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CRUZ CE. Characterization of proteinases involved in the generation of antimicrobial peptides in the gut of Rhipicephalus (Boophilus) microplus [PhD Thesis]. São Paulo. Graduate Program in Parasitology. Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo; 2009. Research data has shown that hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). The first hemoglobinderived antimicrobial peptide characterized in ticks was Hb 33-61. This peptide was purified from midgut contents of the cattle tick Rhipicephalus (Boophilus) microplus and showed activity against both Gram-positive bacteria and fungi (Journal of Biological Chemistry, 274 (36): 25330-34, 1999). There is evidence that hemocidins are generated proteolytically in the tick midgut from hemoglobin. In this work we report the identification and biochemical characterization of an aspartic proteinase, designated BmAP, which was isolated from the tick midgut using three chromatographic steps. Its cDNA was cloned and sequenced, and expression analysis by qPCR revealed that BmAP is predominantly expressed in the midgut when compared with expression levels in salivary glands and ovaries. Electrospray mass spectrometry was employed to map the cleavage specificity of this proteinase using bovine hemoglobin as substrate, which showed that hydrophobic residues are preferentially cleaved at the P1 and P1’ subsites. We also investigated the cleavage specificity of BmCL1, a cathepsin L immunolocalized to the intestine of R. (B.) microplus (Insect Biochemistry and Molecular Biology, 30 (11): 1017-26, 2000), using a combinatorial library of synthetic peptides and through in vitro hemoglobinolysis. BmCL1 hydrolysed bovine hemoglobin preferentially at sites containing aliphatic residues at position P2 and polar residues at P1 and P1’. This enzyme also hydrolysed the α chain of bovine hemoglobin at Ala63/Ala64, generating peptides with a primary structure similar to that of Hb 33-61. Hemoglobinolysis with BmAP and/or BmCL1 resulted in the formation of several peptides with molecular masses between 982 and 3404 Da, some of which showed antimicrobial activity, corroborating the hypothesis that these proteinases are involved in the endogenous generation of hemocidins in the tick gut. We hypothesize that Hb 33-61 is generated by the activity of three enzymes: BmAP, which is responsible for the hydrolysis of the α chain of bovine hemoglobin at Met32/Phe33, BmCL1, which hydrolyses the α chain at Ala63/Ala64 and an exopeptidase, which would remove residues Val62 and Ala63.

Caracterização de proteinases envolvidas na geração de peptídeos antimicrobianos no intestino de Rhipicephalus (Boophilus) microplus.