m1 · m1 at the plasma membrane, enter the cell by endocytosis, and then traffic retrograde into...

Cholesterol dependence of CT action

1

Uncoupling of the cholera toxin GM1 ganglioside-receptor complex from endocytosis,

retrograde Golgi trafficking, and downstream signal transduction by depletion of

membrane cholesterol

Anne A. Wolf*, Yukako Fujinaga*, and Wayne I. Lencer*¥

*GI Cell Biology, the Department of Pediatrics, Children's Hospital and Harvard Medical School, Boston, and the ¥Harvard Digestive Diseases Center

Corresponding Author: Wayne I. Lencer GI Cell Biology Childrens Hospital 300 Longwood Ave. Boston, MA 02115 617-355-8599 617-264-2876 FAX Running Title: Cholesterol dependence of CT action Key Words: cholera toxin, endocytosis, cholesterol, ganglioside GM1, Golgi Apparatus, chloride secretion, lipid rafts, detergent insoluble membrane microdomains, T84 cells

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on February 21, 2002 as Manuscript M109834200 by guest on June 1, 2020

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Summary

To induce toxicity, cholera toxin must first bind ganglioside GM1 at the plasma membrane,

enter the cell by endocytosis, and then traffic retrograde into the ER. We recently proposed that

GM1 provides the sorting motif necessary for retrograde trafficking into the biosynthetic/secretory

pathway of host cells, and that such trafficking depends on association with lipid rafts and lipid

raft function. To test this idea, we examined whether CT action in human intestinal T84 cells

depends on membrane cholesterol. Chelation of cholesterol with 2-hydroypropyl β-cyclodextrin

or methyl β-cyclodextrin reversibly inhibited CT-induced chloride secretion and prolonged the

time required for CT to enter the cell and induce toxicity. These effects were specific to CT, as

identical conditions did not alter the potency or toxicity of anthrax edema toxin that enters the

cell by another mechanism. We found that endocytosis and trafficking of CT into the Golgi

apparatus depended on membrane cholesterol. Cholesterol depletion also changed the density

and specific protein content of CT-associated lipid raft fractions but did not entirely displace the

CT-GM1 complex from these lipid raft microdomains. Taken together these data imply that

cholesterol may function to couple the CT-GM1 complex with raft domains and with other

membrane components of the lipid raft required for CT entry into the cell.

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Introduction

V. cholerae causes worldwide epidemics of life-threatening secretory diarrhea by colonizing

the intestinal lumen and producing cholera toxin (CT)a, a potent enterotoxin that invades the

intestinal epithelial cell as a fully folded protein. Structurally, CT consists of two components.

The pentameric B-subunit binds stoichiometrically to five GM1 gangliosides on the apical

(lumenal) surface of intestinal epithelial cells, and the enzymatic A-subunit activates adenylyl

cyclase inside the cell by catalyzing the ADP-ribosylation of the heterotrimerc GTPase Gs (1).

Activation of adenylyl cyclase in intestinal crypt epithelial cells leads to Cl- secretion, the

fundamental transport event in secretory diarrhea.

To induce disease, both A- and B-subunits must enter the host epithelial cell as a fully

assembled holotoxin by moving retrograde through the biosynthetic pathway into the ER

(Fujinaga and Lencer, unpublished data) where the A-subunit unfolds, dissociates from the B-

pentamer, and translocates to the cytosol, presumably by dislocation through the protein

conducting channel sec61p (2). The time between CT binding to GM1 at the cell surface and

induction of toxicity has been termed the "lag phase". The lag phase corresponds to the time

required for trafficking CT into the ER, unfolding of the A-subunit by interaction with protein

disulfide isomerase, and finally dislocation of the A-subunit into the cytosol (2, 3).

In the model polarized intestinal epithelial cell line T84, CT function depends on B-subunit

binding to (and possibly clustering) ganglioside GM1. Binding to GM1 anchors CT to the host cell

membrane and associates CT with cholesterol-rich detergent insoluble membrane microdomains,

termed DIGs (detergent-insoluble glycosphingolipid rich membrane microdomains) or lipid rafts

(4). GM1 exhibits specificity for CT action in human intestinal epithelial cells as the ganglioside

GD1a does not substitute for GM1 as a receptor for toxin action or for association with lipid rafts in


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this cell type (4,5). Chimeric toxins, for example, containing the A-subunit of CT assembled

with the B-subunit of E. coli toxin LTIIb, bound only to GD1a, did not associate with lipid rafts,

and did not lead to a functional response (4). Based on these studies, we have recently proposed

that the B-subunit-GM1 complex represents the sorting motif necessary for toxin trafficking

retrograde into the Golgi apparatus and possibly ER of host eukaryotic cells. We have also

proposed that such trafficking depends on association with lipid rafts and lipid raft function. To

test this idea, we now examine whether CT action depends on membrane cholesterol.

