title: IrCL1 - the haemoglobinolytic cathepsin L of the hard tick, Ixodes ricinus.
abstract: Intracellular proteolysis of ingested blood proteins is a crucial physiological process in ticks. In our model tick, Ixodes ricinus, cathepsin L (IrCL1) is part of a gut-associated multi-peptidase complex; its endopeptidase activity is important in the initial phase of haemoglobinolysis. We present the functional and biochemical characterisation of this enzyme. We show, by RNA interference (RNAi), that cathepsin L-like activity that peaks during the slow feeding period of females is associated with IrCL1. Recombinant IrCL1 was expressed in bacteria and yeast. Activity profiling with both peptidyl and physiological protein substrates (haemoglobin and albumin) revealed that IrCL1 is an acidic peptidase with a very low optimum pH (3-4) being unstable above pH 5. This suggests an endo/lysosomal localisation that was confirmed by indirect fluorescence microscopy that immunolocalised IrCL1 inside the vesicles of digestive gut cells. Cleavage specificity determined by a positional scanning synthetic combinatorial library and inhibition profile indicated that IrCL1 has the ligand-binding characteristics of the cathepsin L subfamily of cysteine peptidases. A non-redundant proteolytic function was demonstrated when IrCL1-silenced ticks had a decreased ability to feed compared with controls. The data suggest that IrCL1 may be a promising target against ticks and tick-borne pathogens.
authors.affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Ceske Budejovice, CZ 37005, Czech Republic.
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Franta
authors.forename: Daniel
authors.initials: D
authors.lastname: Sojka
authors.forename: Helena
authors.initials: H
authors.lastname: Frantova
authors.forename: Jan
authors.initials: J
authors.lastname: Dvorak
authors.forename: Martin
authors.initials: M
authors.lastname: Horn
authors.forename: Jindrich
authors.initials: J
authors.lastname: Srba
authors.forename: Pavel
authors.initials: P
authors.lastname: Talacko
authors.forename: Michael
authors.initials: M
authors.lastname: Mares
authors.forename: Eric
authors.initials: E
authors.lastname: Schneider
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: James H
authors.initials: JH
authors.lastname: McKerrow
authors.forename: Conor R
authors.initials: CR
authors.lastname: Caffrey
authors.forename: Petr
authors.initials: P
authors.lastname: Kopacek
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D056668:Cathepsin L / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000201:enzymology; D004795:Enzyme Stability; D005260:Female; D015870:Gene Expression; D020868:Gene Silencing; D006454:Hemoglobins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018884:Ixodes / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D059748:Proteolysis; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000418:Albumins; D006454:Hemoglobins; D011994:Recombinant Proteins; D056668:Cathepsin L