Studies of the Ligand Binding to Cholera Toxin, II. The hydrophilic moiety of sialoglycolipids

Choleragen (cholera toxin) agglutinated erythrocytes and liposomes containing the toxin receptor, galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl)-galactosylglucosylceramide (ganglioside GM1) Cells that had been exposed to GM1 were agglutinated, but agglutination was not observed when cells had been exposed to other gangliosides (GM2, GM3, GDla, GDlb). Choleragen-dependent agglutination of liposomes was slightly less specificbecause liposomes containing either GM1 or GDlb, but neither GM2, GDla, nor GM3 were agglutinated. The oligosaccharide isolated from GM1 inhibited both the agglutination of cells and liposomes containing GM1 and the binding of choleragen to liposomes containing GM1. Galactose and sialic acid were less effective inhibitors of liposomal agglutination and did not inhibit cellular agglutination or binding of choleragen to liposomes. Liposomal agglutination was dependent on choleragen concentration and occurred with the B but not the A protomer of choleragen. These results suggest that choleragen, through its binding to the oligosaccharide portion of a glycolipid, exhibits lectinlike activity, which results in agglutination of liposomes and erythrocytes.Lectins are proteins that are characterized by their ability to bind to sugars and agglutinate cells, particularly erythrocytes. Many lectins are mitogenic and also induce patching and capping on lymphocytes (1). The lectins originally described were plant proteins (1), but in recent years proteins exhibiting these properties have been isolated from several mammalian tissues (2-6). Specific binding of plant lectins to glycolipids, including galactosyl1N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (ganglioside GM1), also has been described (7-10).Choleragen is a bacterial toxin consisting of two protomers, A and B, of which the B protomer, composed of five identical polypeptide units, contains the determinants for binding to cells and membranes (11,12). Previous studies have demonstrated that the receptor for choleragen is ganglioside GM1 (11-18). Binding of choleragen to lymphocytes resulted in patching and capping of membrane GM1 (19-21). In addition, lymphocyte-bound choleragen has been found to crosslink the lymphocytes to agarose beads containing covalently attached gangliosides (21). Choleragen also bound to liposomes containing GM1; this binding resulted in membrane damage (22,23), and membrane damage occurred only with the B protomer (24). Because choleragen could bind specifically to the oligosaccharide portion of GM1 (17,(25)(26)(27) and each toxin molecule could bind more than one molecule of this oligosaccharide (26, 27), we hypothesized that choleragen might exhibit lectinlike behavior at a membrane surface. In this paper we show that choleragen can agglutinate cells and liposomes containing GM1, as would be expected of a lectin or lectinlike protein.EXPERIMENTAL PROCEDURES Sheep erythrocytes, washed and standardized as described (28) Liposomes contained dimyristoyl phosphatidylcholine; cholestero...

Section: Discussionsupporting

confidence: 60%

mentioning

confidence: 99%

Choleragen (cholera toxin): a bacterial lectin.

Richards¹,

Moss²,

Alving³

et al. 1979

“…Furthermore, substituting the N-acetyl of the acetamido group with N-glycolyl does not affect binding ( Table 1), arguing that this moiety also is noncrucial for binding to occur. Moreover, reduction of the carboxyl group of NeuAca3 to the corresponding alcohol yields a substantially lowered binding to CT as compared with native GM1 (31). However, more recent measurements using the TLC overlay assay show, at least in this system, that this derivative still is a relatively good receptor, indicating that a charged group is not a prerequisite for binding to occur (37).…”

mentioning

confidence: 85%

Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes.