Lipid rafts are highly enriched in cholesterol, GPI-linked proteins, and glycosphingolipids,

including ganglioside GM1, the receptor for CT. Abundant evidence indicates that raft structure

and function in cellular metabolism, including certain forms of ligand-induced signal

transduction, protein and lipid sorting, endocytosis, and transcytosis, depend critically on

cholesterol (4, 6-15). Demonstration of such sensitivity to membrane cholesterol has been taken

widely as evidence for dependence on raft function in these cellular processes. With respect to

CT and aerolysin toxin, another bacterial enterotoxin that binds membrane receptors located in

lipid rafts, disruption of membrane cholesterol by the sterol binding molecule filipin or by

chelation with β-cyclodextrin has been shown to effect endocytosis and toxin action (16, 17). On

the other hand, whether lipid rafts function directly in CT endocytosis has recently been

questioned (18). In some cellular systems, clathrin-mediated endocytosis displays sensitivity to

disruption of membrane cholesterol (19, 20), and although CT binds GM1 located both in lipid

rafts and clathrin-coated pits, the toxin enters hippocampal neurons and A431 epithelial cells by

clathrin-mediated mechanisms (18, 19).

In the current study, we utilize 2-hydroxy and methyl β-cyclodextrin to examine CT

trafficking and function in model epithelial cells acutely depleted in cholesterol (22-24). To


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control for general effects of cholesterol depletion on membrane dynamics, we utilize anthrax

edema toxin (EdTx). Like CT, anthrax EdTx is a two component toxin. Both CT and EdTx

enter polarized monolayers of intestinal T84 cells by receptor-mediated endocytosis, and both

toxins induce an identical cAMP-dependent Cl- secretory response, but by different mechanisms.

The enzymatic component of EdTx, termed edema factor (EF), is itself a potent calmodulin-

dependent adenylyl cyclase that unlike CT translocates to the cytosol directly across the

endosome membrane. Membrane translocation of EF and the induction of toxicity depend

critically on entry of EdTx into an acidic endosomal compartment. Retrograde transport into

Golgi cisternae or ER is not required (25). Also unlike CT, the membrane binding component of

EdTx, termed protective antigen (PA), recognizes a receptor on T84 cell membranes that displays

strict basolateral polarity, and the EdTx-receptor complex does not fractionate with lipid rafts

(25).

Our data show that endocytosis and trafficking of CT into Golgi cisternae, as well as CT-

induced Cl- secretion, depend on membrane cholesterol. Cholesterol depletion altered lipid raft

structure and presumably function, and only partially displaced the CT- GM1 complex from lipid

raft microdomains. Cholesterol depletion had no detectable affect on entry of anthrax EdTx into

acidic endosomes as assessed as an EdTx induced Cl- secretory response. These data indicate

that CT trafficking depends on toxin association with lipid rafts, and imply that cholesterol may

function in lipid raft structure to couple the CT-GM1 complex with raft domains and with other

membrane components involved in membrane sorting or downstream signal transduction

required for CT entry into the cell.


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Experimental Procedures

Materials and Antibodies. Cholera toxin and cholera toxin B-subunit coupled to horseradish

peroxidase (HRP) were obtained from Calbiochem (San Diego, CA) or were recombinantly

expressed. Anthrax edema toxin was a kind gift from Dr. R. John Collier (Microbiology,

Harvard Medical School). 2-hydroxy β−cyclodextrin, methyl β -cyclodextrin, cholesterol and

cholesterol analogues were obtained from Sigma Chemical Co. (St. Louis, MO). Mouse

monoclonal antibody to pp60src was obtained from Upstate Biotechnology (Lake Placid, NY)

rabbit polyclonal antibody to caveolin-1 from Santa Cruz Biotechnology (Santa Cruz, CA) and

mouse monoclonal antibody to ecto-5’ nucleotidase from Linda Thompson, Oklahoma Medical

Research Foundation (Oklahoma City, Oklahoma). Rabbit polyclonal antibodies to CT B-

subunits were previously described (26). All other chemicals were from Sigma Chemical Co.

(St. Louis, MO) unless otherwise stated.

Cell Culture. T84 cells obtained from American Type Culture Collection (Rockville, MD)

were cultured and passaged as previously described (27). Passages 80-92 were used for these

studies. Monolayers used for electrophysiology and for toxin binding studies were grown on

0.33 cm2 polycarbonate filters (Costar, Corning, NY) and used 12-14 days after plating.

Monolayers used for isolation of lipid rafts were grown on 45 cm2 filters and used 21 days after

plating. Hanks balanced salt solution (HBSS) to which 10mM HEPES was added and PH

adjusted to 7.4 was used for all assays unless otherwise noted.

Cholesterol Depletion and Electrophysiology. Initial studies showed that cyclodextrins

induced a progressive concentration and time dependent decrease in transepithelial resistance.

We thus defined conditions for acute cholesterol depletion that preserved monolayer resistance


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using either 16.5mM 2OHβ-CD or 4mM mβ−CD applied to apical or basolateral cell surfaces

of T84 cell monolayers for one hour.

To assay for cholesterol depletion, total cell lipids from T84 cell monolayers were extracted

in chloroform, dried and then resuspended in minimal volume (3 µl) of chloroform.

Cholesterol content was analyzed per mg total cell protein by colorimetric assay using the

Infinity Cholesterol Reagent according to the manufacture’s directions (Sigma Diagnostics, St.

Louis, MO). Absorbance was read at 500nM on a Spectronic21D spectrophotometer (Milton

Roy Products).

