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 (HlLAP). Endogenous HlLAP was detected in the soluble fraction of adult tick extracts by immunoblotting. Immunohistochemical studies demonstrated that endogenous HlLAP expression mainly took place in the cytosol of midgut epithelial cells. Furthermore, expression of HlLAP was induced by a blood-feeding process. A functional recombinant HlLAP expressed in Escherichia coli efficiently hydrolyses synthetic substrates for aminopeptidase, a leucyl (with the Km value 0.19 +/- 0.011 mM and Vmax value 157.2 +/- 3.17 nmol/min/mgprotein) and a methionyl substrate (with the Km value 0.12+/-0.0052 mM and Vmax value 171.9 +/- 2.31 nmol/min/mgprotein). Enzyme activity was found to be optimum at pH 8 and 35 degrees C. The recombinant HlLAP enzyme activity was strongly dependent on metal divalent cations, Mn2+, and was inhibited by bestatin. These results indicate that HlLAP play an important role for host's blood digestion process.
Hatta
Takeshi
T
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Kazama
Kouji
K
Miyoshi
Takeharu
T
Umemiya
Rika
R
Liao
Min
M
Inoue
Noboru
N
Xuan
Xuenan
X
Tsuji
Naotoshi
N
Fujisaki
Kozo
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019149:Bioreactors; D018076:DNA, Complementary / Q000032:analysis; D004063:Digestion; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D004926:Escherichia coli; D015151:Immunoblotting; D007150:Immunohistochemistry; D007422:Intestines / Q000201:enzymology*; D026863:Ixodidae / Q000201:enzymology* / Q000502:physiology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000032:analysis* / Q000235:genetics; D008345:Manganese; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
D018076:DNA, Complementary; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D008345:Manganese; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex