title: Two domains of cytotoxic necrotizing factor type 1 bind the cellular receptor, laminin receptor precursor protein.
abstract: 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, DeltaN63, DeltaN545, and DeltaC469, 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.
authors: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA.
authors: Beth A
authors: BA
authors: McNichol
authors: Susan B
authors: SB
authors: Rasmussen
authors: Humberto M
authors: HM
authors: Carvalho
authors: Karen C
authors: KC
authors: Meysick
authors: Alison D
authors: AD
authors: O'Brien
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D018613:Microscopy, Confocal; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D011498:Protein Precursors / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017474:Receptors, Laminin / Q000378:metabolism*; D017384:Sequence Deletion; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D011498:Protein Precursors; D017474:Receptors, Laminin; C086995:cytotoxic necrotizing factor type 2; C060296:cytotoxic necrotizing factor type 1; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein
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