Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain.
Zonula occludens toxin (Zot) is an enterotoxin elaborated by Vibrio cholerae that increases intestinal permeability by interacting with a mammalian cell receptor with subsequent activation of intracellular signaling leading to the disassembly of the intercellular tight junctions. Zot localizes in the bacterial outer membrane of V. cholerae with subsequent cleavage and secretion of a carboxyl-terminal fragment in the host intestinal milieu. To identify the Zot domain(s) directly involved in the protein permeating effect, several zot gene deletion mutants were constructed and tested for their biological activity in the Ussing chamber assay and their ability to bind to the target receptor on intestinal epithelial cell cultures. The Zot biologically active domain was localized toward the carboxyl terminus of the protein and coincided with the predicted cleavage product generated by V. cholerae. This domain shared a putative receptor-binding motif with zonulin, the Zot mammalian analogue involved in tight junction modulation. Amino acid comparison between the Zot active fragment and zonulin, combined with site-directed mutagenesis experiments, confirmed the presence of an octapeptide receptor-binding domain toward the amino terminus of the processed Zot.
Division of Pediatric Gastroenterology and Nutrition and Gastrointestinal Pathophysiology Section, Center for Vaccine Development, Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
M
M
Di Pierro
R
R
Lu
S
S
Uzzau
W
W
Wang
K
K
Margaretten
C
C
Pazzani
F
F
Maimone
A
A
Fasano
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins; D004847:Epithelial Cells / Q000378:metabolism; D017353:Gene Deletion; D007421:Intestine, Small / Q000166:cytology; D008297:Male; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D019108:Tight Junctions / Q000737:chemistry*
D004731:Endotoxins; C077728:zonula occludens toxin, Vibrio cholerae; D002772:Cholera Toxin