Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis.
ABSTRACT We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; k(cat)/K(m)=0.19 and 0.0023 M(-1) S(-1), respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks.
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ABSTRACT: Ticks, as obligate hematophagous ectoparasites, impact greatly on animal and human health because they transmit various pathogens worldwide. Over the last decade, several cystatins from different hard and soft ticks were identified and biochemically analyzed for their role in the physiology and blood feeding lifestyle of ticks. All these cystatins are potent inhibitors of papain-like cysteine proteases, but not of legumain. Tick cystatins were either detected in the salivary glands and/or the midgut, key tick organs responsible for blood digestion and the expression of pharmacologically potent salivary proteins for blood feeding. For example, the transcription of two cystatins named HlSC-1 and Sialostatin L2 was highly upregulated in these tick tissues during feeding. Vaccinating hosts against Sialostatin L2 and Om-cystatin 2 as well as silencing of a cystatin gene from Amblyomma americanum significantly inhibited the feeding ability of ticks. Additionally, Om-cystatin 2 and Sialostatin L possessed strong host immunosuppressive properties by inhibiting dendritic cell maturation due to their interaction with cathepsin S. These two cystatins, together with Sialostatin L2 are the first tick cystatins with resolved three-dimensional structure. Sialostatin L, furthermore, showed preventive properties against autoimmune diseases. In the case of the cystatin Hlcyst-2, experimental evidence showed its role in tick innate immunity, since increased Hlcyst-2 transcript levels were detected in Babesia gibsoni-infected larval ticks and the protein inhibited Babesia growth. Other cystatins, such as Hlcyst-1 or Om-cystatin 2 are assumed to be involved in regulating blood digestion. Only for Bmcystatin was a role in tick embryogenesis suggested. Finally, all the biochemically analyzed tick cystatins are powerful protease inhibitors, and some may be novel antigens for developing anti-tick vaccines and drugs of medical importance due to their stringent target specificity.Ticks and Tick-borne Diseases 04/2012; 3(3):117-27. · 2.35 Impact Factor
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ABSTRACT: Ticks employ a battery of proteases to digest the contents of host blood meals. Host hemoglobin degradation is facilitated by proteolytic networks in the midgut, the first major region of the body where ingested blood comes into contact with the tick's internal tissues. Our previous studies indicated that HlCPL-A, a cathepsin L-like cysteine protease isolated from the midgut of the ixodid tick Haemaphysalis longicornis, is a potent hemoglobinase, and plays important roles in the digestion of blood acquired from a host. In this paper, we report the effects of silencing of the HlCPL-A gene in H. longicornis using RNA interference (RNAi). We observed that the survival of HlCPL-A-silenced ticks was reduced compared with that of controls during blood digestion, most likely due to the compromised ability of ticks to digest blood. The morphological analysis results of midgut lumen were different between HlCPL-A-silenced ticks and controls, indicating that HlCPL-A plays a crucial role in hemolysis in the midgut of ticks. The expression level was analyzed using quantitative RT-PCR-based endogenous expression approach. Compared to that in malE double stranded RNA (dsRNA)-treated ticks, in the midgut of HlCPL-A dsRNA-treated ticks, some proteases and inhibitors related to the hemoglobin digestive cascade were up-regulated while the others were down-regulated. These results suggest that HlCPL-A is related to the multi-enzyme cascade and protease network for hemoglobin digestion. These findings suggest that the hemoglobin digestive cascade may assemble in the midgut of ticks.Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 02/2013; · 3.22 Impact Factor
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ABSTRACT: Blood-protein digestion is a key physiological process providing essential nutrients for ticks and is a prerequisite for the transmission of tick-borne pathogens. Recently, substantial progress has been made in determining the proteolytic machinery in tick gut tissue, which is based on a dynamic multienzyme network capable of processing a vast amount of host blood. In this article we summarize our current knowledge of the molecular mechanisms of tick hematophagy and their similarities to those of Platyhelminthes, nematodes, and Plasmodium. Future research perspectives, including the potential for rational control of ticks and transmitted diseases, are also discussed.Trends in Parasitology 05/2013; · 5.51 Impact Factor