Serpil BAŞPINAR, Venhar ÇELİK, Dilek Turgut BALIK, Mustafa KAPLAN

Regional Differences in the Amino Acid Sequences of Fasciola hepatica Cathepsin L2 From a Turkish Isolate

Objective: This study describes cloning and sequence analysis of the gene coding for the cathepsin L2 gene from a F. hepatica isolate from Turkey. Methods: The adult form of F. hepatica was collected from the infected cattle liver and cDNA was obtained following total RNA isolation. After amplification of the gene by PCR, ligation and transformation was performed. Finally, DNA sequencing and phylogenetic analysis were performed. Results: The gene consists of an open reading frame of 981 nucleotides corresponding for 327 amino acids. There are five amino acid positions where Firat F. hepatica cathepsin L2 differs from the F. hepatica cathepsin L2 Dublin strain. This sequence was the first cathepsin L2 gene from a Turkey isolate and the second cathepsin L2 sequence entered into the database. A comparative amino acid sequence analysis indicated that there is about 99% homology with the F. hepatica cathepsin L2 reported in the literature, 98% homology with the F. hepatica cathepsin L-like protease, and 85% homology with the F. hepatica cathepsin L1 isolated in the Elazığ region at the nucleotide level. Conclusion: The amino acid differences between sequences could be evaluated for further immunization and diagnosis studies.

Keywords:

