Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis.
Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz-L-Arg-pNA at the rate of 30.2 micromol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.
Miyoshi
Takeharu
T
Laboratory of Parasitic Diseases, National Agricultural Research Organization, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
Tsuji
Naotoshi
N
Islam
M Khyrul
MK
Kamio
Tsugihiko
T
Fujisaki
Kozo
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D004926:Escherichia coli; D005260:Female; D020869:Gene Expression Profiling; D026863:Ixodidae / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C029486:intrinsic factor-cobalamin receptor; D012697:Serine Endopeptidases