ther evidence for the receptor function of GM, is indicated by the finding that incubation of fat cells with GM, increased the cell membrane (13).

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 7, pp , 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 small intestinal mucosa of man, pig, and beef and amounted to 0.1, 2.0, and 43 nmol per g fresh weight, respectively. These differences in GMI content were associated with a quantitatively differing ability of the mucosal cells to bind cholera toxin. Human cells bound about 15,000 toxin molecules when saturated with the toxin, porcine cells 120,000, and bovine cells 2,600,000 molecules. The association constant (KA) of the cholera toxin binding was, for cells of all three species, about 109 liters/mol. Exogenously added GM1 ganglioside was incorporated in intestinal mucosal cells as well as in intact rabbit small bowel. The increment in GM1 was associated with a correspondingly increased number of binding sites for cholera toxin, whereas KA was unchanged. GM! incorporation increased the sensitivity of the rabbit small bowel to the diarrheogenic action of cholera toxin. Vibrio cholerae sialidase hydrolyzed isolated intestinal diand trisialogangliosides to GM,. However, the enzyme did not change the ganglioside pattern of intestinal mucosa, had very little influence on the number of toxin binding sites on intestinal cells, and. did not alter the sensitivity of the small bowel to the diarrheogenic action of the toxin. These results demonstrate a relationship in the intestinal mucosa between the GM, ganglioside concentration, the number of binding sites for cholera toxin, and the sensitivity to the biologic action of the toxin. Thus, the study strongly supports the concept that the GM, ganglioside is the intestinal binding receptor for cholera toxin. Diarrhea in cholera is due to the action on the small intestine of a protein exotoxin produced by the infecting Vibrio cholerae (2). The isolated cholera toxin, molecular weight 84,000 (3), consists of two types of noncovalently linked subunits, one heavy (H) and several light (L) ones (4-6). The complex of L subunits gives a rapid, tight binding of toxin to the outer membrane of mammalian cells, which is followed by a slower biologic action probably mediated by the H subunit (4-7). The key action of toxin on cellular metabolism seems to be activation of adenylate cyclase, and in the intestine the resulting cyclic AMP (camp) accumulation causes body-tolumen secretion of chloride, which leads to diarrhea (8). The monosialosylganglioside GM1 was recently found to bind and inactivate cholera toxin with an affinity and specificity which suggested that this ganglioside, believed to be a normal constituent of the plasma membrane of most mammalian cells, might be the membrane binding receptor for toxin (9-11). The observation that an inactive toxin derivative, choleragenoid toxoid, which binds as efficiently as toxin to target cells (7, 12), also had toxin-like binding properties to the GMi ganglioside in vitro (10) supports this view. Fur- Abbreviations: The nomenclature of Svennerholm (ref. 1) is used to designate the gangliosides: GM3, monosialylgalactosylglucosylceramide; GD3, disialylgalactosylglucosylceramide; GM1, galactosyl-nacetylgalactosaminyl-(sialyl)-galactosylglucosylceramide; GD1, disialylgalactosyl-n-acetylgalactosaminylgalactosylglucosylceramide; GTI, trisialylgalactosyl-n-acetylgalactosaminylgalactosylglucosylceramide; GA1, ganglio-n-tetraose, galactosyl-n-acetylgalactosaminylgalactosylglucosylceramide. ther evidence for the receptor function of GM, is indicated by the finding that incubation of fat cells with GM, increased the toxin binding ability of the cells and also the lipolytic response to the toxin, which suggested that the exogenous ganglioside was incorporated as functional receptor in the cell membrane (13). In the present study, we investigate the binding properties of cholera toxin to intestinal mucosa cells of various species, including man, and examine how these properties relate to the ganglioside pattern of the mucosa, in particular to the content of GM1 ganglioside. We further directly demonstrate incorporation of GM, ganglioside in intestinal cells and analyze the effects of such incorporation on both binding of toxin to the cells and on the diarrheogenic action of cholera toxin on the intact small bowel. It