For assessment of Cl- secretion (measured as a short circuit current, Isc), cells were treated

as above with the appropriate cyclodextrin analogue for one hour at 37°C before adding CT 20

nM or 1nM to apical or basolateral reservoirs respectively in the continued presence or absence

of cyclodextrin. The time course of toxin-induced Cl- secretion (Isc) and transepithelial

resistance were measured as previously described (28). To provide two point calibration for

all studies, the cAMP-agonist vasoactive intestinal peptide (VIP, 10 nM) was applied to

basolateral reservoirs at the end of each experiment (either 60 or 85 min after application of

toxin). To replete monolayers with cholesterol, cholesterol and related sterols were complexed

with mβ-CD as previously described and used at a final concentration of 0.2 mM of sterol (29).

Toxin Binding. Specific toxin binding to apical membranes of T84 cell monolayers treated or

not treated with 16.5mM 2OHβ-CD at 37°C was measured. All incubations were done in

buffer made of HBSS containing 0.1% BSA. After pretreatment, monolayers were cooled to

4°C, and incubated with buffer alone or100 nM CT B-subunit (to measure nonspecific binding)

in buffer for 20 min. Monolayers were then washed and incubated apically with 20 nM CT B-

subunit-HRP in buffer for 30 minutes on ice. Unbound toxin was removed by three washes


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with ice cold HBSS. Cell surface bound CT B-HRP was assessed by colorimetric assay using

30% hydrogen peroxide in 1 mM 2,2’ azino-di(3-ethylbenzthiazoline sulfonic acid in 100mM

citrate-phosphate buffer, pH 4.2 as previously described (4). Specific binding of toxin to the

cell surface was determined by subtracting nonspecific binding from total surface bound toxin.

Endocytosis. T84 cells grown for two weeks on 0.33 cm2 filter supports were treated with 4mM

mβ -CD or buffer alone for 1 h at 37°C, brought to 4°C in HBSS, and incubated with 20 nM

CT in the presence of 4mM mβ -CD or buffer alone for 45 min at 4°C. Monolayers were then

warmed to 37°C in the presence of apical 20 nM CT 16.5mM mβ-CD or buffer for the

indicated times, or kept at 4°C for the duration of the experiment. All monolayers were then

washed in ice cold buffer to remove unbound toxin. Where indicated, cell surface bound CT

was removed by immmersing each monolayer in HBSS pH 7.4 at 37°C for 10 s (to further

release unbound toxin at the cell surface) and then immediately transferred to HBSS pH 2.5 at

4°C for 5 min. Each monolayer was then immersed for 5 min in HBSS pH 7.4 at 4°C, and

incubated again in HBSS pH 2.5 at 4°C for an additional 5 min. 97.5% +/- 16.5mM (n=4) of

CT was stripped from the cell surface using this procedure. After removal of cell-surface

bound toxin, monolayers were cut from their filter supports, immersed in 5% SDS and boiled

for analysis of total cell associated CT B-subunit.

Sucrose Equilibrium Density Centrifugation. Sucrose Equilibrium Density Centrifugation

was performed as described previously (4). One or two confluent 45 cm2 monolayers of T84

cells were used for isolation of detergent insoluble membranes. All steps were performed at

4°C. Cells were scraped into 2 ml of ice cold 50mM Tris buffered saline containing

0.16.5mM Tween-20 (TTBS) and a protease inhibitor tablet (containing EDTA, Pancreas-

extract, pronase, thermolysin, chymotrypsin, trypsin, papain) from Boehringer Mannheim


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(GmbH, Germany), and homogenized by five strokes in a tight-fitting dounce on ice. The

homogenate was adjusted to 40% sucrose by adding 2 ml of 80% sucrose in TTBS, layered

under a continuous 5-30% sucrose gradient and centrifuged at 39,0000 rpm for 16-20 h in a

swinging bucket rotor (model SW41; Beckman Instruments, Palo Alto, CA). The presence of a

floating membrane fraction was noted visually, and 0.5 ml fractions were collected from the

top. For step gradients, the cell homogenates in 4 ml of 40% sucrose were layered first under

5 ml of 30% sucrose , and then 5 ml of 5% sucrose was added to the top of the gradient. The

step gradients were centrifuged as described above. Raft fractions were collected from the 30-

5% interface and combined. Soluble material remained in the 40% sucrose fraction at the

bottom of the gradient. For all gradients, fractions were normalized for protein content (Pierce

BCA protein assay, Pierce, Rockford, Illinois), analyzed by SDS PAGE, Western blotted for

the indicated proteins as previously described (30), and quantified by densitometry using a

Kodak Digital Science Image Station 440 CF (NEN Life Sciences, Boston, MA). All signals

were in the linear range. Raft and soluble fractions from some experiments were analyzed for

5’nucleotidase (CD73) activity as previously described (31).

Retrograde Golgi Transport Assay. CT holotoxin was engineered to contain the sulfation

consensus motif SAEDYEYPS at the C-terminus of its B-subunits (Y. Fujinaga and W. I.

