Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki 305-8572, Japan. Parasitology International
(Impact Factor: 1.86).
06/2009; 58(3):232-7. DOI: 10.1016/j.parint.2009.05.003
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.
Available from: Mehrdad Moosazadeh Moghaddam
- "Cathepsin L which is synthesized as a proenzyme is one of the cysteine proteases belonging to the family C1 (cathepsin L-and cathepsin B-like). The mature form of cathepsin L is shown to be involved in tissue remodeling, immune system as well as modulation and alteration of cell function (Dickinson 2002), and its functional activities have been described in several tick species such as R. annulatus (Taheri et al. 2014a), Rhipicephalus (Boophilus) microplus (Renard et al. 2000, 2002; Seixas et al. 2003; Estrela et al. 2007), Ixodes ricinus (Franta et al. 2011), Haemaphysalis longicornis (Tsuji et al. 2008; Yamaji et al. 2009) and Ornithodoros moubata (Fagotto 1990). Several other pathogenic proteins and enzymes of R. annulatus ticks such as serine protease, vitellogenin and tropomyosin have been studied (Nikpay et al. 2012; Nabian et al. 2013, 2014; Ranjbar et al. 2013, 2014, 2015; Taheri et al. 2014b). "
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ABSTRACT: The tick Rhipicephalus (Boophilus) annulatus is one of the most important ectoparasites of bovines and is responsible for the transmission of different pathogens such as Babesia and Anaplasma. Cysteine proteases are involved in several host-tick interactions including invasion of host tissues, immune evasion, pathogen transmission, embryogenesis and blood digestion. In this study, the gene encoding R. annulatus cathepsin L-like enzyme (RaCL1) was cloned into pTZ57R/T vector, sequenced and analyzed using bioinformatics approaches. The nucleotide length of RaCL1 was 999 bp. Bioinformatics analysis showed 332 amino acids with an approximate molecular weight of 36.3 kDa which contained a signal peptide sequence (18 amino acids), pro-region (97 amino acids) and mature enzyme (217 amino acids). Multiple sequence alignment of the RaCL1 revealed high similarity to cathepsin L-like cysteine proteases from other tick species such as Rhipicephalus (Boophilus) microplus and Amblyomma variegatum. Based on bioinformatics analyses, results of this work suggest that RaCL1 can be a suitable candidate for the development of vaccine against R. annulatus.
Available from: Michail Kotsyfakis
- "Tick species Name of inhibitor Type a MW (kDa)* pI* NCBI number Tissue specificity Cysteine protease inhibition Cystatin function References A. americanum Cystatin 2 15.0 5.5* – M, SG (p) – Regulation of host immune response (t) Karim et al. (2005) A. variegatum Cystatin 2 10.5 1 6.9 1 DAA34288 SG (s) – – Ribeiro et al. (2011) D. variabilis DvM602 1 11.1* 5.8* ACF35512 M (s) – – Anderson et al. (2008) DvM334 2 9.7* 1 4.5* 1 ACF35514 M (s) – – Anderson et al. (2008) H. longicornis Hlcyst-1 1 11.0 5.5 ABZ89553 M (s) Papain cath B, H, L HlCPL-A Regulation of hemoglobin digestion (r) Zhou et al. (2006) (2009) and Yamaji et al. (2010) Hlcyst-2 2 12.9 8.5 ABC94582 M (s, p) H, SG, O, F (p) Papain cath L, H HlCPL-A Regulation of hemoglobin digestion and tick innate immunity (r) Zhou et al. (2006) and Yamaji et al. (2009a,b, 2010 "
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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.
Available from: Zdenek Franta
- "e l s e v i e r . c o m / l o c a t e / i j p a r a by reports on individual genes and/or enzymes in different tick species (Renard et al., 2000; Boldbaatar et al., 2006; Sojka et al., 2007; Tsuji et al., 2008; Yamaji et al., 2009; Cruz et al., 2010). "
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ABSTRACT: Intracellular proteolysis of ingested blood proteins is a crucial physiological process in ticks. In our model tick, Ixodes ricinus, cathepsin L (IrCL1) is part of a gut-associated multi-peptidase complex; its endopeptidase activity is important in the initial phase of haemoglobinolysis. We present the functional and biochemical characterisation of this enzyme. We show, by RNA interference (RNAi), that cathepsin L-like activity that peaks during the slow feeding period of females is associated with IrCL1. Recombinant IrCL1 was expressed in bacteria and yeast. Activity profiling with both peptidyl and physiological protein substrates (haemoglobin and albumin) revealed that IrCL1 is an acidic peptidase with a very low optimum pH (3-4) being unstable above pH 5. This suggests an endo/lysosomal localisation that was confirmed by indirect fluorescence microscopy that immunolocalised IrCL1 inside the vesicles of digestive gut cells. Cleavage specificity determined by a positional scanning synthetic combinatorial library and inhibition profile indicated that IrCL1 has the ligand-binding characteristics of the cathepsin L subfamily of cysteine peptidases. A non-redundant proteolytic function was demonstrated when IrCL1-silenced ticks had a decreased ability to feed compared with controls. The data suggest that IrCL1 may be a promising target against ticks and tick-borne pathogens.
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