is well known that GM1, which by itself is resistant to sialidase, can be generated in vitro from more complex gangliosides by the action of V. cholerae sialidase (1). The possibility that such hydrolysis of membrane-located di- and trisialogangliosides could occur as a contributory pathogenic event in cholera infection has been recognized (9, 10). Consequently, we tried to evaluate the ganglioside-converting effect of V. cholerae sialidase on intestinal ganglioside preparations, cells, and intact small bowel mucosa as well as the possible influence of enzyme action for binding and diarrheogenicity of cholera toxin. MATERIALS AND METHODS Cholera Toxin and Toxoid. Culture filtrate of V. cholerae (lot 4493 G) and highly purified toxin (lot 0572) were received from Dr. Carl Miller, Cholera Research Program, NIAID, Bethesda, Md. Purified choleragenoid toxoid was a gift from Dr. R. A. Finkelstein, Dallas, Texas. The purified toxin and toxoid had been isolated as described (14). Neither the purified nor crude toxin had demonstrable sialidase activity, as tested with GD1a ganglioside as substrate. 125I-Labeled toxin was prepared with Na 125I and the Chloramine T coupling method as described (7), and the fraction of protein similar in size to native toxin was isolated by column filtration through Sephadex G-100. More than 90% of the radioactivity of this fraction showed specific binding to cells (mouse thymocytes) or to the ganglioside GM, (coupled to polystyrene tubes or to agarose beads), and the binding properties of the labeled toxin were indistinguishable from those of unlabeled toxin. The biologic activity was also unaffected by the labeling (permeability increase of rabbit skin and thymocyte camp generation). Glycosphingolipids. Pure GM1 ganglioside, the sialic acid-free derivative GA1, and GD1a ganglioside were prepared from human brain and characterized as described (15). [3H]GMl ganglioside was prepared by sequential treatment with galactose oxidase and sodium [3H]borohydride (16). The radioactive ganglioside was purified by column 2520

2 Biochemistry: Ho1mgren et al. chromatography on Sephadex G-25 and on silicic acid. The ganglioside was finally purified by preparative thin-layer chromatography on Silica Gel G in a solvent system of chloroform/methanol/water 60:32:7 (by volume). The specific activity was 2.0 X 106 dpm/nmol. Sephadex G-25 and DEAE-Sephadex A-25 were purchased from Pharmacia Ltd., Uppsala, Sweden; Silica Gel G and H from Fluka AG, Buchs, Switzerland. V. cholerae sialidase (500 U/ml) was purchased from Behringswerke AG, Marburg-Lahn, West Germany. Intestinal Preparations for Binding and Activity Studies. Human cells were prepared from surgically removed segments of jejunum, inches (38-50 cm) from the pylorus, of three patients. Porcine and bovine cells were prepared from 1.5-m segments taken immediately after slaughter from the middle of the small intestines of adult animals. The gut segments were placed on ice, cut open longitudinally, and with Ringer's solution gently washed free from visible mucus. After incubation with shaking at 370 for 30 min in Ringer's solution, the cell-rich fluid was poured through a nylon mesh. The passed cells were sedimented at 200 X g for 10 min and washed once with Ringer's solution. About 80-90% of the human cells were viable and more than 90% of the animal cells, as judged by trypan blue exclusion and phase contrast microscopy. They were suspended in Eagle's minimal essential medium supplemented with 1 mg/ml of CaCl2 to give cell densities of 5 X 106 per ml for the human cells and 2 X 106 per ml for the porcine and bovine cells. Aliquots of the cell suspensions were incubated at 370 for 25 min with GM1 or [3H]GM1 ganglioside in concentrations ranging from ,gM ( ,g/ml), V. cholerae sialidase (1-50 U/ml), or with no additive. The cells were then washed four times in minimal medium by centrifugation at 200 X g for 10 min and resuspended to the same densities in minimal medium supplemented with 0.2 mg/ml of freshly dissolved bovine serum albumin. Small bowel segments were prepared in live, anesthetized 8- to 12-week-old rabbits, that had been starved for hr. Starting 50 cm from the pylorus, about 100 cm of the gut was ligated, and via connecting tubes to each end of this segment it was rinsed with borate/gelatin buffer, ph 7.5 (7), supplied under mild hydrostatic pressure until the