Uncovering the essential roles of glutamate carboxypeptidase 2 orthologs in Caenorhabditis elegans.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans to genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. The present study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to physiology of this protease group and in extension to human GCP2 involvement in pathophysiological processes.

Characterization of glutamate carboxypeptidase 2 orthologs in trematodes.

BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.

An Activity-Based Probe for Cathepsin K Imaging with Excellent Potency and Selectivity.

The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (kinac/Ki = 35,300 M-1s-1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.

Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization.

BACKGROUND: The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host-parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. METHODOLOGY: Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. RESULTS: FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. CONCLUSIONS: The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host-parasite interactions.

Sensitive Fluorescence In Situ Hybridization on Semithin Sections of Adult Schistosoma mansoni Using DIG-Labeled RNA Probes.

In situ hybridization is a tool for evaluation of gene expression within tissues or single cells. This protocol describes optimized sensitive fluorescence detection of gene transcripts (mRNAs) in semithin sections of Schistosoma mansoni adult worms using specifically designed and labeled RNA probes. Due to improved methodologies in tissue preservation, sectioning, amplification of fluorescent signal, and prehybridization tissue treatment, it is possible to detect transcripts in the fine structures of schistosomes. The protocol is sensitive enough to detect very low abundance targets. This procedure is optimized for tissues derived from S. mansoni adult worms; however, it can be successfully applied to other trematode species.

Collection of Excretory/Secretory Products from Individual Developmental Stages of the Blood Fluke Schistosoma mansoni.

Individual developmental stages of blood fluke Schistosoma mansoni excrete or secrete a different set of molecules. Here we describe optimized protocols for collection of excretory/secretory products (E/S products) from cercariae, schistosomula, adult worms, and eggs. These E/S products are essential for successful parasitism functioning at the host-parasite interface, enabling invasion into the host and contributing to the survival of the parasite by modulation of host physiology and immune responses. Collection of sufficient amounts of E/S products is required for detailed research of these processes.

A novel Kunitz protein with proposed dual function from Eudiplozoon nipponicum (Monogenea) impairs haemostasis and action of complement in vitro.

Serine peptidases are involved in many physiological processes including digestion, haemostasis and complement cascade. Parasites regulate activities of host serine peptidases to their own benefit, employing various inhibitors, many of which belong to the Kunitz-type protein family. In this study, we confirmed the presence of potential anticoagulants in protein extracts of the haematophagous monogenean Eudiplozoon nipponicum which parasitizes the common carp. We then focused on a Kunitz protein (EnKT1) discovered in the E. nipponicum transcriptome, which structurally resembles textilinin-1, an antihemorrhagic snake venom factor from Pseudonaja textilis. The protein was recombinantly expressed, purified and biochemically characterised. The recombinant EnKT1 did inhibit in vitro activity of Factor Xa of the coagulation cascade, but exhibited a higher activity against plasmin and plasma kallikrein, which participate in fibrinolysis, production of kinins, and complement activation. Anti-coagulation properties of EnKT1 based on the inhibition of Factor Xa were confirmed by thromboelastography, but no effect on fibrinolysis was observed. Moreover, we discovered that EnKT1 significantly impairs the function of fish complement, possibly by inhibiting plasmin or Factor Xa which can act as a C3 and C5 convertase. We localised Enkt1 transcripts and protein within haematin digestive cells of the parasite by RNA in situ hybridisation and immunohistochemistry, respectively. Based on these results, we suggest that the secretory Kunitz protein of E. nipponicum has a dual function. In particular, it impairs both haemostasis and complement activation in vitro, and thus might facilitate digestion of a host's blood and protect a parasite's gastrodermis from damage by the complement. This study presents, to our knowledge, the first characterisation of a Kunitz protein from monogeneans and the first example of a parasite Kunitz inhibitor that impairs the function of the complement.

SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

BACKGROUND: Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: SmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting. CONCLUSIONS/SIGNIFICANCE: The data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy.

Cysteine peptidases of Eudiplozoon nipponicum: a broad repertoire of structurally assorted cathepsins L in contrast to the scarcity of cathepsins B in an invasive species of haematophagous monogenean of common carp.

BACKGROUND: Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored. METHODS: Sequences of cathepsins L and B (EnCL and EnCB) were mined from E. nipponicum transcriptome and analysed bioinformatically. Genes encoding two EnCLs and one EnCB were cloned and recombinant proteins produced in vitro. The enzymes were purified by chromatography and their activity towards selected substrates was characterised. Antibodies and specific RNA probes were employed for localisation of the enzymes/transcripts in tissues of E. nipponicum adults. RESULTS: Transcriptomic analysis revealed a set of ten distinct transcripts that encode EnCLs. The enzymes are significantly variable in their active sites, specifically the S2 subsites responsible for interaction with substrates. Some of them display unusual structural features that resemble cathepsins B and S. Two recombinant EnCLs had different pH activity profiles against both synthetic and macromolecular substrates, and were able to hydrolyse blood proteins and collagen I. They were localised in the haematin cells of the worm's digestive tract and in gut lumen. The EnCB showed similarity with cathepsin B2 of Schistosoma mansoni. It displays molecular features typical of cathepsins B, including an occluding loop responsible for its exopeptidase activity. Although the EnCB hydrolysed haemoglobin in vitro, it was localised in the vitelline cells of the parasite and not the digestive tract. CONCLUSIONS: To our knowledge, this study represents the first complex bioinformatic and biochemical characterisation of cysteine peptidases in a monogenean. Eudiplozoon nipponicum adults express a variety of CLs, which are the most abundant peptidases in the worms. The properties and localisation of the two heterologously expressed EnCLs indicate a central role in the (partially extracellular?) digestion of host blood proteins. High variability of substrate-binding sites in the set of EnCLs suggests specific adaptation to a range of biological processes that require proteolysis. Surprisingly, a single cathepsin B is expressed by the parasite and it is not involved in digestion, but probably in vitellogenesis.