Ångström

Teneberg

Karlsson

1994

Binding studies of various glycolipids, mainly belonging to the gangio series, to the toxins isolated from Vibrio cholerae, Eschienchia coil, and Closridium tetan have been performed, using the microtiter well assay. By using the found binding preferences in conjunction with minimum-energy conformations obtained from molecular modeling of the various ligands, binding epitopes on the natural receptor glycolipids for the toxins have been defined. The binding preferences for the cholera toxin and the heat-labile E. coil toxin are very similar, with the gangoide GM1 being the most efient lgd. The tetanus toxin binds strongly to ganglides of the Glb series, with GT1b as the most efficient igand. It is found that the binding epitope on GM1 for the cholera and heat-labile toxins to a large extent overlaps with the epitope on GQ1b for the tetanus toxin.The natural carbohydrate ligand for the toxin produced by Vibrio cholerae is the ganglioside GM1, which contains a single sialic acid residue (1-3). Since the discovery by van Heyningen that gangliosides are responsible for toxin binding (4), a wealth ofbiochemical and physicochemical studies have been carried out to define the important structural characteristics which determine the toxin's mode of action. The threedimensional structure of GM1 has been determined by NMR in conjunction with computational studies of its conformation (5-7), thereby allowing an approximation of the binding epitope on this glycolipid to be made. The recent crystal structure of the heat-labile enterotoxin (LT) from Escherichia coli (8) and its complex with lactose (9) provides a further impetus to the type of studies mentioned above due to the strong sequence homology (>80%o) with the cholera toxin (CT) (10, 11).

“…The action of cholera toxin on susceptible mammalian cells begins with binding of the B subunit to a cell surface receptor, the monosialoganglioside GM1, with an association constant of about 109 M-1 (11). The oligosaccharide moiety of GM1 bears the determinants for this interaction (12). Binding is followed by a lag period, during which the Al fragment penetrates the plasma membrane.…”

mentioning

confidence: 99%

Two-dimensional crystals of cholera toxin B-subunit-receptor complexes: projected structure at 17-A resolution.

Ludwig¹,

Ribi²,

Schoolnik³

et al. 1986

The B subunit of cholera toxin forms twodimensional crystals when bound to its membrane receptor, ganglioside GM1, in phospholipid layers. A rectangular crystal lattice gives diffraction extending to 15-A resolution in negative stain, and image-processing of electron micrographs reveals a ring of five protein densities. The diameter of the central hole and the outer diameter of the ring are about 20 and 60 A, respectively. These data are consistent with a pentameric, doughnut-shaped structure of the B subunit that lies flat on a membrane surface. A hexagonal crystal lattice is obtained as well, and results of image processing and chemical crosslinking allow two interpretations: ('l the B subunit may exist in both pentameric and hexameric forms or, more likely, (it) the hexagonal lattice may represent a disordered or liquid crystalline form, in which a pentamer undergoes rotational averaging about its 5-fold axis.The clinical symptoms of cholera are attributable to the effects of a toxin produced by Vibrio cholerae (1-3). Cholera toxin is composed of two subunits, A, cleaved in the active form into A1 (Mr 22,000) and A2 (Mr 5,000) fragments, and B (Mr 11,600) (4-7). The B subunit forms an oligomer, which is generally believed, on the basis of ultracentrifugation and chemical crosslinking studies, to be a pentamer (5,8,9), although other evidence is suggestive of tetrameric or hexameric forms (9, 10). The action of cholera toxin on susceptible mammalian cells begins with binding of the B subunit to a cell surface receptor, the monosialoganglioside GM1, with an association constant of about 109 M-1 (11). The oligosaccharide moiety of GM1 bears the determinants for this interaction (12). Binding is followed by a lag period, during which the Al fragment penetrates the plasma membrane. Finally, Al catalyzes the ADP-ribosylation of adenylate cyclase (13)(14)(15)(16)(17)(18)(19). The events during the lag period and the mechanism of penetration of Al present major challenges for the understanding of toxin action.Although the chemical characterization of cholera toxin has been extensive (4,5,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), there is a lack of the three-dimensional structural information needed to explain the membrane-penetration process. "V-shaped" and "ringlike" particles, 70-105 A in diameter, have been seen in electron micrographs of cholera toxin and B-subunit oligomers (20). Single crystals of cholera toxin suitable for x-ray diffraction analysis have been obtained (30), but the structure has not been reported. We describe here the use of the lipid-layer crystallization technique (31) for imaging B oligomers. The technique exploits binding to a lipid ligand in mono-or multilayers to orient and concentrate a macromolecule. Diffusion within the lipid layers allows the formation of two-dimensional crystals amenable to structure determination by electron microscopy and image processing. This approach gives lower resolution than x-ray diffraction, but it has the advantages that crystals form ...