Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

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.

Figure 1

Purification of aspartic proteinases from tick midgut homogenates. Purification was accomplished in three chromatographic steps: anion exchange chromatography (A), hydrophobic interaction chromatography (B) and pepstatin affinity chromatography (C) in FPLC system. Enzyme activity was assayed with SF 29-35 (-o-). Absorbance was monitored at 280 nm (- -). NaCl gradients (---) were developed as described in Methods. (D) SDS-PAGE of active fraction eluted from the pepstatin affinity chromatography (see also Additional File 1).

Figure 2

Biochemical characterization of BmAP. (A) Effect of pH on BmAP activity. Buffer used: 100 mM citrate-phosphate pH 2.5-7.5. (B) Thermal inactivation of BmAP at 68°C at pH 4.5. (C) and (D) Determination of the cleavage sites of SF 29-35 by BmAP using LC/MS.

Figure 3

Nucleotide and deduced amino acid sequence of R. (B.) microplus aspartic proteinase cDNA. The predicted 20-aa signal peptide is underlined. The conserved residues involved in catalysis are in rectangles. The regions identified by LC-MS/MS are shaded. The cysteine residues involved in dissulfide bond formation are in black. The GenBank accession number for this sequence is FJ655904.

Figure 4

Amino acid sequence alignment of aspartic proteinases. BmAP, Rhipicephalus (B.) microplus aspartic proteinase [GenBank:FJ655904]; HlAP, Haemaphysalis longicornis aspartic proteinase [GenBank:BAE53722]; IrAP, Ixodes ricinus aspartic proteinase [GenBank:EF428204]; THAP, tick heme-binding aspartic proteinase [GenBank:AF286865]; BYC, Boophilus Yolk pro-Cathepsin [GenBank:AY966003]. The catalytic residues are indicated with asterisks.

Figure 5

Biochemical properties of recombinant BmCL1 expressed in Pichia pastoris. (A) SDS-PAGE of BmCL1 purified on ion exchange chromatography, as described in Methods. (B) Thermal inactivation at 68°C at pH 4.5. (C) Determination of the cleavage sites of SF 57-67 by BmCL1 using LC/MS.

Figure 6

Subsite specificity of recombinant BmCL1 using a tetrapeptide library. A P1-P4 positional scanning synthetic combinatorial library (PS-SCL) was used to determine specificities at pH 4.5 (A) and pH 5.5 (B). Activities are represented as the percentage of the maximum activity for each subsite position. Data represent means ± SEM of three runs.

Figure 7

Peptides generated by acid hemoglobinolysis. Peptides were identified by LC-MS/MS after hemoglobinolysis with native BmAP (A), BmCL1 (B) or both enzymes (C). Digestion was performed in 100 mM citrate-phosphate buffer pH 4.5 at 37°C for up to 4 h, as described in Methods.