Lencer, unpublished). Recombinant toxins were produced and purified as previously described

(32). For in vivo sulfation experiments, T84 cells were first washed, incubated in sulfate-free

HBSS for 30 min at 37oC, and then incubated again for an additional 1 h in fresh sulfate-free

HBSS. The monolayers were then incubated with 0.5 mCi/ml Na2

35SO4 in the same buffer for

30 minutes. The CT-variant toxin containing the sulfation motif was added to T84 cells both

apically and basolaterally to a final concentration of 20 nM, incubated for 50 minutes at 37oC,


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and then washed twice with ice-cold HBSS. Following total cell lysis, an immunoprecipitation

using rabbit anti-CT-B antibodies as described previously (33) was performed. Samples were

run on 10-20% denaturing Tris⋅HCl polyacrylamide gels (BioRad, CA), and analyzed with a

PhosphorImager (Molecular Dynamics Inc, Sunnyvale, CA).

Statistics. Data were analyzed using Statview 512+ software (Brainpower, Inc., Calabasa, CA)


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Results

We utilized the cyclodextrin analogues 2-hydroxypropyl β-cyclodextrin (2OHβ-CD) and

methy β-cyclodextrin (mβ-CD) to determine whether the mechanism of CT action depends on

membrane cholesterol. Initial studies showed that treatment of T84 cell monolayers with

2OHβ-CD caused a dose and time-dependent loss of transepithelial resistance (TER),

presumably due to direct effects of cholesterol depletion on tight junction structure (34, 35).

Conditions were thus defined that depleted approximately 50% of total cell cholesterol but

maintained monolayer resistance intact throughout the time course of each experiment. Under

optimal conditions, however, transepithelial resistance still fell progressively to 15% of initial

values by the end of each time course (from 1035 + 54 to 151 + 22 •, n=10), and the cAMP-

agonist VIP was used in all studies to calibrate measurement of Isc. Like CT, VIP increases

intracellular cAMP in T84 cells by activating the heterotrimeric GTPase Gs. All subsequent

events in the Cl- secretory response are identical. The alternative sterol binding agent filipin

could not be used in these experiments to deplete membrane cholesterol as short 15 minute

incubations with filipin (7.6 µM) caused complete loss of monolayer resistance that prevented

assessment of cell function by electrophysiology.

T84 cells treated with 2OHβ-CD exhibited an attenuated response to apically applied CT.

Figure 1A shows a representative time course of CT-induced Cl- secretion in monolayers

depleted or not depleted in cholesterol. VIP was applied at 90 min to control for the effect of

2OHβ-CD on monolayer resistance as described above. 2OHβ-CD prolonged the "lag phase"

by nearly 2-fold (Figures 1A and B) and diminished the apparent rate of toxin-induced Cl-

secretion by >60% (∆Isc= 0.98 +/- .10 vs. 0.39 +/- .08 µA/cm2/min, n=8, control vs. 2OHβ-CD

treated monolayers, p<0.05). Peak Isc responses to apically applied CT were inhibited by 50%


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when calibrated against that induced by VIP (Figures 1A and C). 2OHβ-CD, however, had no

detectable effect on the time course of Isc induced by basolaterally applied CT (not shown).

Two lines of evidence indicated that treatment with 2OHβ-CD did not inhibit CT action by

depleting the toxin’s receptor ganglioside GM1 from cell membranes. First, T84 monolayers

treated or not with 2OHβ-CD exhibited nearly identical densities of apical membrane receptors

for CT (Figure 2A). Second, the effects of 2OHβ-CD were reversed completely by treatment

with cholesterol alone (Figure 2B). For these studies, 2OHβ-CD treated monolayers were

washed free of 2OHβ-CD and incubated for an additional 90 min in the presence or absence of

excess cholesterol or the cholesterol analogues 25-hydroycholesterol or cholestane-3β,5α,6β-

triol. Monolayers depleted in cholesterol and subsequently allowed to recover in buffer alone

exhibited strongly attenuated CT-induced peak Isc's equal to that of T84 monolayers treated

continuously with 2OHβ-CD (Figure 2B, column 3 vs. column 2). In contrast, monolayers

pretreated with 2OHβ-CD and then allowed to recover in the presence of cholesterol exhibited

peak secretory responses equal to that of control monolayers not treated with 2OHβ-CD (Fig

2B. columns 6 vs. 1). Cholesterol treated monolayers also exhibited recovery of the lag phase

consistent with the idea that depletion of membrane cholesterol affected a rate limiting step

required for toxin trafficking into the ER (lag phase =31±4 vs. 48±7 vs. 37±5 min, mean ±SE,

n=7-8, control vs. cholesterol depleted vs. cholesterol repleted monolayers). Monolayers

treated with the inactive cholesterol analogues cholestane-3b,5a,6b-triol or 25-

hydroycholesterol exhibited only partial or no recovery (Figure 2B columns 5 and 4 vs. control

columns 1 and 6). Thus, the effect of 2OHβ-CD on CT-induced Cl- secretion was fully

reversible and specific to cholesterol. Non-specific effects of 2OHβ-CD on GM1-receptor

density, if any, can not explain these results. Taken together, these initial studies indicate that


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CT action on T84 cells, presumably due to toxin entry into the cell or trafficking retrograde

into Golgi cisternae and ER, depends on membrane cholesterol.

To show that the effect of 2OHβ-CD on CT action was not explained by a general

inhibitory effect of cholesterol depletion on membrane dynamics, we utilized anthrax EdTx

toxin. Unlike CT, the enzymatic subunit of EdTx translocates across the limiting membrane of

the acidic endosome to reach the cytosol where it then acts inside the cell to increase

intracellular cAMP (25). Anthrax EdTx, however, can only enter polarized T84 cells by

binding basolateral membrane receptors. Since, basolateral membranes appeared resistant to

cholesterol depletion by 2OHβ-CD, we utilized the more potent cholesterol binding compound

mβ-CD. One hour incubations with 4mM mβ−CD depleted approximately 55% of total

cellular cholesterol with minimal effects on monolayer resistance (TER = 80% control values).

