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.

• Keywords
• Fasciola hepatica, Folate hydrolase, Immunohistochemistry, M28B metalloproteases, NAALADase, Platyhelminth, Prostate specific-membrane antigen, RNA in situ hybridization, Schistosoma mansoni,
• MeSH
• Fasciola hepatica * genetics   MeSH
• Mice MeSH
• Peptide Hydrolases MeSH
• Mammals MeSH
• Schistosoma mansoni MeSH
• Trematoda * genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• glutamate carboxypeptidase MeSH Browser
• Peptide Hydrolases MeSH

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.

• Keywords
• Blood fluke, Fluorescence RNA in situ hybridization, Platyhelminthes, Schistosoma mansoni, Serine proteases, Transcript, mRNA detection,
• MeSH
• Gene Expression * MeSH
• In Situ Hybridization, Fluorescence methods   standards   MeSH
• Helminth Proteins genetics   MeSH
• RNA metabolism   MeSH
• Schistosoma mansoni enzymology   genetics   MeSH
• Serine Proteases genetics   MeSH
• Gene Expression Profiling MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Helminth Proteins MeSH
• RNA MeSH
• Serine Proteases MeSH

The molecular basis for the propensity of a small number of environmental proteins to provoke allergic responses is largely unknown. Herein, we report that mite group 13 allergens of the fatty acid-binding protein (FABP) family are sensed by an evolutionarily conserved acute-phase protein, serum amyloid A1 (SAA1), that promotes pulmonary type 2 immunity. Mechanistically, SAA1 interacted directly with allergenic mite FABPs (Der p 13 and Blo t 13). The interaction between mite FABPs and SAA1 activated the SAA1-binding receptor, formyl peptide receptor 2 (FPR2), which drove the epithelial release of the type-2-promoting cytokine interleukin (IL)-33 in a SAA1-dependent manner. Importantly, the SAA1-FPR2-IL-33 axis was upregulated in nasal epithelial cells from patients with chronic rhinosinusitis. These findings identify an unrecognized role for SAA1 as a soluble pattern recognition receptor for conserved FABPs found in common mite allergens that initiate type 2 immunity at mucosal surfaces.

• MeSH
• Allergens immunology   MeSH
• Rhinitis, Allergic immunology   pathology   MeSH
• Antigens, Dermatophagoides immunology   MeSH
• Asthma immunology   pathology   MeSH
• Adult MeSH
• Epithelial Cells MeSH
• Immunity, Humoral MeSH
• Interleukin-33 metabolism   MeSH
• Cells, Cultured MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Disease Models, Animal MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Lung cytology   immunology   pathology   MeSH
• Primary Cell Culture MeSH
• Immunity, Innate MeSH
• Fatty Acid-Binding Proteins immunology   MeSH
• Receptors, Lipoxin metabolism   MeSH
• Receptors, Formyl Peptide metabolism   MeSH
• Respiratory Mucosa immunology   metabolism   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Serum Amyloid A Protein genetics   metabolism   MeSH
• Signal Transduction immunology   MeSH
• Up-Regulation MeSH
• Animals MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Mice MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Allergens MeSH
• Antigens, Dermatophagoides MeSH
• formyl peptide receptor 2, mouse MeSH Browser
• FPR2 protein, human MeSH Browser
• IL33 protein, human MeSH Browser
• Il33 protein, mouse MeSH Browser
• Interleukin-33 MeSH
• Fatty Acid-Binding Proteins MeSH
• Receptors, Lipoxin MeSH
• Receptors, Formyl Peptide MeSH
• SAA1 protein, human MeSH Browser
• Saa2 protein, mouse MeSH Browser
• Serum Amyloid A Protein MeSH