outflow fluid was clear. The rinsed gut was then tied off into 25- or 50-cm segments, which were incubated for 20 min with 0.5 ml/cm of gut of GM1 ganglioside (0.07 or 0.7,M in borate/ gelatin buffer), V. cholerae sialidase (1, 10, or 20 U/ml in borate/gelatin buffer with 1 mg/ml of CaC12), or, as control, with the buffer alone or, in a few instances, with gangliotetraose GA1 (0.7,M in borate/gelatin buffer). Thereafter the segments were rinsed as above with ml of borate/ gelatin buffer, and further ligated into approximately 4-cm long test loops. Binding of Cholera Toxin. Binding of toxin to the dispersed cells was tested by means of 125I-labeled toxin mixed with multiple concentrations of unlabeled toxin. The test procedure and the Scatchard plot calculations of the association constant, KA, and number, n, of bound toxin molecules when the cells were saturated with toxin have been described for thymus cells (7). Modifications were the use of a greater number of toxin concentrations, and testing aliquots of 4 X 105 porcine and bovine cells as well as 1 X 106 and 5 X 106 human cells. Binding of toxin to rabbit small bowel mucosa was tested by removing the described entire loop-divided intestinal segment, placing it on ice, and incubating the loops for 20 min with 1 ml each of 125I-labeled toxin mixed in minimal Proc. Nat. Acad. Sci. USA 72 (1975) 2521 medium/bovine serum albumin with different concentrations of unlabeled toxin. Each loop was then cut open and dip-washed several times in minimal medium; its radioactivity was determined. Biological Action of Cholera Toxin and Inhibition with Toxoid. The ability of crude toxin (V. cholerae culture filtrate) to cause fluid secretion in the small bowel of live rabbits was quantitated in the prepared intestinal loops (17). Quantitative inhibition of the diarrheogenic action of the crude toxin by choleragenoid (18, 19) was studied by injecting different amounts of choleragenoid in a volume of 0.5 ml into the intestinal loops 15 min before injection of an equal volume containing 0.1 or 1.0 mg of V. cholerae culture filtrate. The assay was the same as that used for the determination of toxin action. Isolation of Gangliosides from Human, Pig, and Bovine Intestine. The same bowel material was used as for the isolation of intestinal cells. The jejunum and ileum were everted over a cold rod, and the mucosa was carefully rinsed from mucus with physiological saline and then scraped off with a surgical knife. Great care was taken not to obtain any of the muscular layer, particularly at the preparation of the human jejunum. Porcine mucosa (555 g), porcine muscular layer (2.0 kg), bovine mucosa (185 g), bovine muscular layer (650 g), human mucosa (8-25 g; 4 samples), and human muscular layer (54 g) were available for ganglioside isolation. To two of the samples of human mucosa 0.1 nmol of [3H]GM1 was added. The mucosa scrapings and the samples of muscular layer, after homogenization in a Waring blendor, were extracted with 6 volumes of chloroform/methanol 1:2 (by volume). The gangliosides, except for ganglioside GM3, were isolated by partition and purified by column chromatography on Sephadex G-25 and Silica Gel G (Svennerholm, in preparation). All the ganglioside preparations, particularly those of human and pig mucosa, were still very impure, and the gangliosides of all the six sources were further purified by mild alkaline hydrolysis, chromatography on Sephadex G-25, DEAE-Sephadex A-25 (20), Sephadex G-25, and finally on Silica Gel H (21). The quantitative determinations of gangliosides were performed on the final eluate of the Silica Gel H column by the resorcinol method (22). The ganglioside pattern was determined as for brain (23). In the human mucosa, the ganglioside concentration was extremely low and total gangliosides were only separated into three fractions: ganglioside GM1, gangliosides GD1 + GT1, and ganglioside GD3. Ganglioside GM, and gangliosides GD1 and GT1 were hydrolyzed with V. cholerae sialidase, and the hydrolyzed gangliosides were semiquantitatively estimated on thin-layer plates sprayed with resorcinol (22). The neutral carbohydrate moiety of the gangliosides of bovine and porcine intestine was characterized in the following manner. The apparent ganglioside GM1 and the gangliosides GD1 and GT1 were isolated by thin-layer chromatography. These fractions were hydrolyzed with V. cholerae sialidase at ph 6.8, and the gangliosides that migrated as ganglioside GM1 were subjected to acid hydrolysis with 1 M formic acid at 1000 for 1 hr (24). The neutral tetraglycosylceramide formed was analyzed with thin-layer chromatography in chloroform/methanol/water 65:25:4 and 60:32:7 (by volume) with neutral glycosylceramides of known chemical structure as reference substances. Sialidase Hydrolysis of Pig Mucosa. Porcine mucosa (100 g) was hydrolyzed for 30 min at 370 with 200 units of V. cholerae sialidase in a total volume of 200 ml of 0.05 M acetate buffer ph S.0 or 0.05 M Tris-maleate buffer ph 6.8.

