Abstract
BackgroundHemoglobin 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.ResultsAn 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'.ConclusionsBmAP 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.
Highlights
Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials
The one-host ixodid tick Rhipicephalus (Boophilus) microplus is a haematophagous ectoparasite of great veterinary importance in tropical and subtropical regions, owing to its involvement in the transmission of diseases to cattle, such as babesiosis and anaplasmosis, that result in severe economic losses [1]
Proteinase activity in R. (B.) microplus midgut homogenate and digestive cell lysate To determine which proteinase classes from the midgut homogenate and digestive cell lysate are responsible for the cleavage of hemoglobin to generate the hemocidin Hb 33-61, we used the fluorescent substrates SF 29-35 (Abz-LERMFLSQ-ethylene diamine-2-4-dinitrophenyl (EDDnp)) and SF 57-67 (Abz-GHGAKVAAALTQQ-EDDnp) containing the amino acid sequences 29-35 and 57-67 of the a-chain of bovine hemoglobin, and flank the N- and C-terminus of Hb 3361, respectively, in combination with specific inhibitors
Summary
Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). (B.) microplus cause anaemia in cattle, as a single engorged female can increase its weight by 100 to 200 times [2]. Such prodigious engorging capacity involves proteolysis of host proteins, which occurs predominantly inside acidic vesicles of digestive cells. The main proteolytic activities in the midgut are acidic aspartic and cysteine proteinases; exopeptidases may participate in the final stages of digestion [6,7,8,9]. A hemoglobinolytic cathepsin L-like enzyme, named BmCL1 [GenBank:AF227957], was characterized and immunolocalized to the midgut cells of partially engorged adult female ticks [8,10]
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