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J. Biol. Chem., Vol. 279, Issue 3, 2316-2323, January 16, 2004

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Molecular Cloning and Characterization of a Protein Farnesyltransferase from the Enteric Protozoan Parasite Entamoeba histolytica^*

Masahiro Kumagai, Asao Makioka, Tsutomu Takeuchi, and Tomoyoshi Nozaki¶||**

From the Department of Tropical Medicine, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan, the Department of Tropical Medicine and Parasitology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan, the ^¶Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan, and the ^||Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 2-20-5 Akebonocho, Tachikawa, Tokyo 190-0012, Japan

Genes encoding - and -subunits of a putative protein farnesyltransferase (FT) from the enteric protozoan parasite Entamoeba histolytica were obtained and their biochemical properties were characterized. Deduced amino acid sequences of the - and -subunit of E. histolytica FT (EhFT) were 298- and 375-residues long with a molecular mass of 35.6 and 42.6 kDa, and a pI of 5.43 and 5.65, respectively. They showed 24% to 36% identity to and shared common signature domains and repeats with those from other organisms. Recombinant - and -subunits, co-expressed in Escherichia coli, formed a heterodimer and showed activity to transfer farnesyl using farnesylpyrophosphate as a donor to human H-Ras possessing a C-terminal CVLS, but not a mutant H-Ras possessing CVLL. Among a number of small GTPases that belong to the Ras superfamily from this parasite, we identified EhRas4, which possesses CVVA at the C terminus, as a sole farnesyl acceptor for EhFT. This is in contrast to mammalian FT, which utilizes a variety of small GTPases that possess a C-terminal CaaX motif, where X is serine, methionine, glutamine, cysteine, or alanine. EhFT also showed remarkable resistance against a variety of known inhibitors of mammalian FT. These results suggest that remarkable biochemical differences in binding to substrates and inhibitors exist between amebic and mammalian FTs, which highlights this enzyme as a novel target for the development of new chemotherapeutics against amebiasis.

Received for publication, October 20, 2003

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB083372 (FT of E. histolytica), AB083373 (FT of E. histolytica), AB112425 (EhRas3), and AB112426 (EhRas4).

^* This work was supported by a Grant for Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (to T. N.), a Health Sciences Research Grant for Research on Emerging and Re-emerging Infectious Diseases from Ministry of Health, Labour, and Welfare (to A. M. and T. N.), Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to A. M., 13670256 and to T. N., 15019120, 15590378), and a grant for the Project to Promote Development of Anti-AIDS Pharmaceuticals from Japan Health Sciences Foundation (to T. N). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel.: 81-3-5285-1111 (ext. 2733); Fax: 81-3-5285-1173; E-mail: nozaki{at}nih.go.jp.

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