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.
- MeSH
- genetická transkripce fyziologie MeSH
- genom fyziologie MeSH
- hemoglobiny genetika metabolismus MeSH
- kathepsin D genetika metabolismus MeSH
- klíště enzymologie genetika MeSH
- posttranslační úpravy proteinů fyziologie MeSH
- proteiny členovců genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- střeva enzymologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- albuminy metabolismus MeSH
- endozomy enzymologie MeSH
- exprese genu MeSH
- fluorescenční mikroskopie MeSH
- hemoglobiny metabolismus MeSH
- kathepsin L chemie genetika metabolismus MeSH
- klíště enzymologie MeSH
- koncentrace vodíkových iontů MeSH
- proteolýza MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- RNA interference MeSH
- stabilita enzymů MeSH
- umlčování genů MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The universal protease inhibitors of the alpha(2)-macroglobulin (alpha(2)M) family are evolutionarily conserved constituents of innate immunity, presumably because they guard organisms against undesired proteolytic attacks of a different origin. Here, we determined the primary structure of alpha(2)-macroglobulin from the hard tick Ixodes ricinus (IrAM) by sequencing of overlapping PCR products. Predicted disulfide and glycosylation patterns, post-translational cleavage and alternative splicing within its 'bait region' demonstrate that IrAM is closely related to the alpha(2)-macroglobulin from the soft tick Ornithodoros moubata. The IrAM message is expressed in all tick developmental stages and tissues, except for the gut, and the protein was detected to be mainly present in the hemolymph. Silencing of IrAM by dsRNA interference markedly reduced the phagocytosis of a potential pathogen, Chryseobacterium indologenes, by tick hemocytes both in vitro and in vivo. In contrast, phagocytosis of the Lyme disease spirochete Borrelia burgdorferi or a commensal bacteria Staphylococcus xylosus was not affected by the IrAM knock-down. Similar results were obtained upon deactivation of all thioester proteins in tick hemolymph by methylamine. We have further demonstrated that phagocytosis of C. indologenes is dependent on an active metalloprotease secreted by the bacteria. These data indicate that interaction of tick alpha(2)-macroglobulin with a protease of an invading pathogen is linked with cellular immune response.
- MeSH
- alfa-makroglobuliny farmakologie genetika chemie imunologie MeSH
- Chryseobacterium imunologie MeSH
- fagocytóza imunologie MeSH
- fenantroliny farmakologie MeSH
- financování organizované MeSH
- hemocyty imunologie mikrobiologie účinky léků MeSH
- hemolymfa imunologie MeSH
- klíště genetika imunologie mikrobiologie MeSH
- metaloproteasy metabolismus účinky léků MeSH
- methylaminy farmakologie MeSH
- molekulární sekvence - údaje MeSH
- RNA interference MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
Ticks are among the most important vectors of a wide range of human and animal diseases. During blood feeding, ticks are exposed to an enormous amount of free iron that must be appropriately used and detoxified. However, the mechanism of iron metabolism in ticks is poorly understood. Here, we show that ticks possess a complex system that efficiently utilizes, stores and transports non-heme iron within the tick body. We have characterized a new secreted ferritin (FER2) and an iron regulatory protein (IRP1) from the sheep tick, Ixodes ricinus, and have demonstrated their relationship to a previously described tick intracellular ferritin (FER1). By using RNA interference-mediated gene silencing in the tick, we show that synthesis of FER1, but not of FER2, is subject to IRP1-mediated translational control. Further, we find that depletion of FER2 from the tick plasma leads to a loss of FER1 expression in the salivary glands and ovaries that normally follows blood ingestion. We therefore suggest that secreted FER2 functions as the primary transporter of non-heme iron between the tick gut and the peripheral tissues. Silencing of the fer1, fer2, and irp1 genes by RNAi has an adverse impact on hatching rate and decreases postbloodmeal weight in tick females. Importantly, knockdown of fer2 dramatically impairs the ability of ticks to feed, thus making FER2 a promising candidate for development of an efficient anti-tick vaccine.
- MeSH
- analýza přežití MeSH
- biologické modely MeSH
- ferritin genetika MeSH
- financování organizované MeSH
- fylogeneze MeSH
- hmyzí geny MeSH
- hmyzí proteiny genetika metabolismus MeSH
- intracelulární prostor metabolismus MeSH
- klíšťata fyziologie genetika růst a vývoj MeSH
- klonování DNA MeSH
- messenger RNA genetika metabolismus MeSH
- morčata MeSH
- proteosyntéza MeSH
- regulace genové exprese MeSH
- rozmnožování MeSH
- stanovení celkové genové exprese MeSH
- stravovací zvyklosti MeSH
- umlčování genů MeSH
- western blotting MeSH
- železo metabolismus MeSH
- zvířata MeSH
- Check Tag
- morčata MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates and inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and to determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.
- MeSH
- cysteinové endopeptidasy metabolismus MeSH
- endopeptidasy metabolismus MeSH
- hemoglobiny chemie metabolismus MeSH
- inhibitory enzymů farmakologie MeSH
- katalytická doména MeSH
- kathepsin B metabolismus MeSH
- kathepsin C metabolismus MeSH
- kathepsin D metabolismus MeSH
- kathepsin L metabolismus MeSH
- klíště enzymologie MeSH
- koncentrace vodíkových iontů MeSH
- molekulární sekvence - údaje MeSH
- proteomika metody MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite's ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterised a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which we believe is the first such characterisation of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and EM localised IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH > or = 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 -- an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites.
- MeSH
- cysteinové endopeptidasy genetika chemie izolace a purifikace MeSH
- financování organizované MeSH
- fluorescenční protilátková technika nepřímá MeSH
- fylogeneze MeSH
- hemoglobiny metabolismus MeSH
- kathepsin B metabolismus MeSH
- klíště enzymologie genetika MeSH
- klonování DNA MeSH
- messenger RNA biosyntéza genetika MeSH
- molekulární sekvence - údaje MeSH
- Pichia genetika metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- transmisní elektronová mikroskopie MeSH
- trávicí systém MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
