Choleragen

Moss J, Vaughan M Activation of adenylate cyclase by choleragen. Annu Rev Biochem 1979 48 581-600. 

In recent work on CHO cells, it had been suggested that the effects of butyrate are mediated by cyclic AMP (18). We found, however, that cyclic AMP (2 mM), its mono- (1 mM and dibutyryl (0.5 mM) derivatives, theophylline and prostaglandins did not cause an elevation in si alyl transferase activity (1). Choleragen, which is a potent and persistant activator of adenylate cyclase (see below), also did not elevate sialyl transferase activity in HeLa cells (Table I) or alter cell morphology (unpublished observations). Thus, it is unlikely that these effects of butyrate are mediated by elevation of cyclic AMP levels. 

Experiment Number Treatment Time (h) Sialyl transferase Activity Control Butyrate Choleragen ... 

Figure 4. Effect of butyrate treatment on choleragen binding to HeLa cells...

A Cells exposed to medium containing 5mM sodium butyrate for times indicated. B After 48 hr, as in A, medium replaced with fresh medium without butyrate. C Cells exposed for 48 hr to medium containing the indicated concentrations of butyrate. Specific binding of, 2sI-choleragen determined as described in Ref. 5. (Data from Ref. 5J... 

Figure 5. Effect of labeled and unlabeled choleragen concentrations on n5I-choleragen binding to control and butyrate-treated HeLa cells...

The induction of toxin receptors by butyrate also occurred in serum-free medium (5). Serum contains gangliosides including GM1 and GM1-deficient cells can absorb GM1 from serum and become responsive to choleragen (22). Cells exposed simultaneously to butyrate and cyc1oheximide""cTid not exhibit an increase in choleragen binding (unpublished observations). Thus, butyrate appears to induce toxin receptors de novo in a manner analogous to the induction of GM3. 

Several other cultured cell lines were affected by butyrate (5). These included rat glial C6 cells (12-fold increase) and Friend erythroleukemic cells (4-fold increase). The increase in choleragen receptors in Friend cells was also time dependent (Table III). In addition, butyrate appeared to be specific dimethyl-sulfoxide (DMSO) induces erythroid differentiation (23) as does butyrate (24) but it did not cause an increase in toxin receptors (Table 111)7... 

Table II. Binding of Choleragen to HeLa Cells Treated with Various Fatty Acids ...

Table III. Binding of Choleragen to Friend Erythroleukemic Cells Treated with Sodium Butyrate or Dimethylsulfoxidea...

If GM1 is the choleragen receptor, then butyrate-treated cells should have an increase in GM1 content. This is demonstrated in Table IV. Although GM1 could not be detected in control HeLa cells, they would contain less than 1 pmol per mg protein based on the limits of the sensitivity of the analytical procedure (5). GM1 was quantitated in the butyrate-treated cells (28.5 pmol per mg protein) and this increase is similar to the 32-fold increase in toxin binding observed in cells from the same experiment. The delipidated residue contained less than 1% of the toxin binding found in intact cells (Table IV). In addition, removal of the cells from the culture dishes with trypsin as opposed to the mechanical scraping routinely used had no effect on 125I-cholera-gen binding to either control or butyrate-treated cells (5). 

Table IV. Effect of Sodium Butyrate on Choleragen Receptors and GM1 Content of HeLa Cells...

Table V. Effect of Gangliosides on Binding of Choleragen to Cells ...

The above results indicate that the increased choleragen binding to butyrate-treated HeLa cells is associated with increased GMl content. This was confirmed with Friend erythroleu-kemic cells (Table VII). Untreated Friend cells have measurable... 

HeLa cells were treated with 5 mM butyrate for 48 h or with 1 yM GM1 for 1 h. Both types of treated cells bound similar amounts of 125I-choleragen and about 60-fold more than did control cells (Fig. 6a). When the cells were exposed to a saturating dose of toxin, the time course and extent of cyclic AMP accumulation were similar for both the butyrate-treated and the GM1-treated HeLa cells and more rapid than in the control cells (Fig. 6b). Thus, butyrate induces choleragen receptors in HeLa cells that are functionally equivalent to those created by incorporation of exogenous GM1. 

Liposomes Incubated With 125I-Choleragen Bound (cpm)... 

Figure 6. Comparison of butyrate and GM1 treatment of HeLa cells on choleragen...

Table VIII. Effect of Cycloheximide on Butyrate-Induced Choleragen Receptors In HeLa Cellsa...

Additions 0-48 h to Medium 48-96 h Choleragen Receptors (fmol/mq protein)... 

Little is known about the function of gangliosides. Gangliosides may serve as cell surface receptors (45) and as biotransducers of membrane-mediated information ). The ganglioside GM1 has been implicated as the receptor for choleragen. Our studies clearly indicate that butyrate induces toxin receptors and GM1 in parallel. In addition, the toxin receptors induced by butyrate are functionally indistinguishable from exogenous GM1 that has been absorbed by the cells. We believe that these observations are the quintessential evidence that GM1 alone is the receptor for choleragen. 

Brady, R.O. Functional incorporation of ganglioside into intact cells Induction of choleragen responsiveness. Proc. Natl. Acad. Sci., (U.S.A.), 1976, 73, 1031-1037. 

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