Gut tissue extract prepared from partially engorged I. ricinus females (5th day of feeding) was tested on degradation of the biologically relevant substrate hemoglobin. Proteolysis was analyzed using a fluorescence assay incorporating AMC-hemoglobin. Optimal proteolysis was measured at acidic pH between 3.0 and 4.5 (Fig. 1). No substantial degradation occurred above pH 6.0. At optimum pH (~4.0), hemoglobinolytic activity was inhibited by the small molecule inhibitors E64 and pepstatin that selectively target cysteine and aspartic peptidases, respectively. A combined application of both compounds resulted in nearly complete blockage of the activity (~97% inhibition), the individual treatment showed about 80% inhibition (~83% and ~78% for E64 and pepstatin, respectively). In contrast, inhibitors of serine peptidases and metallopeptidases Pefabloc and EDTA, respectively, were ineffective (data not shown).

Next, we focused on dissecting the major component peptidases in gut extracts responsible for the acidic degradation of blood meal using peptidase selective substrates and inhibitors (Fig. 2). At pH 4.0, hydrolytic activity cleaving the substrate Z-Arg-Arg-AMC (i.e., suggestive of cathepsin B activity) was inhibited 90% by the cathepsin B inhibitor, CA-074. Likewise, activity against Z-Phe-Arg-AMC was inhibited 90% by Z-Phe-Phe-DMK. Dipeptidyl peptidase activity of cathepsin C was measured with Gly-Arg-AMC and inhibited > 95% by Gly-Phe-DMK. Asparaginyl endopeptidase activity as measured with Z-Ala-Ala-Asn-AMC was inhibited > 95% by the azapeptide, Aza-N-11a. Cathepsin D-like activity measured with Abz-Lys-Pro-Ala-Glu-Phe-Nph-Ala-Leu was effectively inhibited (~98%) by pepstatin.

Thus, five significant endo- and exopeptidase activities of the cysteine and aspartic classes of peptidases were profiled in the gut tissue of I. ricinus. The identified activities include (i) the CA clan (papain-type) cysteine peptidases: cathepsins B, L, and C and a Clan CD asparaginyl endopeptidase (legumain), and (ii) a Clan AA aspartic peptidase activity: cathepsin D.

Single stranded cDNA derived from the gut of I. ricinus was used as a template for identification and cloning of genes encoding cysteine and aspartic peptidase precursors. Based on results of the functional screening of peptidase activities, multiple protein and cDNA alignments were performed to identify conserved domains in cathepsins B, L, D and C as shown for the schistosomal orthologues in Table 1. Degenerate primers derived from these motifs are listed together with expected PCR product lengths, optimal annealing temperatures, number of sequenced clones and number of identified isoforms (Table 1). Two isoforms of cathepsin B and one form each of cathepsins L, C and D were identified.

Hybridization screening of the gut cDNA library with radio-labeled PCR amplicons had been previously used to obtain the full-length coding sequence for IrAE 13. Here the same approach succeeded in identifying full length cDNA sequences for cathepsins B, L and D. The full cDNA sequence of cathepsin C was generated by overlapping PCR, 5' and 3' RACE PCR fragments.

For the sake of consistency, we adopted a nomenclature for these enzymes previously used for schistosomal peptidases 1518 – a nomenclature already applied to the I. ricinus asparaginyl endopeptidase (IrAE) 13. Thus, we designated the novel peptidases as IrCB for cathepsin B (form 1), IrCL for cathepsin L, IrCC for cathepsin C and IrCD for cathepsin D.

The three clan CA cysteine peptidases, namely IrCB, IrCL and IrCC could be clearly classified using multiple sequence alignments followed by a phylogenetic analysis (Fig. 3). The GenBank blast program blastp 19 search of the IrCD sequence revealed the closest relation (55% identity) as longepsin, the aspartic peptidase from H. longicornis 12.

