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J. Biol. Chem., Vol. 279, Issue 28, 29849-29856, July 9, 2004

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Cloning and Characterization of a Novel Mannose-binding Protein of Acanthamoeba^*

Marco Garate, Zhiyi Cao, Erik Bateman, and Noorjahan Panjwani¶||

From the Department of Ophthalmology, Center for Vision Research and the New England Eye Center, the ^¶Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111 and the Department of Microbiology, Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405

Acanthamoebae produce a painful, blinding infection of the cornea. The mannose-binding protein (MBP) of Acanthamoeba is thought to play a key role in the pathogenesis of the infection by mediating the adhesion of parasites to the host cells. We describe here the isolation and molecular cloning of Acanthamoeba MBP. The MBP was isolated by chromatography on the mannose affinity gel. Gel filtration experiments revealed that the Acanthamoeba lectin is a 400-kDa protein that is constituted of multiple 130-kDa subunits. Cloning and sequencing experiments indicated that the Acanthamoeba MBP gene is composed of 6 exons and 5 introns that span 3.6 kb of the amoeba genome and that MBP cDNA codes for a precursor protein of 833 amino acids. That the cloned cDNA encodes authentic MBP was demonstrated by showing that: (i) recombinant MBP possesses mannose binding activity, and (ii) polyclonal antibodies prepared against Acanthamoeba MBP bound to the recombinant protein. Sequence analysis revealed that the MBP contains a large N-terminal extracellular domain, a transmembrane domain, and a short C-terminal cytoplasmic domain. Despite extensive BLAST searches using the MBP sequence, no significant matches were retrieved. The most striking feature of the Acanthamoeba MBP sequence is the presence of a cysteine-rich region containing 14 CXCXC motifs within the extracellular domain. In summary, we have isolated, cloned, and characterized a novel MBP from Acanthamoeba. Because the presence of antibodies to MBP in tears provides protection against infection, the availability of the MBP cDNA sequence and rMBP should help develop: (i) a tear-based test to identify individuals who are at risk of developing the keratitis and (ii) strategies to immunize high-risk individuals.

Received for publication, March 2, 2004 , and in revised form, April 26, 2004.

^* This work was supported by National Institutes of Health Grants EY09349 (to N. P.) and EY08706 (to E. B.), core Grant EYP30-13078 for Vision Research, the Massachusetts Lions Eye Research Fund, and the New England Corneal Transplant Research Fund. 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 Ophthalmology, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6776; Fax: 617-636-0348; E-mail: Noorjahan.Panjwani{at}tufts.edu.

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