Initial studies showed that, in contrast to 2OHβ-CD, mβ−CD inhibited by 70% and 30% the

peak Isc induced by apically and basolaterally applied CT respectively [Figure 3D, solid bars

(not treated) vs. open bars (mβ−CD treated)]. The time course and lag phase for toxin-induced

Isc were also affected [Figure 3A and C, solid bars (not treated) vs. open bars (treated)].

Inhibition of CT induced Isc by mβ-CD was fully reversed by subsequent treatments with

cholesterol indicating specificity for cholesterol depletion (Figure 3D, shaded bar). In contrast

to CT, however, treatment of T84 monolayers with mβ−CD had no detectable effect on the

cAMP-dependent Isc induced by anthrax EdTx or on the time course of EdTx action [Figure

3B and panels C and D, solid bars (not treated) vs. open bars (mβ−CD treated)]. Thus,

movement of EdTx from its binding site on the cell surface into the basolateral acidic

endosome was not affected by mβ−CD. These data indicate that the inhibitory effects of

2OHβ-CD and mβ−CD on CT-induced Cl- secretion cannot be due to non-specific effects on


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membrane dynamics in general.

Cholesterol depletion reduced the protein content of raft fractions isolated from T84 cells by

50% (Figure 4B), but did not preferentially displace the CT-GM1 from lipid raft microdomains.

In some experiments, treatment with cyclodextrin displaced a fraction of CT into detergent–

soluble membrane domains (Figure 4F ), but always the CT-GM1 complex remained enriched

in lipid rafts (Figure 4 A, C, E and Table 1). Even in monolayers treated for 24 h with 2OHβ-

CD, all of the detectable CT-GM1 complex continued to fractionate with lipid rafts (Fig 4A).

The GPI-anchored 5’nucleotidase (5’NT or CD73) was also not displaced from lipid rafts in

T84 monolayers as assessed by enzymatic assay (data not shown). Similar results were

obtained in monolayers treated with 2OHβ-CD for only two hours. These conditions were the

same as those used for physiologic studies described above. Unlike CT, however, the function

of CD73 that acts enzymatically at the cell surface was not affected by cholesterol depletion as

CD73 continued to convert 5’AMP to adenosine at maximal rates (104+/-4 vs. 99+/-1 % of

maximal 5’AMP-induced Isc, control vs. cholesterol depleted, n = 2). While both CT and

CD73 were retained and enriched in raft fractions after cholesterol depletion, some proteins

were not. Two other raft-associated proteins, caveolin-1, an intrinsic membrane protein

oriented towards the cell cytosol, and pp60src, a cytoplasmic lipid-anchored kinase, were

selectively solubilized (Figures 4 E and F and Table 1). Thus two cytoplasmically-oriented

proteins were preferentially displaced from raft fractions by cholesterol depletion, but the

extracellularly oriented CT-GM1 complex and GPI-anchored CD73 remained enriched.

To demonstrate that cholesterol depletion affected the protein content of rafts containing

the CT-GM1 complex specifically, we examined raft density. Figures 4C and D show that CT-

containing lipid rafts isolated from monolayers depleted in cholesterol fractionated in a


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continuous gradient in sucrose at lower apparent densities (20-22 vs. 22-25 % sucrose, 2OHβ-

CD treated vs. untreated, n = 2). Soluble fractions contained no detectable CT, even when

analyzed at 100-fold excess total protein (data not shown). The effect of cholesterol depletion

on raft structure was also readily apparent by visual inspection of sucrose gradients. Lipid rafts

prepared from monolayers treated with 2OHβ-CD formed two distinct light scattering bands on

sucrose gradients with one band exhibiting lighter density relative to the single more diffuse

band of lipid rafts obtained from control monolayers not depleted in cholesterol. Thus,

cholesterol depletion affected the apparent density and presumably the protein/lipid ratio of

isolated raft microdomains containing CT.

Finally, to explain why depletion of membrane cholesterol caused inhibition of CT action,

we examined whether the toxin entered the cell. Figure 5A shows that at all time points

examined, control monolayers not depleted in cholesterol internalized more CT than

monolayers treated with mβ−CD. This is best visualized after 30 min of continuous uptake

where eight fold less CT was detected in endosomes of monolayers depleted of cholesterol

(Figure 5B). To test the idea that retrograde trafficking of CT into Golgi cisternae (and

presumably ER) was also affected, we utilized a toxin variant engineered to contain the

sulfation consensus motif SAEDYEYPS (36,37). Sulfation at this site can be measured after

toxin entry into Golgi cisternae (Y. Fujinaga and W. I. Lencer, unpublished data). Figure 5C

shows that sulfation of CT-B-SAEDYEYPS was strongly attenuated in monolayers depleted in

cholesterol by treatment with mβ−CD (17.85% +/- 7.3% of control sulfation, n=2). These data

provide direct evidence that endocytosis or both endocytosis and trafficking of CT into Golgi

cisternae depend on membrane cholesterol.


