Molecular and immunological characterization of Fasciola antigens recognized by the MM3 monoclonal antibody

Muiño, Laura; Perteguer, María Jesús; Gárate, Teresa; Martínez-Sernández, Victoria; Beltrán, Ana Rosa; Romarís, F.; Mezo, Mercedes; González-Warleta, Marta; Ubeira, Florencio M.

doi:10.1016/j.molbiopara.2011.06.003

Cited by 35 publications

(31 citation statements)

References 47 publications

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“…The sample was then reduced with 10 mM DTT in 25 mM ammonium bicarbonate for 30 min at 56°C and subsequently alkylated with 55 mM iodoacetamide in 25 mM ammonium bicarbonate for 15 min in the dark. Finally, samples were digested overnight with 12.5 ng/l sequencing grade trypsin (Roche Applied Science) in 25 mM ammonium bicarbonate (pH 8.5) at 37°C, and the resulting peptides were sequenced in a 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA) under the same experimental conditions as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

“…The slides were finally washed with TBS and mounted with glass coverslips in PBS-glycerol (1:1). The slides were examined, photographed, and then immersed in 70% ethanol, to remove the coverslips, and stained with Wheatley's trichrome, as described previously (26,28). Briefly, the slides were placed in trichrome stain for 10 min and then dipped twice in 90% ethanol containing 0.5% acetic acid, dipped twice in 90% ethanol, dehydrated through an increasing ethanol gradient and xylene (5 min each), and mounted in Entellan (Electron Microscopy Sciences, Hatfield, PA).…”

Section: Cloning and Expression Of The Gene Encoding The Recognized Pmentioning

confidence: 99%

“…For better identification of the Fasciola structures, we also stained all slides with Wheatley's trichrome stain (28). For comparative purposes, we stained sections with mAb MM3, which stains L-cathepsins from the L1 and L2 clades (26). We show some representative images obtained after staining sagittal sections, cross-sections, and frontal sections of the parasite in Fig.…”

Section: The Mf6p/fhhdm-1 Protein Is Produced and Distributed By Parementioning

confidence: 99%

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The MF6p/FhHDM-1 Major Antigen Secreted by the Trematode Parasite Fasciola hepatica Is a Heme-binding Protein

Martínez-Sernández

Mezo²,

González-Warleta³

et al. 2014

Journal of Biological Chemistry

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Background:The mechanisms involved in heme handling in trematodes are poorly understood. Results: The biochemical and functional characteristics of a new family of small proteins (MF6p/FhHDM-1) secreted by Fasciola and other trematodes are reported. Conclusion:The Fasciola MF6p/FhHDM-1 major antigen is a heme-binding protein. Significance:Our results provide new insights into the biology of hematophagous trematodes.

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Section: Methodsmentioning

confidence: 99%

Section: Cloning and Expression Of The Gene Encoding The Recognized Pmentioning

confidence: 99%

Section: The Mf6p/fhhdm-1 Protein Is Produced and Distributed By Parementioning

confidence: 99%

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The MF6p/FhHDM-1 Major Antigen Secreted by the Trematode Parasite Fasciola hepatica Is a Heme-binding Protein

Martínez-Sernández

Mezo²,

González-Warleta³

et al. 2014

Journal of Biological Chemistry

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“…To date, various diagnostic techniques for the identification and discrimination of Fasciola spp. have been developed, including the traditional detection of eggs in feces directly or after Kato-Katz based sedimentation (19,43), the morphological examination of adults (38), the lateral flow immunoassay for serodiagnosis (28), and the enzyme-linked immunosorbent assay (indirect ELISA) (12,13,32,36).…”

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confidence: 99%

Development and Evaluation of a Single-Step Duplex PCR for Simultaneous Detection of Fasciola hepatica and Fasciola gigantica (Family Fasciolidae, Class Trematoda, Phylum Platyhelminthes)

Nguyen

et al. 2012

J Clin Microbiol

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A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.

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“…In a previous study, Bozas et al (33) isolated a fraction from adult F. hepatica containing a KT inhibitor with activity against trypsin. Furthermore, an F. hepatica KT inhibitor was detected among a preparation that was isolated by affinity purification of adult F. hepatica excretory-secretory products using anti-cysteine protease monoclonal antibodies (MM3) (49). These studies show that the mature parasite also expresses an FhKT and suggest that the parasite produces more than one KT inhibitor that is possibly temporally regulated alongside the secreted cathepsin Ls.…”

Section: Discussionmentioning

confidence: 99%

Unexpected Activity of a Novel Kunitz-type Inhibitor

Smith¹,

Tikhonova

Jewhurst³

et al. 2016

Journal of Biological Chemistry

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Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu15/Arg15) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu15 in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host.

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Molecular and immunological characterization of Fasciola antigens recognized by the MM3 monoclonal antibody

Cited by 35 publications

References 47 publications

The MF6p/FhHDM-1 Major Antigen Secreted by the Trematode Parasite Fasciola hepatica Is a Heme-binding Protein

The MF6p/FhHDM-1 Major Antigen Secreted by the Trematode Parasite Fasciola hepatica Is a Heme-binding Protein

Development and Evaluation of a Single-Step Duplex PCR for Simultaneous Detection of Fasciola hepatica and Fasciola gigantica (Family Fasciolidae, Class Trematoda, Phylum Platyhelminthes)

Unexpected Activity of a Novel Kunitz-type Inhibitor

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