Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia

Abstract

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951-962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (Kd approximately 5 nM), only CT elicited a cAMP-dependent Cl- secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.

Figure 1

CT and LTIIb bind specifically with high affinity to intact T84 monolayers. All binding assays were performed in HBSS containing 5% bovine serum albumin. Nonspecific signals in the absence of applied toxins were nearly identical to background (0.09 O.D. units). (A) Binding isotherms for CT and LTIIb applied at 4°C to T84 cells grown on plastic. Polyclonal anti-CT or anti-LTIIb antibodies were used to detect toxin bound at the cell surface. (B) Steady-state binding of biotin-labeled LTIIb (5 nM) or CT (5 nM) to T84 cells at 4°C in the presence of excess unlabeled LTIIb (1 μM), LTG33D B subunit (500 nM), purified anthrax protective antigen (200 nM), chicken IgY (1 μM), or unlabeled CT (1 μM) as indicated. Biotin-labeled toxins are indicated by *. In these studies, enzyme-linked avidin was used to detect toxin bound at the cell surface.

Figure 2

Ganglioside- enriched lipid extracts of T84 cell monolayers contain both GM1 and GD1a. (A) Lipid extracts prepared from 1,500 cm² confluent T84 cells (third lane) and purified GM1 (first lane) and GD1a (second lane) standards (300 ng each) were applied to activated silica gel plates, processed as described in Materials and Methods, and ligand blotted with CT (1 nM, top) or LTIIb (1 nM, bottom). Toxin binding to ganglioside extracts was assessed by application of specific antitoxin antibodies, followed by HRP-conjugated secondary antibody, and development using enhanced chemiluminescence. (B) Size resolution of T84 cell gangliosides by thin layer chromatography and resorcinol spray. GM1 standard (first lane), GD1a standard (second lane), T84 cell (4,500 cm²) lipid extract lower phase Folch cut (third lane), and upper phase Folch cut (fourth lane). In this preparation, the bulk of acidic glycolipids including GD1a remained associated with the lower lipid soluble phase because of conditions of Folch extraction (low salt and pH). Gangliosides migrating with GD1a are apparent in the third lane (and faintly in the fourth). Gangliosides migrating with GM1 are apparent in the fourth lane.

Figure 3

Signal transduction by CT and LTIIb in polarized human intestinal T84 monolayers and mouse Y1 adrenal cells. (A) Time course of Cl⁻ secretion induced by application of CT (20 nM) or LTIIb (20 nM) to apical reservoirs of T84 cell monolayers. The viability of monolayers exposed to LTIIb was demonstrated by application of the cAMP-agonist VIP to basolateral reservoirs at 120 min. (B–E) Bright field micrographs of mouse Y1 adrenal cells treated for 180 min at 37°C with the cAMP-agonist forskolin (10 μM, C), CT (20 nM, D), LTIIb (20 nM, E), or buffer alone (B).

Figure 3

Signal transduction by CT and LTIIb in polarized human intestinal T84 monolayers and mouse Y1 adrenal cells. (A) Time course of Cl⁻ secretion induced by application of CT (20 nM) or LTIIb (20 nM) to apical reservoirs of T84 cell monolayers. The viability of monolayers exposed to LTIIb was demonstrated by application of the cAMP-agonist VIP to basolateral reservoirs at 120 min. (B–E) Bright field micrographs of mouse Y1 adrenal cells treated for 180 min at 37°C with the cAMP-agonist forskolin (10 μM, C), CT (20 nM, D), LTIIb (20 nM, E), or buffer alone (B).