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Discussion

The results of these studies show that early steps in endocytosis and retrograde trafficking of

CT into Golgi cisternae of human intestinal epithelial cells depend on membrane cholesterol.

Cholesterol depletion of T84 cell monolayers affected the trafficking of CT specifically, as

under identical conditions, anthrax EdTx entered the basolateral endosome and induced

toxicity with no detectable loss in potency. The defect in membrane trafficking of CT caused

by cholesterol depletion correlated with a loss of toxin function and with a change in the

density and specific protein content of CT-associated lipid raft fractions isolated from T84

cells. Cholesterol depletion, displaced some CT into soluble fractions in some experiments,

but left the bulk of the CT-GM1 complex enriched in lipid raft microdomains. These data imply

that cholesterol may function to couple the CT-GM1 complex with other membrane components

of the lipid raft required for CT entry into the cell.

Cholesterol functions critically in the biogenesis of eukaryotic cell membranes and is

particularly enriched in lipid raft microdomains (38 and reviewed in 39). Cholesterol dictates

the physical state of the lipid bilayer. In model membrane systems, increasing amounts of

cholesterol will induce formation of liquid ordered (lo) domains that exhibit detergent

insolubility (40). Thus, the lo phase may describe the physical state of lipid rafts in vivo.

Some membrane-associated proteins depend on cholesterol (and presumably the lo phase) for

association with or displacement from raft domains, and this may regulate protein function (17,

29, 41-45). Cholesterol may also effect the activity of membrane associated proteins by direct

interaction, or indirectly by affecting membrane fluidity (46-48).


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In this study, we find that trafficking of the CT-GM1 complex, and toxin function exhibit

dependency on membrane cholesterol. In some experiments, CT shifts partially into the

soluble fractions, but the bulk of CT remains enriched in lipid rafts following cholesterol

depletion. Abundant data indicate that sphingolipids such as GM1, which typically exhibit long

chain fully saturated sphingosine and fatty acid domains, self-assemble into lipid ordered (lo)

microdomains in vitro (13, 14, 49). Such a tendency to self-aggregate may explain the strong

association of the CT-GM1 complex with lipid rafts in native cell membranes, and why the CT-

GM1 complex remains enriched in lipid raft domains in T84 cells after cholesterol depletion.

The GPI-anchored CD73 was also not displaced from lipid raft fractions after cholesterol

depletion, suggesting that CD73 in T84 cells may be anchored to the membrane by a similarly

long and saturated acyl domain. Alternatively, it is possible that CT may induce its own

unique form of lo domain by clustering GM1 via binding by the pentameric CT B-subunit.

Clustering of GM1 could stabilize the assembly or induce the coalescence of raft domains

carrying CT just as cross-linking of the GPI-anchored folate receptor in 3T3 fibroblasts may

stabilize the association between this protein and caveolae (50). Further support for this idea

can be taken from studies on shiga toxin. Shiga toxin also exhibits a pentameric subunit that

binds multivalently to glycolipid receptors for retrograde trafficking into the ER and the

induction of toxicity (51). Also, such clustering of GM1 by binding to the B-subunit of CT (or

clustering of glycolipid Gb3 by binding the B-subunit of shiga toxin) may induce the formation

of a lipid rafts with unique structure and function. Available evidence indicate that lipid rafts

isolated from a single cell type exhibit heterogeneity in structure and function (52), and we

have recently found that the CT-GM1 complex fractionates with a discreet raft domain in T84

cells (5).


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Even though, cholesterol depletion did not displace the bulk of the CT-GM1 complex from

raft domains in T84 cells, two other cytoplasmically oriented raft-associated proteins were

depleted. These results correlated with a loss of toxin function and implied to us that

cholesterol may be required for stable coupling of the CT-GM1 complex with raft domains or

with other components of the lipid raft required for toxin entry into the cell, or both. Thus, we

hypothesize that CT, which is anchored only to the extra-cytoplasmic surface of the plasma

membrane by binding GM1, must interact with cytoplasmically oriented membrane-associated

proteins or lipids that form or communicate with the machinery for endocytosis, or sorting into

Golgi, or both. Our data indicate that such interactions depend on the structure and function of

lipid rafts. This idea was strengthened by two observations. First, the total protein content and

the relative enrichments of caveolin-1 and pp60src in lipid raft fractions isolated from T84 cells

were altered by cholesterol depletion, and second, the apparent density or protein/lipid ratio of

CT-associated rafts was affected specifically.

The block in CT function caused by cholesterol depletion can be explained by inhibition of

endocytosis and trafficking of the CT-GM1complex into the Golgi apparatus. Our results are

consistent with those previously reported by Orlandi and Fishman(17). In the human intestinal

Caco2 cell line, sequestration of membrane cholesterol by chelation with filipin or cyclodextrin

inhibited endocytosis of CT and blocked toxin function. Cholesterol depletion of Caco2 cells,

however, did not affect the activity of diptheria toxin that likely enters host cells by clathrin-

dependent mechanisms (17, 53). In the current study, we also find that treatment of T84 cells

with cyclodextrins inhibited endocytosis and function of CT, but had no detectable affect on

the action of anthrax EdTx that does not bind receptors localized in lipid rafts.


