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Volume 272, Number 24, Issue of June 13, 1997 pp. 15562-15568
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells
SIGNAL TRANSDUCTION BY A PROTEASE-RESISTANT TOXIN VARIANT

(Received for publication, January 31, 1997, and in revised form, March 24, 1997)

Wayne I. Lencer , Carita Constable , Signa Moe , Paul A. Rufo , Anne Wolf , Michael G. Jobling , Steve P. Ruston , James L. Madara ^** , Randall K. Holmes and Timothy R. Hirst

From the  Combined Program in Pediatric Gastroenterology and Nutrition, Children's Hospital, ^** Gastrointestinal Pathology, Brigham and Women's Hospital, and the Departments of Pediatrics and Pathology, Harvard Medical School, and the Harvard Digestive Diseases Center, Boston, Massachusetts 02115, the ^** Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262, and the  Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom

Cholera and Escherichia coli heat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed "nicking"). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 µA/cm²). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type toxins with trypsin in vitro restored the "basolateral" secretory responses to "apical" levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.

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