Isolation and characterization of digestive aspartic and cysteine proteinases involved in the generation of the antimicrobial peptide Hb 33-61 from the tick Boophilus microplus
Cruz, C.E.1; Fogaça, A.C.2; Miranda, A.3; Daffre, S.1
1 Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo
2 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo
3 Departamento de Biofísica, Universidade Federal de São Paulo
The peptide Hb 33-61 is a proteolytic product of the a-chain of bovine hemoglobin purified from the gut contents of the tick Boophilus microplus (J. Biol. Chem. 1999 274: 25330-25334). Previous studies suggested the presence of aspartic and cysteine proteinases involved in the generation of this peptide. In order to further characterize these digestive proteinases, enzyme specific activities were initially determined in the gut contents as well as in homogenates of digestive cells. These activities were determined at pH 4 and pH 7 using chemically synthesized fluorogenic substrates containing the aminoacid sequences 29-35 (named SF 29-35) and 57-67 (named SF 57-67) of the a-chain of bovine hemoglobin. Using SF 29-35, aspartic proteinase activity was detected at pH 4 in both preparations, but was higher in the digestive cells. Besides, these activities were inhibited by pepstatin. Cysteine proteinase activity, inhibited by E-64, was observed at pHs 4 and 7 using SF 57-67. For the purification of these proteinases, we used a total gut homogenate, considering that, during tissue dissection, epithelial cells were not completely freed from the luminal contents. Firstly, a gut homogenate was precipitated with three ammonium sulfate concentrations (30%, 55% and 80%). After solubilization and salt removal, the 80% ammonium sulfate precipitate showed the highest specific activity for both classes of proteinases and thus was used in further chromatographic steps. Through a FPLC system, this fraction was applied onto a Mono Q HR 5/5 column (Pharmacia) previously equilibrated with 50 mM Tris-HCl pH 7.4 and eluted with a 0-0.5 M NaCl gradient in the same buffer. This chromatography yielded two aspartic proteinase peaks that cleaved SF 29-35 at pH 4 and a major cysteine proteinase activity peak, increased by DTT, which cleaved SF 57-67 at pHs 4 and 7. These proteinases are being purified through gel filtration and ion exchange chromatographies. The sites of aminoacid cleavage of SF 29-35 and SF 57-67 will be analyzed through liquid chromatography coupled to mass spectrometry. We are also working on the kinetic characterization of these proteinases. Supported by FAPESP and CNPq.
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