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Based on these data, however, we cannot identify the exact mechanism of endocytosis for

CT in either cell type. While we find a significant affect of cholesterol depletion on

endocytosis in T84 cells, both clathrin-mediated and non-clathrin mediated mechanisms of

endocytosis have been shown to depend on membrane cholesterol (6, 19, 20, 54), and recently,

CT was shown to enter COS-7 cells by both clathrin-dependent and clathrin-independent

mechanisms (55). In hippocampal neurons, CT may enter the cell in clathrin-coated pits while

still bound to lipid raft microdomains (18). Our data do show, however, that inhibition of toxin

endocytosis and trafficking cannot be explained by a general non-specific effect of cholesterol

depletion on membrane dynamics. Anthrax EdTx requires entry into the basolateral acidic

endosome to induce toxicity, and this was not affected by depletion of membrane cholesterol.

Anthrax EdTx represents almost a perfect control for our studies on trafficking of CT as both

toxins induce a Cl- secretory response in T84 cells. Thus, non-specific effects of the

cyclodextrins on T84 cell function would affect equally the Cl- secretory response induced by

both toxins. Finally, that cholesterol depletion had no effect on the action of EdTx, which does

not bind receptors located in lipid rafts, provides indirect evidence that CT may traffic

retrograde into the biosynthetic pathway of T84 cells by lipid raft dependent mechanisms.

In support of this idea, we find direct evidence that cholesterol depletion inhibits transport

of CT into Golgi cisternae. Multiple trafficking steps en route from plasma membrane to Golgi

cisternae, including the initial endocytosis of the CT-GM1 complex, are likely affected. Our

data fit closely with the results of recent studies showing that retrograde trafficking of shiga

toxin into the Golgi apparatus of HeLa cells also depends on association with functional lipid

rafts (51). Such retrograde transport into Golgi cisternae and then ER is required for both CT

and shiga toxin function. That lipid rafts may function in protein sorting has been in the


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literature for many years (56). For example, sorting of apical protiens (15, 57), endocytosis

(19), endosome to Golgi trafficking (10, 58, 59) and even Golgi structure (57) have all been

shown to depend on membrane cholesterol and presumably on lipid raft structure and function

as discussed above.

In summary, our results show that trafficking of the CT- GM1 complex into the Golgi

apparatus of polarized intestinal epithelial cells, and thus the induction of toxicity, depends on

membrane cholesterol. We propose that cholesterol may function to couple the ganglioside

GM1-CT complex with raft microdomains and with other membrane components required for

CT entry into the cell. This likely depends on lipid raft structure and function.

Acknowledgements

These studies were supported by NIH grant DK48106 to WIL, by an individual NRSA F32

DK09863, Charles A. Janeway Award from Children’s Hospital and Research Scholar Award

from the American Digestive Health Association to AAW, and NIH grant DK34854 to the

Harvard Digestive Diseases Center . We thank Margaret Ferguson Maltzaman and Heidi Wheeler

for expert assistance with tissue culture, Dr. Linda Thompson for performing enzymatic assays

of 5’ nucleotidase, Dr. John Collier for the gift of anthrax edema toxin, and members of the

Lencer laboratory for critical reading of this manuscript.


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Footnotes:

aAbbreviations used in the text: cholera toxin (CT), ganglioside GM1 (GM1 ), ganglioside GD1a (GD1a ), 2-hydroypropyl β-cyclodextrin (2OHβ -CD), methy β-cyclodextrin (mβ-CD), anthrax edema toxin (EdTx), anthrax protective antigen (PA), vasoactive intestinal peptide (VIP), short circuit current (Isc), Hanks balanced salt solution (HBSS), transepithelial resistance (TER), 5’nucleotidase (5’NT)


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Figure Legends

Figure 1: Effect of 2OHβ -CD on CT-induced chloride secretion.

A) Chloride secretory response induced by CT in T84 monolayers treated or not treated with

2OHβ-CD. Monolayers pretreated for 1 h at 37°C with 16.5mM 2OHβ-CD (squares) or buffer

(circles) were exposed to 20 nM CT at time =0 min (open symbols). Toxin-induced chloride

secretion was measured as a short circuit current (mean ± S.D. of duplicate monolayers,

representative of n=8 independent experiments). Monolayers not exposed to CT (closed

symbols), either treated or not with 2OHβ-CD, were treated with VIP approximately 15 min

prior to the end of the time course.

B). Effect of 2OHβ -CD on the lag phase of the chloride secretory response induced by apical 20

nM CT (mean +/- S.E, n = 8 independent experiments in duplicate, * indicates p< 0.05).

C) Effect of treatment with 2OHβ -CD on the maximal Isc induced by apically applied CT

(shaded bar) or VIP (solid bar) (mean +/- S.E., n=8, * indicates p< 0.01). Cells were treated as

described in Panel A.

Figure 2: Effect of 2OHβ -CD on GM1 receptor density and reversibility of inhibition by

treatment with exogenous cholesterol.

A) Binding of CT to T84 monolayers treated (shaded bar) or not treated (solid bar) with 2OHβ -

CD (mean+/- S.E., n=8, p< 0.05).