Figure 4

Association of the CT–GM1 and LTIIb–GD1a complexes with detergent-insoluble apical membrane domains in human T84 and mouse Y1 cells. Polarized human intestinal T84 or nonpolarized mouse Y1 adrenal cell monolayers were exposed apically to CT (20 nM) or LTIIb (20 nM) at 4°C for 30 min, and unbound toxins were removed by washing. The monolayers were homogenized in 1% TTBS at 4°C, adjusted to 40% sucrose, and layered under a 5–30% continuous sucrose gradient as described in Materials and Methods. Fractions from left to right (top to bottom) represent linear sucrose gradient from 5–32% sucrose. (A) SDS-PAGE and Coomassie stain (top) and Western blot (bottom) of sucrose gradient fractions from T84 cells. Fractions 2–15 represent linear sucrose gradient from 5–32% sucrose. Less than 1% total cellular protein floats into the gradient (fractions 6–9, representing 18.2–22.8% sucrose). This fraction is highly enriched for the CT–GM1 complex as assessed by Western blot. Fractions 16–19 represent Triton-soluble proteins at 40% sucrose. (B) Sucrose gradient of T84 cells exposed apically to biotin-labeled CT or LTIIb as described above and analyzed for CT or LTIIb B subunits by avidin blot. For gradients labeled CT and LTIIb, fractions 10 and 11 represent 19.4 –21% sucrose. Fractions 18–25 represent solubilized proteins at 40% sucrose. (C) Sucrose gradient of Y1 cells exposed to CT or LTIIb as described above and analyzed for CT or LTIIb A subunits by Western blot. For CT, fractions 11–13 represent 19.5–23.9% sucrose. For LTIIb, fractions 9–11 represent 19–24% sucrose. In both gradients, fractions 18–25 represent solubilized proteins at 40% sucrose. Lanes C indicate 1 ng of purified toxins run as controls. In all studies, A and B subunits for CT and LTIIb fractionated together.

Figure 5

Time course of CT and LTIIb chimeric toxins on cAMP-dependent Cl⁻ secretion in polarized T84 monolayers. (A) Time courses of Cl⁻ secretion (Isc) induced by application of purified LTIIb, LTIIb(S193M), or chimeric toxin 184 (LTIIb_A/ CT_B) to apical reservoirs of T84 cell monolayers. (B) Time courses of Cl⁻ secretion induced by application of chimeric toxin 184 (LTIIb_A/CT_B) to apical reservoirs of T84 cell monolayers in the absence or presence of purified GM1 (10 μM) or GD1a (10 μM) as competitive inhibitors. (C) Time courses of Cl⁻ secretion induced by application of rCT, or chimeric toxin 179 (CT_A/ LTIIb_B) to apical reservoirs of T84 cell monolayers. All toxins were applied in equivalent concentrations of 5,000 U/ml as assessed by bioassay on Y1 cells.

Figure 6

Effect of CT and LTIIb chimeric toxins on cAMP-dependent Cl⁻ secretion in polarized T84 monolayers. (A) ΔIsc (peak Isc mean ± SEM, 120 min after toxin addition corrected for baseline currents) for wt LTIIb(S193M), chimeric toxin 187, or chimeric toxin 184 (LTIIb_A/CT_B) applied to apical reservoirs of T84 cell monolayers in the presence or absence of purified GM1, GD1a, rCT B subunit, and rLTIIB as competitive inhibitors. ANOVA, P = 0.0001. * indicates conditions significantly different from chimera 184 alone (third column) at P ≤ 0.05 by multiple comparison procedures. (B) ΔIsc (peak Isc mean ± SEM, 120 min after toxin addition corrected for baseline currents) for wt rCT and chimeric toxin 179 (CT_A/LTIIb_B) to apical reservoirs of T84 cell monolayers in the presence or absence of GM1 or rCT B subunit as competitive inhibitors. ANOVA, P = 0.0001. * indicates conditions significantly different from rCT alone (third column) at P ≤ 0.05 by multiple comparison procedures. In all studies, mean ± SEM baseline currents were 1.1 ± 0.2 μA/cm², n = 25.

Figure 7

Association of chimeric toxins 184 LTIIb_A/CT_B (A) and 179 CT_A/LTIIb_B (B) with detergent-insoluble apical membrane domains in human T84 cells. Sucrose gradient of T84 cells exposed apically to chimeric toxins 184 and 179 and analyzed for CT or LTIIb B subunits by Western blot. For gradient labeled chimera 184, fractions 13–16 represent 19.2–24.8% sucrose. Fractions 20–25 represent solubilized proteins at 40% sucrose. For gradient labeled chimera 179, fractions 12–16 represent 19–28% sucrose. Fractions 20–25 represent solubilized proteins at 40% sucrose. Association of toxin with detergent-insoluble membranes depends on binding GM1—a function of the toxin's B subunit.