Gene-specific PCR primer sets for the newly identified cDNAs and IrAE cDNA (Table 2) were used to amplify the relevant peptidase genes from different tick developmental stages and tissues. Semi-quantitative RT-PCR of whole-body homogenates revealed that IrCL, IrCC and IrAE are abundantly present in all developmental stages including eggs (Fig. 8A). In contrast, messages for IrCB and IrCD were absent from tick eggs. No apparent differences were observed for any enzyme expression between un-fed and freshly attached females suggesting that the enzyme messages are not changed in the initial feeding phase. Once partially engorged, it is possible to reliably dissect individual organs of females for RT-PCR tissue profiling (Fig. 8B) and the data demonstrate that all the peptidases of interest are co-expressed in the gut towards the end of the slow feeding period 7 what indicates their simultaneous action in a putative cascade or network. Moreover, IrCB, IrAE and IrCD seem to be strictly gut-specific, whereas messages for IrCL and IrCC were also found in other tick tissues. Negative controls in which the template cDNA was replaced by sterile distilled water gave no PCR products (data not shown).

I. ricinus ticks were collected by flagging in woodland localities around České Budějovice in the Czech Republic. Adult males and females were kept separately in glass vials in wet chambers with humidity of about 95% and temperature 26°C. If not stated otherwise, the females were allowed to feed naturally for 5 days on laboratory guinea pigs, carefully removed by forceps and referred to as partially engorged ticks in experiments described below. Laboratory animals were treated in accordance with the Animal Protection Law of the Czech Republic no. 246/1992 Sb.

The 7-amino-4-methylcoumarin (AMC)-conjugated bovine hemoglobin was prepared according to Partanen et al. 39. All peptidyl AMC-substrates were from Bachem, Abz-Lys-Pro-Ala-Glu-Phe-Nph-Ala-Leu (Abz, aminobenzoic acid; Nph, 4-nitrophenylalanine) substrate was prepared as described in Máša et al. 40. Peptidase inhibitors were from Bachem, Gly-Phe-diazomethyl ketone (DMK) was prepared as described in Green and Shaw 41. The aza-peptide Michael acceptor (CBz-Ala-Ala-(aza-Asn)-CH = CH-COOEt) further referred to as Aza-N-11a 42 was kindly donated by Dr. J.C. Powers of the School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia.

For an experiment, tissues were dissected from 10 partially engorged I. ricinus females. The gut contents were carefully removed with a special care not to disrupt the epithelium. Cleaned guts were washed in phosphate-buffered saline solution (PBS) and pooled. The gut tissue extract (150 μg protein/ml) was prepared by homogenization of the gut tissue (without contents) in 1 ml of 0.1 M Na-acetate, pH 4.5, 1% CHAPS, 2.5 mM DTT using teflon-glass homogenizer on ice. The homogenate was centrifuged for 10 min at 10000 × g, the supernatant was filtered with Micropure-0.22 Separator (Millipore) and stored at -80°C.

Hemoglobinolytic activity was assayed using AMC-hemoglobin as a fluorogenic substrate 43. Digestion of fluorogenic AMC-hemoglobin (0.5 μg supplemented with 2 μg of bovine hemoglobin) was performed at 35°C with the gut tissue extract (20-fold diluted stock solution) in 0.1 M Na-citrate-phosphate, pH 2.5–8.0 including 2.5 mM DTT, 25 mM NaCl and 0.05% Tween 20 in a reaction mixture of 100 μl. The proteolytic fragmentation of AMC-hemoglobin results in an increase of the fluorescence intensity that was continuously monitored to determine the relative reaction rate. The fluorescence signal was measured using a GENios Plus reader (TECAN) at 360 nm excitation and 465 nm emission wavelengths. For the hemoglobinolytic assay in the presence of a peptidase inhibitor, an aliquot of the extract was preincubated (15 min at 35°C) in the same buffer pH 4.0 with 10 μM E64, 10 μM pepstatin, 1 mM Pefabloc or 1 mM EDTA.