3 2522 Biochemistry: Holmgren et al. MUCOSA MUSCULARIS PIG BEEF REF PIG BEEF. ~~GM1 FIG. 1. Thin-layer chromatogram of the gangliosides from the mucosa and the muscular layers of small intestine of beef and pig. Portions of 20 nmol of N-acetylneuraminic acid were analyzed, and the plate was developed for 18 hr at 230 in propanol/water. The gangliosides were visualized by spraying with the resorcinol reagent (22). REF is a total ganglioside extract of beef brain. The gangliosides were isolated by the same technique as used for the large-scale isolation of gangliosides of porcine intestinal mucosa. G RESULTS Gangliosides in the Intestine of Man, Beef, and Pig. In the intestine there were large differences in the ganglioside concentration of the mucosa between the three species, while the ganglioside concentration of the muscular layer Table 1. Concentration of gangliosides (nmol/g fresh weight) in small intestine of man, pig, and beef Mucosa Ganglioside sialic acid GM, GD, + GT, Man Pig Beef Muscular layer Man Pig Beef Cerebral cortex* Man * Vanier et al. (21). was roughly of the same magnitude (Table 1). Although the total ganglioside content of the mucosa differed manyfold between the species, the concentration of ganglioside GM1 differed much more. Pig had a concentration that was approximately 20 times larger than that in man, and beef a concentration 21 times larger than that in pig, which means that the GM1 concentration of human intestinal mucosa was only 1/400 of that in the bovine mucosa. Human and porcine mucosa contained large concentrations of simple ganglioside mono- and disialosyllactosylceramide (GM3 and GDM). More than 90% of the value for total ganglioside sialic acid of man (Table 1) was accounted for by these two gangli6sides. In the human intestine, particularly in the muscular layer, the glucosamine-containing ganglioside with a neutral lactoneotetraose moiety (25) was also a large fraction. The concentration of the presumptive precursors of ganglioside GM1, gangliosides GD1 and GT1, were low in human but much larger in bovine and porcine mucosa. The ganglioside patterns of porcine and bovine intestine (Fig. 1) differed from those of human brain, because the gangliosides contained both N-acetyl- and N-glycolylneuraminic acid. The gangliosides that migrated slower than GM1 were isolated and hydrolyzed with V. cholerae sialidase at ph 6.8. They were completely hydrolyzed to gangliosides that migrated as ganglioside GM1 with N-acetyl- and N-glycolylneuraminic acid. The identity of GM1 was proven by determination of its components and by acid hydrolysis with 1 M formic acid at 1000 for 1 hr. A neutral tetraglycosylceramide was formed which migrated as N-gangliotetraose (GA1) in chloroform/methanol/water 65:25:4 and 60:32:7 (by volume). Incubation of Porcine Mucosa with V. cholerae Sialidase. Porcine mucosa was subjected to sialidase treatment at ph 5.0 or 6.8. The concentrations of total ganglioside sialic acid were the same in the incubated samples as in the control samples, which indicates that no hydrolysis occurred. This was further proven by quantitative determination of the ganglioside pattern before and after treatment. The pro- Table 2. Proc. Nat. Acad. Sci. USA 72 (1975) Membrane incorporation of GM, ganglioside in intestinal mucosal cells and effect on cholera toxin binding [3H]GM1 Toxin molecules GM, maximally molecules concentra- bound per cell incorporated Species tion* (,M) X 10-4 per cell X 10 Man Pig Beef Rabbit 0 L.Ont nt nt * The cells were incubated at 370 for 25 min with GM, ganglioside in minimal medium/bovine serum albumin and then carefully washed. t The numerical values could not be calculated since the experiment was performed in intestinal segments whose number of cells is unknown.