Fasciola hepatica, cathepsin L2, nucleotide, amino acid sequence,

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Bousses SH, Meunier C, Durand P, Renaud F. Dynamics of host- parasite interactions: the example of population biology of the liver fluke (Fasciola hepatica). Microbes Infect 2001;3:841-9. [CrossRef]
Mas-Coma S, Bargues MD, Valero MA. Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol 2005;35:1255-78. [CrossRef]
Robinson MW, Dalton JP, Donnelly S. Helminth pathogen cathep- sin proteases: it’s a family affair. Trends Biochem Sci 2008;33:601-8. [CrossRef]
Yilmaz H, Gödekmerdan A. Human fasciolosis in Van province, Turkey. Acta Trop 2004;92:161-2. [CrossRef]
Kaya S, Demirci M, Demirel R, Aridogan BC, Ozturk M, Korkmaz M. Seroprevalence of fasciolosis and the difference of fasciolosis between rural area and city center in Isparta, Turkey. Saudi Med J 2006;27:1152-6.
Garcia LS, Bruckner DA, (editors). Diagnostic Medical Parasitol- ogy. (Third Edition). Washington, ASM Press, 1997.
Behm CA, Sangster NC. Pathology, Pathophysiology and Clini- cal Aspects, In: Dalton JP, editor. Fasciolosis. Wallingford, CAB International, 1999.
Hillyer GV. Immunodiagnosis of human and animal fasciolosis. In: Dalton JP, editor. Fasciolosis. Wallingford, CAB International, 1999.
Marcos L, Maco V, Samalvides F, Terashima A, Espinoza JR, Go- tuzzo E. Risk factors for Fasciola hepatica infection in children: a case-control study. Trans R Soc Trop Med Hyg 2006;100:158-66. [CrossRef]
Makay O, Gurcu B, Caliskan C, Nart D, Tuncyurek M, Korkut M. Ectopic fascioliasis mimicking a colon tumor. World J Gastroen- terol 2007;13:2633-5.
Strauss W, O’neill SM, Parkinson M, Angles R, Dalton JP. Short re- port: Diagnosis of human fascioliasis: Detection of anti-cathepsin L antibodies in blood samples collected on filter paper. Am J Trop Med Hyg 1999;60:746-8.
Cornelissen JB, Gaasenbeek CP, Borgsteede FH, Holland WG, Harmsen MM, Boersma W. Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L- like protease. Int J Parasitol 2001;31:728-37. [CrossRef]
Kim K, Yang HJ, Chung YB. Usefulness of 8 kDa protein of Fas- ciola hepatica in diagnosis of fascioliasis. Korean J Parasitol 2003;41:121-3. [CrossRef]
Tort J, Brindley PJ, Knox D, Wolfe KH, Dalton JP. Proteinases and associated genes of parasitic helminths. Adv Parasitol 1999;43:161-6. [CrossRef]
Spithill TW, Dalton JP. Progress in Development of Liver Fluke Vaccines. Parasitol Today 1998;14:224-7. [CrossRef]
Piacenza L, Acosta D, Basmadjian I, Dalton JP, Carmona C. Vac- cination with Cathepsin L Proteinases and with Leucine Amino- peptidase Induces High Levels of Protection against Fascioliasis in Sheep. Infect Immun 1999;67:1954-61.
Liljeqvist S, Stahl S. Production of recombinant subunit vaccines: protein immunogens, live delivery systems and nucleic acid vac- cines. J Biotechnol 1999;73:1-33. [CrossRef]
Kofta W, Mieszczanek J, Plucienniczak G, Wedrychowicz H. Suc- cessful DNA immunisation of rats against fasciolosis. Vaccine 2000;18:2985-90. [CrossRef]
Kofta W, Wedrychowicz H. c-DNA vaccination against para- sitic infections: advantages and disadvantages. Vet Parasitol 2001;100:3-12. [CrossRef]
Li QY, Dong SJ, Zhang WY, Lin RQ, Wang CR, Qian DX, et al. Se- quence-related amplified polymorphism, an effective molecular approach for studying genetic variation in Fasciola spp. of human and animal health significance. Electrophoresis 2009;30:403-9. [CrossRef]
Dosay-Akbulut M, Trudgett A, Stanhope M. Understanding ge- netic diversity of the liver fluke Fasciola hepatica. Z Naturforsch C 2005;60:774-8.
Panaccio M, Trudgett A. Molecular Biology. In: Dalton JP, editor. Fasciolosis. Wallingford, CAB International, 1999.
Cornelissen JB, Gaasenbeek CP, Boersma W, Borgsteede FH, van Milligen FJ. Use of a pre-selected epitope of cathepsin-L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infections in cattle. Int J Parasitol 1999;29:685-96. [CrossRef]
Sambrook J, Russell DW. Molecular cloning: a laboratory manual 3rd ed. New York: Cold Spring Harbor Laboratory, 2001.
Turgut-Balik D, Celik V, Moreton K, Brady L. Overcoming Clon- ing Problems by Staining Agarose Gels with Crystal Violet In- stead of Ethidium Bromide in Lactate Dehydrogenase Gene from Plasmodium vivax and Plasmodium falciparum. Acta Biol Hung 2005;56:3-4. [CrossRef]
Thompson JD, Higgins DG, Gibson TJ. Clustal W. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673-80. [CrossRef]
Dowd AJ, Tort J, Roche L, Ryan T, Dalton JP. Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae. Mol Biochem Parasitol 1997;88:163-74. [CrossRef]
Heussler VT, Dobbelaere DA. Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction. Mol Bio- chem Parasitol 1994;64:11-23. [CrossRef]
Kuk S, Kaplan M, Ozdarendeli A, Tonbak S, Felek S, Kalkan A. Fasciola hepatica cathepsin L1 from a Turkish isolate is related to Asiatic isolates. Acta Parasitol 2005;50:244-8.
Kaplan M, Kuk S, Kalkan A. [Elazığ ELET A.Ş. Kesimhanesinde 1998-2000 Yılları Arasında Kesilen Hayvanlarda Fasciolosis Görül- me Sıklığı] Incidence of Fasciolosis in Animals slaughtered Between 1998-2000 in Slaughterhouse of Elazığ ELET Company and Its Eco- nomic Significance. Fırat medical journal 2002;7:839-42.
Spithill TW, Smooker PM, Sexton JL, Bozas E, Morrison CA, Cre- aney J, Parsons JC. Development of vaccines against Fasciola hepatica. In: Dalton JP, editor. Fasciolosis. Wallingford, CAB In- ternational, 1999.