Excretion/secretion products from Schistosoma mansoni adults, eggs and schistosomula have unique peptidase specificity profiles.

Schistosomiasis is one of a number of chronic helminth diseases of poverty that severely impact personal and societal well-being and productivity. Peptidases (proteases) are vital to successful parasitism, and can modulate host physiology and immunology. Interference of peptidase action by specific drugs or vaccines can be therapeutically beneficial. To date, research on peptidases in the schistosome parasite has focused on either the functional characterization of individual peptidases or their annotation as part of global genome or transcriptome studies. We were interested in functionally characterizing the complexity of peptidase activity operating at the host-parasite interface, therefore the excretory-secretory products of key developmental stages of Schistosoma mansoni that parasitize the human were examined. Using class specific peptidase inhibitors in combination with a multiplex substrate profiling assay, a number of unique activities derived from endo- and exo-peptidases were revealed in the excretory-secretory products of schistosomula (larval migratory worms), adults and eggs. The data highlight the complexity of the functional degradome for each developmental stage of this parasite and facilitate further enquiry to establish peptidase identity, physiological and immunological function, and utility as drug or vaccine candidates.

Prolyl Oligopeptidase from the Blood Fluke Schistosoma mansoni: From Functional Analysis to Anti-schistosomal Inhibitors.

BACKGROUND: Blood flukes of the genus Schistosoma cause schistosomiasis, a parasitic disease that infects over 240 million people worldwide, and for which there is a need to identify new targets for chemotherapeutic interventions. Our research is focused on Schistosoma mansoni prolyl oligopeptidase (SmPOP) from the serine peptidase family S9, which has not been investigated in detail in trematodes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that SmPOP is expressed in adult worms and schistosomula in an enzymatically active form. By immunofluorescence microscopy, SmPOP is localized in the tegument and parenchyma of both developmental stages. Recombinant SmPOP was produced in Escherichia coli and its active site specificity investigated using synthetic substrate and inhibitor libraries, and by homology modeling. SmPOP is a true oligopeptidase that hydrolyzes peptide (but not protein) substrates with a strict specificity for Pro at P1. The inhibition profile is analogous to those for mammalian POPs. Both the recombinant enzyme and live worms cleave host vasoregulatory, proline-containing hormones such as angiotensin I and bradykinin. Finally, we designed nanomolar inhibitors of SmPOP that induce deleterious phenotypes in cultured schistosomes. CONCLUSIONS/SIGNIFICANCE: We provide the first localization and functional analysis of SmPOP together with chemical tools for measuring its activity. We briefly discuss the notion that SmPOP, operating at the host-parasite interface to cleave host bioactive peptides, may contribute to the survival of the parasite. If substantiated, SmPOP could be a new target for the development of anti-schistosomal drugs.

Trypsin- and Chymotrypsin-like serine proteases in schistosoma mansoni-- 'the undiscovered country'.

BACKGROUND: Blood flukes (Schistosoma spp.) are parasites that can survive for years or decades in the vasculature of permissive mammalian hosts, including humans. Proteolytic enzymes (proteases) are crucial for successful parasitism, including aspects of invasion, maturation and reproduction. Most attention has focused on the 'cercarial elastase' serine proteases that facilitate skin invasion by infective schistosome larvae, and the cysteine and aspartic proteases that worms use to digest the blood meal. Apart from the cercarial elastases, information regarding other S. mansoni serine proteases (SmSPs) is limited. To address this, we investigated SmSPs using genomic, transcriptomic, phylogenetic and functional proteomic approaches. METHODOLOGY/PRINCIPAL FINDINGS: Genes encoding five distinct SmSPs, termed SmSP1 - SmSP5, some of which comprise disparate protein domains, were retrieved from the S. mansoni genome database and annotated. Reverse transcription quantitative PCR (RT- qPCR) in various schistosome developmental stages indicated complex expression patterns for SmSPs, including their constituent protein domains. SmSP2 stood apart as being massively expressed in schistosomula and adult stages. Phylogenetic analysis segregated SmSPs into diverse clusters of family S1 proteases. SmSP1 to SmSP4 are trypsin-like proteases, whereas SmSP5 is chymotrypsin-like. In agreement, trypsin-like activities were shown to predominate in eggs, schistosomula and adults using peptidyl fluorogenic substrates. SmSP5 is particularly novel in the phylogenetics of family S1 schistosome proteases, as it is part of a cluster of sequences that fill a gap between the highly divergent cercarial elastases and other family S1 proteases. CONCLUSIONS/SIGNIFICANCE: Our series of post-genomics analyses clarifies the complexity of schistosome family S1 serine proteases and highlights their interrelationships, including the cercarial elastases and, not least, the identification of a 'missing-link' protease cluster, represented by SmSP5. A framework is now in place to guide the characterization of individual proteases, their stage-specific expression and their contributions to parasitism, in particular, their possible modulation of host physiology.