4 E E IL z z Ia Biochemistry: Holmgren et al CRUDE TOXI N. mg b -XA INHIBITING ~1 I CHOLERAGENOID. ug FIG. 2. Effect of pretreating rabbit small intestine with GM1 ganglioside on the sensitivity of the bowel to the diarrheogenic action of toxin (a) and to choleragenoid toxoid inhibition of the toxin action (b). (a) Hatched bars are mean values (+SEM) of fluid accumulation in four to five differently positioned small bowel segments of different animals, preincubated with 0.7 gm GM1 for 20 min and then carefully washed before challenge with the indicated amounts of crude toxin; open bars are corresponding values for buffer-treated control segments. The GM1 treatment enhances the intestinal sensitivity to toxin. (b) Corresponding values of GM1- treated and buffer-treated segments exposed to the indicated amounts of choleragenoid for 15 min before challenge with 0.1 mg of crude toxin. Choleragenoid inhibits the toxin action in the control (open bars) but not in the GMl-treated segments (hatched bars). portions of di- and trisialogangliosides were unchanged after sialidase hydrolysis. Binding of Cholera Toxin to Small Intestine. By means of '25I-labeled toxin in mixture with unlabeled toxin, specific saturable binding was demonstrated of toxin to dispersed small bowel mucosal cells of human, porcine, and bovine origin. The association constant, KA, in the binding to the different cell types was similar, about 109 liters/mol, but the number of receptors per cell differed much between the species. When saturated, the human cells bound, on the average, 15,000 toxin molecules per cell, the porcine cells 120,000 molecules, and the bovine cells 2,600,000 molecules (Table 2). Specific binding of toxin to intact rabbit small bowel mucosa was also demonstrated. Since the number of cells per intestinal loop was unknown, the number of bound toxin molecules per mucosal cell could not be estimated. The binding affinity, however, was found to be similar to that observed with dispersed cells of the other species. GM1 Ganglioside Incorporation and Cholera Toxin Binding. By means of [3H]GM1 it could be directly demonstrated that this ganglioside, by mere incubation at 370 for 25 min, can be firmly attached to intestinal cells, probably by incorporation in the outer membrane. This was qualitatively demonstrated with all cells tested, i.e., human, porcine, and bovine mucosal cells, but great quantitative differences were noted between the porcine and bovine cells (Table 2). Incubation of intestinal cells with GM1 ganglioside did not change the KA values in the binding of toxin to the cells, but led to an increase in the number of toxin molecules that could bind to the cells. This would indicate an increased number of receptors of identical qualities as the natural ones. Table 2 shows such GM1 concentration-dependent increases in the number of receptors for toxin on human and porcine intestinal mucosa cells and in rabbit small bowel segments; on the GMl-rich bovine intestinal cells the effect was much less. Influence of Sialidase on Cholera Toxin Binding. The toxin-binding properties of buffer-treated and sialidase- Proc. Nat. Acad. Sci. USA 72 (1975) 2523 CRUDE TOXIN mg INHIBITING CHOLERAGENOID ug Effect of treating rabbit small intestine with V. cholerae FIG 3. sialidase on the sensitivity of the bowel to the diarrheogenic action of toxin (a) and to choleragenoid toxoid inhibition of the toxin action (b). (a) mean values (+SEM) of fluid accumulation in 9 to 12 randomly positioned small bowel segments of different animals, preincubated with 10 U/ml of sialidase for 20 min and then washed before challenge with the indicated amounts of crude toxin; 0, corresponding values for buffer-treated control segments. (b) Mean values of fluid accumulation in sialidase-treated (filled symbols) and buffer-treated (open symbols) segments exposed to the indicated amounts of choleragenoid for 15 min before challenge with 0.1 (A, A) or 1.0 mg (-, 0) of crude toxin. In no instance do the values for the sialidase- and buffer-treated segments differ significantly (Student's test). treated intestinal cells or small bowel segments were also compared. Only a slight enhancing effect on the number of intestinal binding sites for toxin, much less than that caused by GM1 treatment of the cells, was found after the sialidase treatment. Thus, the increment in toxin binding was for the human, porcine, and bovine cells only about 20, 40, and 10%, respectively, even when the enzyme concentration was very high (40-50 U/ml), and for the intact rabbit mucosa about 30% (10 U/ml of sialidase). The KA values for toxinbinding to sialidase-treated and untreated cells or segments were similar. Biologic Action of Toxin on Small Intestine. Influence of GMl-Ganglioside Incorporation and V. cholerae Sialidase. The sensitivity to the diarrheogenic action of crude toxin (V. cholerae culture filtrate) was compared for buffertreated and GM1 ganglioside-preincubated rabbit small bowel loops (Fig. 2a). It was found that the GM1 ganglioside treatment made the loops responsive tootoxin concentrations that were too low to be effective in the control loops; the maximal response, on the other hand, was unchanged. Treatment of loops with the gangliotetraose GA1 did not change the sensitivity to toxin. To verify that the enhanced toxin sensitivity of the GMItreated intestinal loops could be ascribed to an increased number of toxin-binding sites, we tested whether more choleragenoid toxoid was required to inhibit the secretory response to subsequent toxin challenge in GMlLtreated than in buffer-treated loops. Fig. 2b shows that this was the case. Choleragenoid completely prevented the secretory response of the control loops, but in the tested amounts it had no inhibitory action in the GM1-treated loops. Choleragenoid binds as efficiently as cholera toxin to both GM1 ganglioside in vitro (10) and to mammalian cells (7, 12), which indicates that it inhibits the secretory response to toxin by occupying the mucosal GM1 receptors for the toxin. In a similar manner the influence of treating rabbit intestinal loops with V. cholerae sialidase was evaluated (Fig. 3). The sialidase-treated loops showed no different sensitivity than the control loops to the diarrheogenic action of toxin (Fig. 3a), nor did they require more choleragenoid for inhi-

5 2524 Biochemistry: Holmgren et al. bition of fluid production to toxin challenge (Fig. Sb). The quantitative inhibition by choleragenoid of two different doses of challenge toxin gives further support for the concept of receptor competition with similar affinity between the toxoid and toxin. To study a possible delayed influence of sialidase on the intestinal sensitivity to toxin, the incubation time with the enzyme in the intestine was extended to 200 min in three animals. This, however, did not change the sensitivity for toxin of the enzyme-treated segments from that of the control segments incubated with borate/gelatin buffer. Finally, it was found that incubation of the intestine in mvo with 10 U/ml of sialidase for 20 min did not result in liberation of more free sialic than incubation with buffer; the enzyme was fully active after the incubation, however, as tested with GDla ganglioside as substrate. DISCUSSION The concept, derived from the observations described in the introduction, that the GM1 ganglioside is the binding receptor for cholera toxin in the small bowel has gained much support from the present study. The occurrence of GM1 is demonstrated in the small bowel mucosa of various mammalian species, including man, and the marked differences in the content of this ganglioside among the species are found to correspond to different numbers of toxin molecules that can bind per intestinal cell. Further, it is shown by means of [3H]GMS that the exogenously added GM1 can be incorporated in intestinal mucosal cells