Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis.
We characterize here a cDNA from the ixodid tick Haemaphysalis longicornis, which encodes an asparaginyl endopeptidase, legumain (HlLgm), that was present as a functional molecule in the midgut of this tick. Endogenous HlLgm was detected as a 38-kDa antigen in H. longicornis extracts and was seen throughout all developmental stages. Endogenous HlLgm was mainly localized in the midgut epithelium by immunohistochemistry, and was shown to be up-regulated by the host blood-feeding process. Recombinant HlLgm (rHlLgm) produced in Escherichia coli was shown to hydrolyze the synthetic substrate Z-Ala-Ala-Asn-MCA at the rate of 6.42x10(-4)mumol/min/mg protein. Its activity was inhibited by the thiol blocking reagents iodoacetamide and N-ethylmaleimide. The enzyme was shown to possess a unique feature of having an autocatalyzed cleavage at asparagines(364-365) at the C-terminus of both endogenous HlLgm and rHlLgm. rHlLgm degraded bovine hemoglobin and bovine serum albumin (BSA) showing its strict specificity for hydrolysis of the peptide on the carboxyl side of the asparagines, as demonstrated by internal amino acid sequence analysis of proteolytic product of BSA cleavage. These results suggest that HlLgm plays an important role in host blood-meal digestion and may be critical for the final process of digestion of blood components.
Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, Tsukuba, Ibaraki 305-0856, Japan.
M
M
Abdul Alim
Naotoshi
N
Tsuji
Takeharu
T
Miyoshi
M
M
Khyrul Islam
Xiaohong
X
Huang
Maki
M
Motobu
Kozo
K
Fujisaki
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000032:analysis; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D026863:Ixodidae / Q000145:classification / Q000201:enzymology*; D007564:Japan; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000099240:Asparaginyl Endopeptidase
D000941:Antigens; D003546:Cysteine Endopeptidases; D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase