title: Characterization of gut-associated cathepsin D hemoglobinase from tick Ixodes ricinus (IrCD1).
abstract: To identify the gut-associated tick aspartic hemoglobinase, this work focuses on the functional diversity of multiple Ixodes ricinus cathepsin D forms (IrCDs). Out of three encoding genes representing Ixodes scapularis genome paralogs, IrCD1 is the most distinct enzyme with a shortened propeptide region and a unique pattern of predicted post-translational modifications. IrCD1 gene transcription is induced by tick feeding and is restricted to the gut tissue. The hemoglobinolytic role of IrCD1 was further supported by immunolocalization of IrCD1 in the vesicles of tick gut cells. Properties of recombinantly expressed rIrCD1 are consistent with the endo-lysosomal environment because the zymogen is autoactivated and remains optimally active in acidic conditions. Hemoglobin cleavage pattern of rIrCD1 is identical to that produced by the native enzyme. The preference for hydrophobic residues at the P1 and P1' position was confirmed by screening a novel synthetic tetradecapeptidyl substrate library. Outside the S1-S1' regions, rIrCD1 tolerates most amino acids but displays a preference for tyrosine at P3 and alanine at P2'. Further analysis of the cleavage site location within the peptide substrate indicated that IrCD1 is a true endopeptidase. The role in hemoglobinolysis was verified with RNAi knockdown of IrCD1 that decreased gut extract cathepsin D activity by >90%. IrCD1 was newly characterized as a unique hemoglobinolytic cathepsin D contributing to the complex intestinal proteolytic network of mainly cysteine peptidases in ticks.
authors.affiliation: From the Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, České Budějovice, CZ 370 05, Czech Republic.
authors.forename: Daniel
authors.initials: D
authors.lastname: Sojka
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Franta
authors.forename: Helena
authors.initials: H
authors.lastname: Frantová
authors.forename: Pavla
authors.initials: P
authors.lastname: Bartosová
authors.forename: Martin
authors.initials: M
authors.lastname: Horn
authors.forename: Jana
authors.initials: J
authors.lastname: Váchová
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: O'Donoghue
authors.forename: Alegra A
authors.initials: AA
authors.lastname: Eroy-Reveles
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Giselle M
authors.initials: GM
authors.lastname: Knudsen
authors.forename: Conor R
authors.initials: CR
authors.lastname: Caffrey
authors.forename: James H
authors.initials: JH
authors.lastname: McKerrow
authors.forename: Michael
authors.initials: M
authors.lastname: Mares
authors.forename: Petr
authors.initials: P
authors.lastname: Kopácek
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism*; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D016678:Genome / Q000502:physiology; D006454:Hemoglobins / Q000235:genetics / Q000378:metabolism*; D007422:Intestines / Q000201:enzymology*; D018884:Ixodes / Q000201:enzymology* / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology
chemical_list: D060829:Arthropod Proteins; D006454:Hemoglobins; D011994:Recombinant Proteins; D002402:Cathepsin D