title: A hemocyte-derived Kunitz-BPTI-type chymotrypsin inhibitor, HlChI, from the ixodid tick Haemaphysalis longicornis, plays regulatory functions in tick blood-feeding processes.
abstract: Inhibitors of proteases play key roles in the biological processes of vertebrate and invertebrate animals, including arthropod parasites. Here, we describe a cDNA that encodes a functionally active chymotrypsin inhibitor of the BPTI/Kunitz family of serine protease inhibitors from the hemocytes of the ixodid tick, Haemaphysalis longicornis, herein called HlChI. HlChI sequence is evolutionarily conserved and contains six cysteine residues and three disulfide bonds with a calculated molecular weight of 9.1 kDa. HlChI-specific mRNA was expressed in all developmental stages of ticks and the expression was up-regulated by host's blood-feeding processes. Endogenous HlChI was localized mainly in the hemocytes. HlChI potently inhibited bovine pancreatic α-chymotrypsin for hydrolyzing the fluorogenic substrate (IC(50) 8.32 nM, K(d) 5.35 ± 1.01 nM) and bovine casein digestion. However, HlChI weakly inhibited bovine pancreatic trypsin and could not affect the porcine elastase activity, suggesting its narrow specificity to chymotrypsin. HlChI was stable over the pH range 2-11 and heating up to 70 °C at pH 8. HlChI was highly stable to 8 M urea and 2% SDS at pH 8.0, when treated for 24 h at 37 °C. However, 0.2 M 2-mercaptoethanol caused complete but reversible inactivation of HlChI. Knockdown of HlChI gene by RNA interference (RNAi) caused death of the feeding ticks, failure of ticks to engorge and significantly reduced body weight gain. RNAi also resulted in significantly decreased egg conversion ratio and fecundity. These results suggest that HlChI is a chymotrypsin-specific inhibitor with high stability and may play regulatory functions in host's blood-feeding processes and tick reproduction.
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural and Food Research Organization, Tsukuba, Ibaraki 305-0856, Japan.
authors.forename: M Abdul
authors.initials: MA
authors.lastname: Alim
authors.forename: M Khyrul
authors.initials: MK
authors.lastname: Islam
authors.lastname: Anisuzzaman
authors.forename: Takeharu
authors.initials: T
authors.lastname: Miyoshi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hatta
authors.forename: Kayoko
authors.initials: K
authors.lastname: Yamaji
authors.forename: Makoto
authors.initials: M
authors.lastname: Matsubayashi
authors.forename: Kozo
authors.initials: K
authors.lastname: Fujisaki
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Tsuji
mesh_terms: D000818:Animals; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D018076:DNA, Complementary; D005247:Feeding Behavior; D005260:Female; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006434:Hemocytes / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D026863:Ixodidae / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D012098:Reproduction
chemical_list: D018076:DNA, Complementary; D019476:Insect Proteins; D002918:Chymotrypsin