or intact small bowel, and that such incorporation increases the number of toxin molecules that can bind per cell as well as the sensitivity of the intestine to the diarrheogenic action of toxin. Large differences were found in the concentration of GM1 ganglioside in the intestinal mucosa of man, pig, and beef. It is striking that the 21-fold higher GM1 concentration in beef as compared with pig mucosa was associated with a 22-fold higher average number of binding sites to toxin in the bovine than in the porcine mucosal cell. It is also notable that the naturally GM1-rich bovine cells incorporated many fewer [3H]GM1 molecules than the porcine cells, and parallelly did not increase the number of toxin binding sites per cell as much as the porcine cells. In consideration of the complicated experimental procedures, the rather close agreement in the porcine cells between the number of [3H]GM1 molecules incorporated per cell and the increase in number of toxin molecules that were bound by a toxin-saturated cell, the mean ratio being 1.3, suggests that almost every GM1 molecule incorporated is in a position to attach one toxin molecule. Moreover, the association constant in binding is similar, around 109 liters/mol, in the GMl-treated cells as in the untreated ones, further indicating that the incorporated GM1 molecules have identical toxin-binding properties as the natural receptors. V. cholerae sialidase can convert isolated di- and trisialogangliosides to the sialidase-resistant GM1 ganglioside, as shown previously (1) and in the present study. However, when permitted to act in vitro on intestinal cells or mucosa or in ivo in the small bowel, the enzyme did not change the ganglioside pattern of the mucosa, had little influence on the number of toxin-binding sites per intestinal cell, and altered neither the sensitivity of the small bowel to the diarrheogenic action of toxin nor the amount of choleragenoid toxoid re- Proc. Nat. Acad. Sci. USA 72 (1975) quired to inhibit the toxin action on the gut. These findings contradict a major pathogenetic role of V. cholerae sialidase production as a toxin-receptor-creating enzyme in the intestine. In contrast to our findings, King and van Heyningen (9) found with sialidase treatment an 8-fold increase of the toxin-inactivating capacity of rabbit and bovine intestinal mucosa scrapings; they also claimed that the ganglioside patterns of bovine brain and intestinal mucosal scrapings did not show any remarkable differences. Our Fig. 1 illustrates that the ganglioside pattern of the intestinal muscular layer is similar to that of brain, which is not unexpected since the gangliosides of the muscular layer are partly derived from the nerve cells in this tissue. Contamination of the mucosal scrapings tested by King and van Heyningen with muscular layer tissue might possibly explain the divergent findings as compared with those of the present study. We thank Dr. Tore Schersten for the specimens of human small intestine and Dr. Harald Almgren, Scan, Uddevalla, for the facilities he put at our disposal for the work with beef and pig intestine. This work was supported by grants from the Swedish Medical Research Council (no. 6X-3383 and 3X-627), the W.H.O., and the Hesselman Foundation. 1. Svennerholm, L. (1963) J. Neurochem. 10, Pierce, N. F., Greenough, W. B., III & Carpenter, C. C. (1971) Bacteriol. Rev. 35, LoSpalluto, J. J. & Finkelstein, R. A. (1972) Biochim. Biophys. Acta 257, Lonnroth, I. & Holmgren, J. (1973) J. Gen. Microbiol. 76, Cuatrecasas, P., Parikh, I. & Hollenberg, D. (1973) Biochemistry 12, Holmgren, J. & Lonnroth, I. (1975) J. Gen. Microbiol 86, Holmgren, J., Lindholm, L. & Lonnroth, I. (1974) J. Exp. Med. 139, Field, M. (1971) N. Engl. J. Med. 284, King, C. A. & van Heyningen, W. E. (1973) J. Infect. Dis. 127, Holmgren, J., Lonnroth, I. & Svennerholm, L. (1973) Infect. Immun. 8, Cuatrecasas, P. 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