Peptidase activities were identified and characterized by hydrolysis of the following fluorogenic substrates: 25 μM Z-Arg-Arg-AMC for cathepsin B 44, 25 μM Z-Phe-Arg-AMC for cathepsin L 44, 30 μM Gly-Arg-AMC for cathepsin C 45, 40 μM Abz-Lys-Pro-Ala-Glu-Phe-Nph-Ala-Leu for cathepsin D 40, and 30 μM Z-Ala-Ala-Asn-AMC for asparaginyl endopeptidase 46 as described previously 4047. The activity measurement was performed at 35°C using an aliquot of the gut tissue extract (20 to 200-fold diluted stock solution) in 0.1 M Na-acetate, pH 4.0 including 2.5 mM DTT (for cysteine peptidases) and 25 mM NaCl (for cathepsin C). For the activity assay in the presence of peptidase inhibitors, an aliquot of the extract was pre-incubated (15 min at 35°C) in the same buffer with the inhibitor: 10 μM CA-074 for cathepsin B 48, 10 μM Z-Phe-Phe-DMK for cathepsin L 49, 1 μM Gly-Phe-DMK for cathepsin C 41, 10 μM pepstatin for cathepsin D 50 or 1 μM Aza-N-11a for asparaginyl endopeptidase 42. Hydrolytic activity was continuously measured after addition of substrate in a fluorescence reader GENios Plus at 320 nm excitation and 420 nm emission wavelengths (for Abz-containing substrate) or at 360 nm excitation and 465 nm emission wavelengths (for AMC-containing substrates). Assays of asparaginyl endopeptidase and cathepsin L were measured in the presence of 10 μM CA-074 to prevent confounding hydrolysis by cathepsin B 1647.

Tissues (gut, salivary glands, ovaries and Malpighian tubules) were dissected from partially engorged females in a wax filled Petri dish with phosphate-buffered saline (PBS) under a binocular dissection microscope. The whole body homogenates from different developmental stages were prepared by crushing the appropriate number of eggs, larvae, nymphs, males, unfed females and females removed from guinea pigs one day after attachment using mortar and pestle and repeated freezing under liquid nitrogen. For total RNA isolation, the samples were further homogenized in a micro-tube with a plastic pestle in the TRI Reagent^® solution (Sigma) at 1 ml per 50–100 mg of wet tissue and processed according to the instructions provided with the TRI-reagent kit (Sigma). Isolated total RNA was stored at -80°C and further used for preparing single stranded cDNA templates using Superscript II (Invitrogen) and oligo(dT) primers, following the instructions provided by the manufacturer or for RT-PCR experiments described below.

Degenerate primers were designed from conserved domains of Schistosoma mansoni, S. japonicum, mosquito, rat and human peptidases. Protein and nucleotide sequences downloaded from the NCBI GenBank web site were used for multiple ClustalW alignments in DNAstar MegAlign software (Lasergene). The oligonucleotides are listed in Table 1.

Mastercycler gradient (Eppendorf) was used to optimize PCR amplifications. Amplicons were purified, ligated into vector plasmid pCR 4-TOPO using the TOPO TA^® Cloning Kit (Invitrogen) and transformed into E. coli TOP 10 cells (Invitrogen). Clones containing ligated PCR products were sequenced using an automated sequencer model ABI Prism 3130 XL and the BigDye^® Terminator sequencing kit (Applied Biosystems) with appropriate sequencing primers. Sequence data were compared by blastn 51 against the NCBI GenBank database records. To obtain complete cDNA sequences, 3' RACE PCR was performed using a modified protocol for SMART™ cDNA Library Construction Kit (Clontech, BD Biosciences) described previously 52. The N-terminal sequences including signal peptides and the 5' untranslated regions were determined using the Invitrogen 5'RACE system and instructions provided by the manufacturer.

The protocol for construction of the I. ricinus gut-derived cDNA library with SMART™ cDNA Library Construction Kit (Clontech, BD Biosciences) and the Gigapack^® III Gold packaging extract (Stratagene) as well as the method of cDNA library screening by [P³²]dATP radio-labeled gene-specific probes have been described previously 13.

The primary sequences used for phylogenetic analysis comprised the conserved domains spanning across the mature enzyme sequences without pro-domains. Sequences were obtained from the MEROPS database 53 and aligned in the program ClustalX 1.81 54. The alignment was manually checked using the BioEdit program 55. Tree reconstruction employed the Neighbor Joining (NJ) method 56 in the program MEGA 2.1 57. Nodal supports were calculated with 1000 replications.

Gene specific PCR primer pairs (listed in Table 2) were designed for each peptidase type with DNAstar PrimerSelect software (Lasergene). Two-step RT-PCR was performed using total RNA templates (prepared as described above; 50 ng/μl final concentration) and the Enhanced Avian HS RT-PCR Kit (Sigma) according to the protocol provided by the manufacturer. Amplification of the ferritin mRNA, previously shown to be presented in all tick tissues and expressed independently of feeding 58, was used as a loading control.