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1.  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.
Author Summary
Schistosomes are blood flukes that live in the blood system and cause chronic and debilitating infection in hundreds of millions of people. Proteolytic enzymes (proteases) produced by the parasite allow it to survive and reproduce. We focused on understanding the repertoire of trypsin- and chymotrypsin-like Schistosoma mansoni serine proteases (SmSPs) using a variety of genomic, bioinformatics, RNA- and protein-based techniques. We identified five SmSPs that are produced at different stages of the parasite's development. Based on bioinformatics and cleavage preferences for small peptide substrates, SmSP1 to SmSP4 are trypsin-like, whereas SmSP5 is chymotrypsin-like. Interestingly, SmSP5 forms part of a ‘missing link’ group of enzymes between the specialized chymotrypsin-like ‘cercarial elastases’ that help the parasite invade human skin and the more typical chymotrypsins and trypsins found in the nature. Our findings form a basis for further exploration of the functions of the individual enzymes, including their possible contributions to influencing host physiology.
doi:10.1371/journal.pntd.0002766
PMCID: PMC3967958  PMID: 24676141
2.  The crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata 
The Biochemical journal  2010;429(1):103-112.
Synopsis
The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help overcome the host’s defense during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease-vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 Å and used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the proinflammatory cytokines TNF-α and IL-12, and proliferation of antigen-specific CD4+ T cells. This suggests that OmC2 may suppress the host’s adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.
doi:10.1042/BJ20100280
PMCID: PMC3523712  PMID: 20545626
cathepsin; cystatin; immune cells; structure-activity relationship; parasite; peptidase inhibitor
3.  Crystallization and diffraction analysis of the serpin IRS-2 from the hard tick Ixodes ricinus  
Cleavage of the serpin IRS-2 from the hard tick I. ricinus by contaminating proteolytic activity mimicked the specific processing of the serpin by its target protease and resulted in a more stable form of the serpin which produced crystals that diffracted to 1.8 Å resolution.
IRS-2 from the hard tick Ixodes ricinus belongs to the serpin family of protease inhibitors. It is produced in the salivary glands of the tick and its anti-inflammatory activity suggests that it plays a role in parasite–host interaction. Recombinant IRS-2 prepared by heterologous expression in a bacterial system was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the primitive tetragonal space group P43 and diffracted to 1.8 Å resolution. Mass-spectrometric and electrophoretic analyses revealed that IRS-2 was cleaved by contaminating proteases during crystallization. This processing of IRS-2 mimicked the specific cleavage of the serpin by its target protease and resulted in a more stable form (the so-called relaxed conformation), which produced well diffracting crystals. Activity profiling with specific substrates and inhibitors demonstrated traces of serine and cysteine proteases in the protein stock solution.
doi:10.1107/S1744309110032343
PMCID: PMC3001646  PMID: 21045293
serpins; ticks; proteolysis
4.  Enzymatic activity and immunoreactivity of Aca s 4, an alpha-amylase allergen from the storage mite Acarus siro 
BMC Biochemistry  2012;13:3.
Background
Enzymatic allergens of storage mites that contaminate stored food products are poorly characterized. We describe biochemical and immunological properties of the native alpha-amylase allergen Aca s 4 from Acarus siro, a medically important storage mite.
Results
A. siro produced a high level of alpha-amylase activity attributed to Aca s 4. This enzyme was purified and identified by protein sequencing and LC-MS/MS analysis. Aca s 4 showed a distinct inhibition pattern and an unusual alpha-amylolytic activity with low sensitivity to activation by chloride ions. Homology modeling of Aca s 4 revealed a structural change in the chloride-binding site that may account for this activation pattern. Aca s 4 was recognized by IgE from house dust mite-sensitive patients, and potential epitopes for cross-reactivity with house dust mite group 4 allergens were found.
Conclusions
We present the first protein-level characterization of a group 4 allergen from storage mites. Due to its high production and IgE reactivity, Aca s 4 is potentially relevant to allergic hypersensitivity.
doi:10.1186/1471-2091-13-3
PMCID: PMC3306266  PMID: 22292590
Aca s 4; Acarus siro; α-amylases; group 4 mite allergens; storage mites
5.  Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus 
Parasites & Vectors  2010;3:119.
Background
Ticks are vectors of a wide variety of pathogens causing severe diseases in humans and domestic animals. Intestinal digestion of the host blood is an essential process of tick physiology and also a limiting factor for pathogen transmission since the tick gut represents the primary site for pathogen infection and proliferation. Using the model tick Ixodes ricinus, the European Lyme disease vector, we have previously demonstrated by genetic and biochemical analyses that host blood is degraded in the tick gut by a network of acidic peptidases of the aspartic and cysteine classes.
Results
This study reveals the digestive machinery of the I. ricinus during the course of blood-feeding on the host. The dynamic profiling of concentrations, activities and mRNA expressions of the major digestive enzymes demonstrates that the de novo synthesis of peptidases triggers the dramatic increase of the hemoglobinolytic activity along the feeding period. Overall hemoglobinolysis, as well as the activity of digestive peptidases are negligible at the early stage of feeding, but increase dramatically towards the end of the slow feeding period, reaching maxima in fully fed ticks. This finding contradicts the established opinion that blood digestion is reduced at the end of engorgement. Furthermore, we show that the digestive proteolysis is localized intracellularly throughout the whole duration of feeding.
Conclusions
Results suggest that the egressing proteolytic system in the early stage of feeding and digestion is a potential target for efficient impairment, most likely by blocking its components via antibodies present in the host blood. Therefore, digestive enzymes are promising candidates for development of novel 'anti-tick' vaccines capable of tick control and even transmission of tick-borne pathogens.
doi:10.1186/1756-3305-3-119
PMCID: PMC3016361  PMID: 21156061
6.  Hemoglobin digestion in Blood-Feeding Ticks: Mapping a Multi-Peptidase Pathway by Functional Proteomics 
Chemistry & biology  2009;16(10):1053-1063.
SUMMARY
Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates/inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.
doi:10.1016/j.chembiol.2009.09.009
PMCID: PMC2801564  PMID: 19875079
7.  Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases 
Background
Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets.
Results
Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain), and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood.
Conclusion
Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.
doi:10.1186/1756-3305-1-7
PMCID: PMC2289814  PMID: 18348719

Results 1-7 (7)