Orientation of cholera toxin bound to target cells.
Cholera toxin (CT) consists of a pentameric B subunit that binds to specific cell surface receptors identified as ganglioside GM1 and an A subunit that activates adenylylcyclase. The A subunit consists of A1 and A2 peptides linked by a disulfide bond; A2 acts to connect A to B, whereas A1 is an ADP-ribosyltransferase that modifies the alpha subunit of the stimulatory G protein (Gs). How the toxin is oriented when it binds to the cell surface and the related issue of the mechanism by which A1 gains access to Gs alpha are not known. In the present study, we used subunit-specific antibodies and their corresponding Fab fragments to assess their affects on holotoxin binding to target cells and their immunoreactivity to cell-bound toxin. Our results suggest that CT binds with A1 facing away from the membrane. Our hypothesis is further supported by the ability to assemble active CT on the cell surface of cultured human intestinal and neurotumor cells by the sequential addition of purified B and A subunits. We also observed that when cells containing bound CT were incubated at 37 degrees C, both subunits rapidly became inaccessible to their respective antibodies. We propose that the holotoxin binds with its A subunit facing away from the membrane and must enter the cell in order for A1 to be released, gain access to Gs alpha, and activate adenylylcyclase.
Orlandi
P A
PA
Membrane Biochemistry Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Fishman
P H
PH
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007700:Kinetics; D011487:Protein Conformation; D013696:Temperature; D014407:Tumor Cells, Cultured
D007106:Immune Sera; D007140:Immunoglobulin Fab Fragments; D002772:Cholera Toxin