Identification and characterisation of a leucine aminopeptidase from the hard tick Haemaphysalis longicornis

Aminopeptidases responsible for blood digestion have yet to be identified in haematophagous ticks. We report here the cloning and molecular characterisation of a cDNA encoding leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from the hard tick Haemaphysalis longicornis (HILAP). Endogenous HILAP was detected in the soluble fraction of adult tick extracts by immunoblotting. Immunohistochemical studies demonstrated that endogenous HILAP expression mainly took place in the cytosol of midgut epithelial cells. Furthermore, expression of HILAP was induced by a blood-feeding process. A functional recombinant HILAP expressed in Escherichia coli efficiently hydrolyses synthetic substrates for aminopeptidase, a leucyl (with the K-m value 0.19 +/- 0.011 mM and V-max, value 157.2 +/- 3.17 nmol/min/mg protein) and a methionyl substrate (with the K-m value 0.12 +/- 0.0052 mM and Vmax value 171.9 +/- 2.31 nmol/min/mg protein). Enzyme activity was found to be optimum at pH 8 and 35 degrees C. The recombinant HILAP enzyme activity was strongly dependent on metal divalent cations, Mn2+, and was inhibited by bestatin. These results indicate that HILAP play an important role for host's blood digestion process. (c) 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.