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Interaction of Cholera Toxin and Membrane GM1 Ganglioside of Small Intestine Proc. Nat. Acad. Sci. USA Vol. 72, No. 7, pp. 2520-2524, July 1975 Biochemistry Interaction of cholera toxin and membrane GM1 ganglioside of small intestine (mucosal receptors/[3HJGMl ganglioside/ Vibrio cholerae sialidase/diarrhea) JAN HOLMGREN*, IVAR LONNROTH*, JAN-ERIC MANSSONt, AND LARS SVENNERHOLMt * Institute of Medical Microbiology and t Department of Neurochemistry, University of G~teborg, Goteborg, Sweden Communicated by Saul Roseman, March 31, 1975 ABSTRACT Ganglioside GM, was isolated from the ther evidence for the function of small intestinal mucosa of man, receptor GM, is indicated pig, and beef and amounted by the finding that incubation of fat cells with GM, in- to 0.1, 2.0, and 43 nmol per g fresh weight, respectively. creased These differences in GMI content were associated with a the toxin binding ability of the cells and also the li- quantitatively differing ability of the mucosal cells to bind polytic response to the toxin, which suggested that the exog- cholera toxin. Human cells bound about 15,000 toxin mole- enous ganglioside was incorporated as functional receptor in cules when saturated with the toxin, porcine cells 120,000, the cell membrane and bovine (13). cells 2,600,000 molecules. The association con- In the present study, we investigate the binding properties stant (KA) of the cholera toxin binding was, for cells of all of cholera to three species, about 109 liters/mol. Exogenously added GM1 toxin intestinal mucosa cells of various species, ganglioside was incorporated in intestinal mucosal cells as including man, and examine how these properties relate to well as in intact rabbit small bowel. The increment in GM1 the ganglioside pattern of the mucosa, in particular to the was associated with a correspondingly increased number of content of GM1 ganglioside. We further directly demonstrate binding sites for cholera toxin, whereas KA was unchanged. incorporation of GM, ganglioside in intestinal cells and ana- GM! incorporation increased the sensitivity of the rabbit the effects of small bowel to lyze such incorporation on both binding of the diarrheogenic action of cholera toxin. toxin to the on Vibrio cholerae sialidase hydrolyzed isolated intestinal di- cells and the diarrheogenic action of cholera and trisialogangliosides to GM,. However, the enzyme did toxin on the intact small bowel. It is well known that GM1, not change the ganglioside pattern of intestinal mucosa, had which by itself is resistant to sialidase, can be generated in very little influence on the number of toxin binding sites on vitro from more complex gangliosides by the action of V. intestinal cells, and. did not alter the sensitivity of the small cholerae sialidase The that such bowel to (1). possibility hydrolysis of the diarrheogenic action of the toxin. membrane-located di- and could occur These results demonstrate a relationship in the intestinal trisialogangliosides mucosa between the GM, ganglioside concentration, the as a contributory pathogenic event in cholera infection has number of binding sites for cholera toxin, and the sensitivity been recognized (9, 10). Consequently, we tried to evaluate to the biologic action of the toxin. Thus, the study strongly the ganglioside-converting effect of V. cholerae sialidase on supports the concept that the GM, ganglioside is the intesti- intestinal ganglioside preparations, cells, and intact small nal binding receptor for cholera toxin. bowel mucosa as well as the possible influence of enzyme ac- tion for and of cholera Diarrhea in cholera is due to the action on the small intestine binding diarrheogenicity toxin. of a protein exotoxin produced by the infecting Vibrio chol- erae (2). The isolated cholera toxin, molecular weight 84,000 MATERIALS AND METHODS (3), consists of two types of noncovalently linked subunits, one heavy (H) and several light (L) ones (4-6). The complex Cholera Toxin and Toxoid. Culture filtrate of V. cholerae of L subunits gives a rapid, tight binding of toxin to the (lot 4493 G) and highly purified toxin (lot 0572) were re- outer membrane of mammalian cells, which is followed by a ceived from Dr. Carl Miller, Cholera Research Program, slower biologic action probably mediated by the H subunit NIAID, Bethesda, Md. Purified choleragenoid toxoid was a (4-7). The key action of toxin on cellular metabolism seems gift from Dr. R. A. Finkelstein, Dallas, Texas. The purified to be activation of adenylate cyclase, and in the intestine the toxin and toxoid had been isolated as described (14). Neither resulting cyclic AMP (cAMP) accumulation causes body-to- the purified nor crude toxin had demonstrable sialidase ac- lumen secretion of chloride, which leads to diarrhea (8). tivity, as tested with GD1a ganglioside as substrate. 