title: Major acid endopeptidases of the blood-feeding monogenean Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae).
abstract: In parasitic flatworms, acid endopeptidases are involved in crucial processes, including digestion, invasion, interactions with the host immune system, etc. In haematophagous monogeneans, however, no solid information has been available about the occurrence of these enzymes. Here we aimed to identify major cysteine and aspartic endopeptidase activities in Eudiplozoon nipponicum, an invasive haematophagous parasite of common carp. Employing biochemical, proteomic and molecular tools, we found that cysteine peptidase activities prevailed in soluble protein extracts and excretory/secretory products (ESP) of E. nipponicum; the major part was cathepsin L-like in nature supplemented with cathepsin B-like activity. Significant activity of the aspartic cathepsin D also occurred in soluble protein extracts. The degradation of haemoglobin in the presence of ESP and worm protein extracts was completely inhibited by a combination of cysteine and aspartic peptidase inhibitors, and diminished by particular cathepsin L, B and D inhibitors. Mass spectrometry revealed several tryptic peptides in ESP matching to two translated sequences of cathepsin L genes, which were amplified from cDNA of E. nipponicum and bioinformatically annotated. The dominance of cysteine peptidases of cathepsin L type in E. nipponicum resembles the situation in, e.g. fasciolid trematodes.
authors.affiliation: Department of Parasitology, Faculty of Science,Charles University in Prague,Viničná 7,128 44 Prague 2,Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Jedličková
authors.affiliation: Department of Parasitology, Faculty of Science,Charles University in Prague,Viničná 7,128 44 Prague 2,Czech Republic.
authors.forename: Hana
authors.initials: H
authors.lastname: Dvořáková
authors.affiliation: Department of Parasitology, Faculty of Science,Charles University in Prague,Viničná 7,128 44 Prague 2,Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Kašný
authors.affiliation: Department of Botany and Zoology,Faculty of Science,Masaryk University,Kamenice 753/5 (A31),625 00 Brno,Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Ilgová
authors.affiliation: RG Proteomics,Central European Institute of Technology,Masaryk University,Brno,Kamenice 753/5 (A26),625 00 Brno Bohunice,Czech Republic.
authors.forename: David
authors.initials: D
authors.lastname: Potěšil
authors.affiliation: RG Proteomics,Central European Institute of Technology,Masaryk University,Brno,Kamenice 753/5 (A26),625 00 Brno Bohunice,Czech Republic.
authors.forename: Zbyněk
authors.initials: Z
authors.lastname: Zdráhal
authors.affiliation: Department of Parasitology, Faculty of Science,Charles University in Prague,Viničná 7,128 44 Prague 2,Czech Republic.
authors.forename: Libor
authors.initials: L
authors.lastname: Mikeš
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D056668:Cathepsin L / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid; D057056:Cysteine Proteases / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000378:metabolism; D010986:Platyhelminths / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011480:Protease Inhibitors / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry
chemical_list: D018076:DNA, Complementary; D005456:Fluorescent Dyes; D010455:Peptides; D011480:Protease Inhibitors; D057056:Cysteine Proteases; D010450:Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L; D002402:Cathepsin D
keywords: aspartic peptidase; cathepsin B; cathepsin D; cathepsin L; common carp; cysteine peptidase; fish parasite; haematophagous monogenea; protease