Ganglioside cholera toxin

SPR imaging was also employed to look at the same cholera toxin-GMl interaction on a supported bilayer by Philips et al. in array experiments [74]. Another investigative technique applied to the model ganglioside-cholera toxin system was total internal reflection fluorescence microscopy applied to spatially addressable supported bilayer membranes [75]. The recognition by HIV-1 surface glycoprotein gpl20 of glycosphingolipids partitioned within an SBM has also been looked at by total internal reflection fluorescence [76]. 

Figure 14-13. G i ganglioside, a monosialoganglio-side, the receptor in human intestine for cholera toxin.

Glycosphingolipids are constituents of the outer leaflet of plasma membranes and are important in cell adhesion and cell recognition. Some are antigens, eg, ABO blood group substances. Certain gangliosides function as receptors for bacterial toxins (eg, for cholera toxin, which subsequently activates adenylyl cyclase). 

Rowe-Taitt C.A., Cras J.J., Patterson C.H., Golden J.P., Ligler F.S., A ganglioside-based assay for cholera toxin using an array biosensor, Anal. Biochem. 2000a 281 123-133. 

A. Bemardi, L. Carrettoni, A. Grosso Ciponte, D. Montib, and S. Sonnino, Second generation mimics of ganglioside GM1 as artificial receptors for Cholera Toxin Replacement of the sialic acid moiety, Bioorg. Med. Chem. Lett., 10 (2000) 2197-2200. 

Two of the most widely spread and well-studied enterotoxigenic forms of bacterial diarrhea are ETEC and Vibrio cholerae. The toxins they produce, labile toxin (LT) and cholera toxin (CT) respectively, are very similar in primary sequence, structure, and mechanism of action [72]. They are homologous multi-subunit proteins in which the non-toxic B subunit mediates GMj ganglioside binding, and thus are candidates for vaccines that can neutralize toxin activity. 

A. A. Wolf, M. G. Jobling, S. Wimer-Mackin, M. Ferguson-Maltzman, J. L. Madara, R. K. Holmes, and W. I. Lencer. Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia. J Cell Biol. 141 917-927 (1998). 

Caveolae can mediate the delivery of CtxB that binds to GM1 ganglioside at the plasma membrane and is delivered to intracellular compartments. Cholera toxin, produced by Vibrio cholerae, consists of five identical subunits B and one A chain. In addition to labeled SV40 and caveolin-1-GFP, CtxB is one of the most commonly used caveolae markers. However, two groups reported that the toxin is internalized by either a clathrin-independent caveolae pathway or a clathrin-dependent uptake, bringing its selectivity/specificity into question (31,81,118). We controlled the suitability of this marker for COS-7 cells pretreated with CPZ, mpCD, and filipin and as expected, the uptake was not influenced by CPZ treatment but was strongly decreased by the latter two (data not shown). 

In both of these cases, the ligand (sialic acid) for the analyte of interest (influenza vims) was covalently linked to the PDA backbone generated upon photopolymerization. Functional sensors based on ligands that are noncovalently incorporated into liposomes have also been reported (Charych et al. 1996 Pan and Charych 1997). Mixed liposomes as well as mixed thin films on glass containing a combination of the ganglioside GMl and diacetylene lipids detect the presence of cholera toxin, a protein that binds to GMl. 

Pan JJ, Charych D. Molecular recognition and colorimetric detection of cholera toxin by poly (diacetylene) hposomes incorporating GMl ganglioside. Langmuir 1997 13 1365-1367. 

Lian, T., T. Bui, and R.J. Ho, Formulation of HFV-envelope protein with lipid vesicles expressing ganglioside GMl associated to cholera toxin B enhances mucosal immune responses. Vaccine, 1999. 18(7-8) 604-11. 

Lian, T., and R.J. Ho, Cholera toxin B-mediated targeting of lipid vesicles containing ganglioside GMl to mucosal epithelial cells. Pharm Res, 1997.14(10) 1309-15. 

Figure 7-5 Stereoscopic view of the B5 pentamer of cholera toxin B. The pentamer, known as choleragenoid, has a central hole of 1.5 nm diameter into which a helix from the A subunit is inserted. As viewed here, the front surface of the pentamer has binding sites for the oligosaccharide chains of ganglioside CM, which serves as the toxin receptor. The back side binds the A subunit. See also Box 11-A. From Zhang et al,31...

Cholera toxin consists of one 240-residue A subunit and a ring of five 103-residue B subunits.c e The latter is a "targeting complex," a bacterial lectin each subunit of which binds to the galactose and sialic acid termini of a single molecule of ganglioside Gm1. 

The three-dimensional structure of cholera toxin.6 Side view of the p subunit pentamer as a ribbon drawing. Bound noncovalently to it are five molecules of the ganglioside Gm1 (compare with the structure in Fig. 7-5). The diacyl glycerol parts of the gangliosides are buried in the membrane that lies below the toxin molecule. Courtesy ofW.G.J. Hoi. 

Ellipsometry can follow the interactions between two types of biological macromolecules, the first of those two bound physically to the surface, the other acting from the solution. The binding of conconavalin A to adsorbed mannan 180) and of cholera toxin to adsorbed ganglioside t83) are examples. The adsorption of complement factors to an antibody-coated surface was monitored by ellipsometry and a modification of the same method was used for quantification of migration inhibition of human polymorphonuclear leucocytes 182). Interaction of proteins and cells with affinity ligands covalently coupled to silicon surfaces has been also studied 183). 

The metabolism of GSLs has been studied in cultured human fibroblasts from normal subjects, patients with lipid storage diseases, and those with FH. The content of the GSLs, as well as activities of the biosynthetic enzymes, the glycosyltransferases and the lysosomal GSL hydrolases,have been studied. Complex gangliosides, such as M1, GDla, have been found in this cell system to serve as receptors for cholera toxin and thyrotropin, respectively (24-26). More recently, GT1 and GDla have been postulated to be receptors for fibronectin in cultured fibroblasts... 

Gangliosides as Receptors for Cholera Toxin, Tetanus Toxin, and Sendai Virus... 

Subsequent studies in several laboratories have provided further evidence that ganglioside GM1 is the natural biological receptor for cholera toxin ... 

Pretreatment of cell membranes with cholera toxin specifically blocked the membrane GM1-ganglioside from reacting with galactose oxidase (13). 

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