title: Multiple cathepsin B isoforms in schistosomula of Trichobilharzia regenti: identification, characterisation and putative role in migration and nutrition.
abstract: Among schistosomatids, Trichobilharzia regenti, displays an unusual migration through the peripheral and central nervous system prior to residence in the nasal cavity of the definitive avian host. Migration causes tissue degradation and neuromotor dysfunction both in birds and experimentally infected mice. Although schistosomula have a well-developed gut, the peptidases elaborated that might facilitate nutrition and migration are unknown. This is, in large part, due to the difficulty in isolating large numbers of migrating larvae. We have identified and characterised the major 33 kDa cathepsin B-like cysteine endopeptidase in extracts of migrating schistosomula using fluorogenic peptidyl substrates with high extinction coefficients and irreversible affinity-labels. From first strand schistosomula cDNA, degenerate PCR and Rapid Amplification of cDNA End protocols were used to identify peptidase isoforms termed TrCB1.1-TrCB1.6. Highest sequence homology is to the described Schistosoma mansoni and Schistosoma japonicum cathepsins B1. Two isoforms (TrCB1.5 and 1.6) encode putatively inactive enzymes as the catalytic cysteine is substituted by glycine. Two other isoforms, TrCB1.1 and 1.4, were functionally expressed as zymogens in Pichia pastoris. Specific polyclonal antibodies localised the peptidases exclusively in the gut of schistosomula and reacted with a 33kDa protein in worm extracts. TrCB1.1 zymogen was unable to catalyse its own activation, but was trans-processed and activated by S. mansoni asparaginyl endopeptidase (SmAE aka. S. mansoni legumain). In contrast, TrCB1.4 zymogen auto-activated, but was resistant to the action of SmAE. Both activated isoforms displayed different pH-dependent specificity profiles with peptidyl substrates. Also, both isoforms degraded myelin basic protein, the major protein component of nervous tissue, but were inefficient against hemoglobin, thus supporting the adaptation of T. regenti gut peptidases to parasitism of host nervous tissue.
authors.affiliation: Department of Parasitology, Faculty of Science, Charles University, Vinicná 7, CZ 12844 Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Dvorák
authors.forename: Melaine
authors.initials: M
authors.lastname: Delcroix
authors.forename: Andrea
authors.initials: A
authors.lastname: Rossi
authors.forename: Václav
authors.initials: V
authors.lastname: Vopálenský
authors.forename: Martin
authors.initials: M
authors.lastname: Pospísek
authors.forename: Miroslava
authors.initials: M
authors.lastname: Sedinová
authors.forename: Libor
authors.initials: L
authors.lastname: Mikes
authors.forename: Mohammed
authors.initials: M
authors.lastname: Sajid
authors.forename: Andrej
authors.initials: A
authors.lastname: Sali
authors.forename: James H
authors.initials: JH
authors.lastname: McKerrow
authors.forename: Petr
authors.initials: P
authors.lastname: Horák
authors.forename: Conor R
authors.initials: CR
authors.lastname: Caffrey
mesh_terms: D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000032:analysis / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004792:Enzyme Precursors / Q000032:analysis; D007150:Immunohistochemistry / Q000379:methods; D007536:Isomerism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism; D018510:RNA, Helminth / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis; D012551:Schistosomatidae / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment / Q000379:methods; D014158:Transcription, Genetic; D014201:Trematode Infections / Q000378:metabolism*; D000099240:Asparaginyl Endopeptidase
chemical_list: D002401:Cathepsin B; D003546:Cysteine Endopeptidases; D004792:Enzyme Precursors; D004676:Myelin Basic Protein; D018510:RNA, Helminth; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase