1. Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis
Takeharu Miyoshi, Naotoshi Tsuji, M Khyrul Islam, Tsugihiko Kamio, Kozo Fujisaki Insect Biochem Mol Biol. 2004 Aug;34(8):799-808. doi: 10.1016/j.ibmb.2004.04.004.
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.
2. Longistatin is an unconventional serine protease and induces protective immunity against tick infestation
Anisuzzaman, M Khyrul Islam, M Abdul Alim, Takeharu Miyoshi, Takeshi Hatta, Kayoko Yamaji, Yasunobu Matsumoto, Kozo Fujisaki, Naotoshi Tsuji Mol Biochem Parasitol. 2012 Mar-Apr;182(1-2):45-53. doi: 10.1016/j.molbiopara.2011.12.002. Epub 2011 Dec 21.
Classical serine proteases use the conserved Ser/His/Asp catalytic triad to hydrolyze substrates. Here, we show that longistatin, a salivary gland protein with two EF-hand domains from the vector tick Haemaphysalis longicornis, does not have the conserved catalytic triad, but still functions as a serine protease. Longistatin was synthesized in and secreted from the salivary glands of ticks, and is injected into host tissues during the acquisition of blood-meals. Longistatin hydrolyzed fibrinogen, an essential plasma protein in the coagulation cascade, and activated plasminogen, into its active form plasmin, a serine protease that dissolves fibrin clots. Longistatin efficiently hydrolyzed several serine protease-specific substrates showing its specificity to the amide bond of Arg. Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperatures and pHs, with the optimum at 37°C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as phenylmethanesulfonyl fluoride (PMSF), aprotinin, antipain, and leupeptin with the estimated IC(50) of 278.57 μM, 0.35 μM, 41.56 μM and 198.86 μM, respectively. In addition, longistatin was also potently inhibited by Zinc (Zn(2+)) in a concentration-dependent manner with an IC(50) value of 275 μM, and the inhibitory effect of Zn(2+) was revived by ethylenediaminetetra acetic acid (EDTA). Immunization studies revealed that longistatin sharply induced high levels of protective IgG antibodies against ticks. Immunization with longistatin reduced repletion of ticks by about 54%, post engorgement body weight by >11% and molting of nymphs by approximately 34%; thus, the vaccination trial was approximately 73% effective against tick infestation. Taken together, our results suggest that longistatin is a new potent atypical serine protease, and may be an interesting candidate for the development of anti-tick vaccines.
3. Molecular and reverse genetic characterization of serine proteinase-induced hemolysis in the midgut of the ixodid tick Haemaphysalis longicornis
Takeharu Miyoshi, Naotoshi Tsuji, M Khyrul Islam, Xiaohong Huang, Maki Motobu, M Abdul Alim, Kozo Fujisaki J Insect Physiol. 2007 Feb;53(2):195-203. doi: 10.1016/j.jinsphys.2006.12.001. Epub 2006 Dec 28.
Enzyme-induced hemolysis has been shown to occur in the midgut of ticks; however, little is known about the molecular basis for hemolytic activity. We report here the molecular and reverse genetic characterization of a hemolytic midgut serine proteinase, HlSP, recently identified from the ixodid tick Haemaphysalis longicornis. Endogenous HlSP was found in the midgut lumen and its contents, indicating that HlSP is extracellularly secreted. Recombinant H. longicornis serine proteinase (rHlSP) expressed in Escherichia coli showed dose-dependent hemolytic activity towards rabbit erythrocytes, with a maximum hemolysis of 94.5% within 1 h in vitro. Tests of pH dependency showed that rHlSP displayed optimal activity at pH 6.0. In binding assays, rHlSP showed high affinity to band 3, which shares the major erythrocyte membrane proteins. Disruption of HlSP-specific mRNA by RNA interference resulted in inhibition of the degradation of host erythrocyte membranes by endogenous HlSP in the knock-down ticks, indicating that HlSP plays a crucial role in the hemolysis in the midgut of haematophagous ticks. Our results suggest that HlSP may be essential for initiating the proteolytic cascade for the degradation of the host blood-meal.
