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Two Domains of Cytotoxic Necrotizing Factor Type 1 Bind the Cellular Receptor, Laminin Receptor Precursor Protein

Beth A. McNichol,^1, Susan B. Rasmussen,¹ Humberto M. Carvalho,^1, Karen C. Meysick,² and Alison D. O'Brien^1*

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814,¹ FDA/CBER, Bethesda, Maryland 20892²

Received 12 January 2007/ Returned for modification 28 February 2007/ Accepted 8 August 2007

Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are highly homologous toxins that are produced by certain pathogenic strains of Escherichia coli. These 1,014-amino-acid toxins catalyze the deamidation of a specific glutamine residue in RhoA, Rac1, and Cdc42 and consist of a putative N-terminal binding domain, a transmembrane region, and a C-terminal catalytic domain. To define the regions of CNF1 that are responsible for binding of the toxin to its cellular receptor, the laminin receptor precursor protein (LRP), a series of CNF1 truncated toxins were characterized and assessed for toxin binding. In particular, three truncated toxins, N63, N545, and C469, retained conformational integrity and in vitro enzymatic activity and were immunologically reactive against a panel of anti-CNF1 monoclonal antibodies (MAbs). Based on a comparison of these truncated toxins with wild-type CNF1 and CNF2 in LRP and HEp-2 cell binding assays and in MAb and LRP competitive binding inhibition assays and based on the results of confocal microscopy, we concluded that CNF1 contains two major binding regions: one located within the N terminus, which contained amino acids 135 to 164, and one which resided in the C terminus and included amino acids 683 to 730. The data further indicate that CNF1 can bind to an additional receptor(s) on HEp-2 cells and that LRP can also serve as a cellular receptor for CNF2.

Published ahead of print on 20 August 2007.

Editor: V. J. DiRita

Present address: FDA/CBER, Bethesda, MD 20892.

Present address: 6700B Rockledge Drive, Bethesda, MD 20892.

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