-mediated regulatory function underscores the potential of these proteases as targets of inhibitory chemotherapies and we hope that the identification of proteases stimulates further research into this area, such as expanding the application of gene silencing to identify the function of various proteases in Schistosoma species [62, 63].
We used the complete set of protease core sequences from the MEROPS (release 7.8) database  to identify putative homologues of known proteases in the S. mansoni genome. Core protease sequences comprise a non-redundant library of the catalytic unit of a protease and exclude all other functional units, such as ATP-binding, or calcium-binding domains. These sequences were used to avoid false positive identification of proteins as proteases due to high homology in non-catalytic parts of the sequence. Core sequences were compared to predicted proteins from the annotated S. mansoni genome. Release 4.0 of the S. mansoni genome includes a first-pass annotation and gene prediction, of which we downloaded the complete database of predicted proteins. In addition, the complete set of ESTs (release 6.0 of the S. mansoni gene index) isolated from 6 life stages of S. mansoni was also acquired.
Using the MEROPS proteases as the database, we used the predicted proteins as the query sequences in a local BLASTP  comparison. Default parameters were used during the search, where all predicted proteins were queried against all members of the protease database. In the comparison, only sequences with similarity scores <1e-04 were retained as S. mansoni protease homologs. From the initial result, query sequences identified as similar to homologous non-protease sequences (protease-like sequences but with mutations in active sites) were culled from the results, as were alternative splice forms of a gene. Additionally, predicted proteins that were shorter than 100 residues or less than 80% of the protease core sequence were not retained. Remaining sequences were subject to full analysis.
Results from the BLASTP query were subject to a number of analyses to characterize the sequences. We first independently sought the predicted function of S. mansoni sequences by searching for conserved motif and domains in the protein sequences. This was done using searches in the Conserved Domain Database (CDD) v. 2.13 of NCBI [66, 67]. CDD searches employ a reverse position-specific BLAST to align query sequence to protein domains from SMART v. 5.0 , Pfam v. 22.0 , and COG . The KEGG automated annotation server (KAAS) was used to assign pathway-based functional orthology to sequences in our dataset . We were also interested in identifying alpha-helix domains that likely span a cellular membrane. The prediction of these transmembrane helices is imprecise, so we report the results of two methods: TMHMM  and TUP .
The expected cellular location (e.g. cytoplasmic, membrane, or mitochondrial) and potential to enter the secretory pathway of a cell is also informative in classifying newly identified proteins. Therefore, we identified signal sequences in the proteins with signalP, using both the neural network and HMM methods [74, 75]. The D score is the average of the maximal Y-score (the most likely location of the cleavage site of the signal sequence) and the mean S-score, and is the best way to discriminate true signal sequences in proteins . Proteins with a D score greater than 55 and HMM greater than 90% were scored as having an N-terminal signal sequence and the Y-score was recorded for these proteins. We used the eukaryote setting with each sequence truncated after 70 residues to avoid false positive detection of signal sequence outside of the N-terminus.
Functionally distinct proteases may show differential phylogenetic clustering. Therefore, we performed phylogenetic comparisons, including mainly species with functionally defined proteases with S. mansoni sequences (accession numbers of sequences used are given in the Additional files). While phylogenetic analysis does detect evidence of divergence, the functional implications of the divergence must be tested experimentally, as such phylogenetic analyses represent an initial assessment of potential functional divergence among multiple S. mansoni proteases within a family. For phylogenetic analysis, homologous protein sequences from selected species were obtained from NCBI GENBANK. Alignments were made using MUSCLE default parameters . Resulting alignments were subject to phylogenetic analysis using MRBAYES . Flexible priors were used by employing a mixed model analysis. Mixed model analysis avoids reliance on a single model of amino acid substitutions (and thus a single prior) by allowing the MCMC sampler to regularly propose and explore the fit of new models during analysis (see MRBAYES documentation for model specifications). Each model contributes to the results in proportion to their posterior probability. We performed two simultaneous but independent runs on each data set, with each run consisting of three heated chains and one cold chain. Runs were continued until the average standard deviation of the split frequencies between the two runs was less than 0.02, with a minimum of 100,000 MCMC generations. Trees were sampled every 50th generation, resulting in at least 2,000 saved trees. The first 25% of trees were discarded as burn-in prior to summarizing sampled trees. Summarizing samples produced a consensus tree with branch bifurcation support (clade credibility) indicated. Clade credibility was calculated for each bifurcation as the proportion of sampled trees with that bifurcation.
We thank the Minchella lab members for support and comments on earlier versions of this manuscript. We thank insightful and constructive editorial and reviewer comments, which have improved this manuscript. This work is funded by National Institute of Health grant #R01-AI-42768 to DJM.
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