125I-La- The monosialosylganglioside GM1 was recently found to beled toxin was prepared with Na 125I and the Chloramine bind and inactivate cholera toxin with an affinity and speci- T coupling method as described (7), and the fraction of pro- ficity which suggested that this ganglioside, believed to be a tein similar in size to native toxin was isolated by column fil- normal constituent of the plasma membrane of most mam- tration through Sephadex G-100. More than 90% of the ra- malian cells, might be the membrane binding receptor for dioactivity of this fraction showed specific binding to cells toxin (9-11). The observation that aninactive toxin deriva- (mouse thymocytes) or to the ganglioside GM, (coupled to tive, choleragenoid toxoid, which binds as efficiently as toxin polystyrene tubes or to agarose beads), and the binding to target cells (7, 12), also had toxin-like binding properties properties of the labeled toxin were indistinguishable from to the GMi ganglioside in vitro (10) supports this view. Fur- those of unlabeled toxin. The biologic activity was also unaf- fected by the labeling (permeability increase of rabbit skin Abbreviations: The nomenclature of Svennerholm (ref. 1) is used to and thymocyte cAMP generation). designate the gangliosides: GM3, monosialylgalactosylglucosylcera- Glycosphingolipids. Pure GM1 ganglioside, the sialic mide; GD3, disialylgalactosylglucosylceramide; GM1, galactosyl-N- acid-free acetylgalactosaminyl-(sialyl)-galactosylglucosylceramide; GD1, disi- derivative GA1, and GD1a ganglioside were pre- alylgalactosyl-N-acetylgalactosaminylgalactosylglucosylceramide; pared from human brain and characterized as described GTI, trisialylgalactosyl-N-acetylgalactosaminylgalactosylglucosyl- (15). [3H]GMl ganglioside was prepared by sequential treat- ceramide; GA1, ganglio-N-tetraose, galactosyl-N-acetylgalactosam- ment with galactose oxidase and sodium [3H]borohydride inylgalactosylglucosylceramide. (16). The radioactive ganglioside was purified by column 2520 Downloaded by guest on September 25, 2021 Biochemistry: Ho1mgren et al. Proc. Nat. Acad. Sci. USA 72 (1975) 2521 chromatography on Sephadex G-25 and on silicic acid. The medium/bovine serum albumin with different concentra- ganglioside was finally purified by preparative thin-layer tions of unlabeled toxin. Each loop was then cut open and chromatography on Silica Gel G in a solvent system of chlo- dip-washed several times in minimal medium; its radioactiv- roform/methanol/water 60:32:7 (by volume). The specific ity was determined. activity was 2.0 X 106 dpm/nmol. Biological Action of Cholera Toxin and Inhibition with Sephadex G-25 and DEAE-Sephadex A-25 were pur- Toxoid. The ability of crude toxin (V. cholerae culture fil- chased from Pharmacia Ltd., Uppsala, Sweden; Silica Gel G trate) to cause fluid secretion in the small bowel of live rab- and H from Fluka AG, Buchs, Switzerland. V. cholerae sial- bits was quantitated in the prepared intestinal loops (17). idase (500 U/ml) was purchased from Behringswerke AG, Quantitative inhibition of the diarrheogenic action of the Marburg-Lahn, West Germany. crude toxin by choleragenoid (18, 19) was studied by inject- Intestinal Preparations for Binding and Activity Stud- ing different amounts of choleragenoid in a volume of 0.5 ies. Human cells were prepared from surgically removed ml into the intestinal loops 15 min before injection of an segments of jejunum, 15-20 inches (38-50 cm) from the py- equal volume containing 0.1 or 1.0 mg of V. cholerae cul- lorus, of three patients. Porcine and bovine cells were pre- ture filtrate. The assay was the same as that used for the de- pared from 1.5-m segments taken immediately after slaugh- termination of toxin action. ter from the middle of the small intestines of adult animals. Isolation of Gangliosides from Human, Pig, and Bovine The gut segments were placed on ice, cut open longitudinal- Intestine. The same bowel material was used as for the iso- ly, and with Ringer's solution gently washed free from visi- lation of intestinal cells. The jejunum and ileum were evert- ble mucus. After incubation with shaking at 370 for 30 min ed over a cold rod, and the mucosa was carefully rinsed in Ringer's solution, the cell-rich fluid was poured through a from mucus with physiological saline and then scraped off nylon mesh. The passed cells were sedimented at 200 X g with a surgical knife. Great care was taken not to obtain any for 10 min and washed once with Ringer's solution. About of the muscular layer, particularly at the preparation of the 80-90% of the human cells were viable and more than 90% human jejunum. Porcine mucosa (555 g), porcine muscular of the animal cells, as judged by trypan blue exclusion and layer (2.0 kg), bovine mucosa (185 g), bovine muscular layer phase contrast microscopy. They were suspended in Eagle's (650 g), human mucosa (8-25 g; 4 samples), and human minimal essential medium supplemented with 1 mg/ml of muscular layer (54 g) were available for ganglioside isola- CaCl2 to give cell densities of 5 X 106 per ml for the human tion. To two of the samples of human mucosa 0.1 nmol of cells and 2 X 106 per ml for the porcine and bovine cells. Al- [3H]GM1 was added.
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