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Zdenek Franta,
Daniel Sojka,
Helena Frantova,
Jan Dvorak,
Martin Horn,
Jindrich Srba,
Pavel Talacko,
Michael Mares, Eric Schneider,
Charles S Craik,
James H McKerrow,
Conor R Caffrey,
Petr Kopacek
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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.
International journal for parasitology 07/2011; 41(12):1253-62. · 3.39 Impact Factor
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Carlos E Cruz,
Andréa C Fogaça,
Ernesto S Nakayasu,
Cláudia B Angeli,
Rodrigo Belmonte,
Igor C Almeida,
Antônio Miranda,
Maria Terêsa M Miranda,
Aparecida S Tanaka,
Glória R Braz,
Charles S Craik, Eric Schneider,
Conor R Caffrey,
Sirlei Daffre
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ABSTRACT: Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins.
An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'.
BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.
Parasites & Vectors 01/2010; 3:63. · 2.94 Impact Factor
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Carlos Cruz,
Andréa Fogaça,
Ernesto Nakayasu,
Cláudia Angeli,
Rodrigo Belmonte,
Igor Almeida,
Antônio Miranda,
Maria Miranda,
Aparecida Tanaka,
Glória Braz,
Charles Craik, Eric Schneider,
Conor Caffrey,
Sirlei Daffre
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ABSTRACT: Abstract
Background
Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins.
Results
An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus ( Boophilus ) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'.
Conclusions
BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro . We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.
Parasites & Vectors. 01/2010;