B) Cholesterol, but not cholestane-3β,5α,6β-triol or 25-hydroxycholesterol, fully reversed the

affect of 2OH-β-CD on CT-induced Cl- secretion. T84 cell monolayers were treated for one


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hour in the absence (-) or presence (+) of 2OHβ-CD at 37° C (1° incubation), washed, and then

treated with either buffer alone (lane 1 and 3) or 2OHβ -CD (lane 2) containing 20 nM CT for

the remainder of the time course or with 0.2mM of the indicated oxysterol (25-OH chol = 25-

hydroycholesterol , cholestane = cholestane-3b,5a,6b-triol). Results are normalized to the

maximal Isc induced by VIP under identical conditions (mean +/- S.E., n=4, * p< 0.05 compared

against lanes 1 and 6).

Figure 3: Cholesterol depletion does not affect toxicity of anthrax edema toxin.

A. mβ-CD inhibits CT-induced Isc. Representative time course showing the effect of treatment

with mβ-CD (closed symbols) on Isc induced by 20 nM apical CT (squares) or 1 nM basolateral

CT (circles), (mean ± SD in duplicate, n = 4 or more independent experiments). The cAMP-

agonist VIP was added to control monolayers 15 min before the end of the time course. The time

course of VIP-induced Isc is offset for visual clarity.

B. mβ-CD has no detectable affect on EdTx-induced Isc. Representative time course showing

the effect of mβ-CD (closed symbols) on Anthrax EdTx-induced chloride secretion. 12 nM

EdTx was added to the basolateral surface of some monolayers at t=0 min.(squares). VIP was

added to control monolayers (triangles) 15 min before the end of the time course.

C. Effect of mβ-CD on lag phase of CT and EdTx. mβ-CD treated monolayers (open bars) and

control monolayers (solid bars). Data are expressed as mean±SE (n=6, p < 0.05).

D. mβ-CD inhibits the peak Isc induced by CT but not by anthrax EdTx. Summary of the effect

of mβ−CD on peak chloride secretion induced by apical and basolateral CT and by anthrax

EdTx. Following the protocol outlined above, monolayers were pretreated for 1 h with 4mM


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mβ−CD (open bars) or buffer alone (closed bars) and then exposed to either 20 nM Apical CT

(n= 10), 1 nM Basolateral CT (n=8), or 12 nM basolateral EdTx (n=6). In some experiments

monolayers pretreated with mβ−CD were allowed to recover in the presence of cholesterol–

mβ−CD complex before exposure to 20 nM apical CT (shaded bar). Results are expressed as

mean fraction of peak Isc (mean maximal toxin-induced Isc/ maximal VIP-induced Isc), +/- S.E.

(n=4, *p< 0.05).

Figure 4: Effect of cholesterol depletion on the protein composition of lipid rafts

A. The CT- GM1 complex remains raft-associated following 24 h treatment with 16.5mM

2OHβ-CD. T84 cell monolayers were pretreated with 16.5mM 2OHβ-CD (upper panel) or media

alone (lower panel) for 24 h at 37°C, after which they were brought to 4°C, washed and

exposed to 20 nM CT. Monolayers were extracted in 1% triton X 100 at 4°C, and layered under

continuous sucrose gradients for equilibrium density centrifugation. Fractions were collected and

protein content and sucrose density were determined for each fraction. Fractions were analyzed

by SDS-PAGE and western blot loaded for equal protein (0.5 µg). * indicates 5ng CT loaded as

control.

B. Protein content of raft fractions isolated from monolayers depleted (open bar) or not depleted

(solid bar) in cholesterol . Results expressed as mean +/-SE, n=8. * indicates p< 0.05.

C. Treatment with 2OHβ-CD decreases the apparent density of lipid rafts containing CT. T84

cell extracts were prepared from monolayers pretreated with 2OHβ-CD for 2 h. The extracts

were layered under a 15-30% continuous gradient for equilibrium density centrifugation. 0.5 ml

fractions were collected from the top of each gradient. Protein content and sucrose density of

each fraction were determined. Equal mass (0.3 µg) of each gradient fraction was run on SDS


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PAGE and analyzed by Western Blot for the CT B-subunit. Western blot data for gradients from

control (upper panel) and 2OHβ-CD treated (lower panel) monolayers are plotted against

percentage of sucrose of each fraction. All soluble fractions are in 40% sucrose. The data are

representative of three experiments.

D. Optical density of gradient fractions shown in panel B. Absolute values of band densities

between the two experiments are not standardized.

E. Treatment with 2OHβ-CD depletes pp60src and caveolin-1 from lipid raft fractions.

Representative Western blots of raft (R) and soluble (S) fractions from step gradients of 2OHβ-

CD treated (right lanes) and control (left lanes) monolayers, loaded for equal protein (0.3µg), and

analyzed for the CT B-subunit, pp60src and caveolin-1.

F. Treatment with 2OHβ-CD shifts pp60src , caveolin-1 and a fraction of the CT B-subunit

into soluble fractions. Representative Western blots of soluble (S) fractions from step gradients

of 2OHβ-CD treated (+) and control (-) monolayers. Each lane was loaded with 15 µg (50-fold

excess protein compared to lanes shown in panel E), and analyzed for the CT B-subunit, pp60src

and caveolin-1.

Figure 5: Effect of mβ−CD on endocytic uptake and Golgi trafficking of CT.

A. Treatment with mβ−CD inhibits endocytosis of CT. Monolayers were pretreated with

mβ−CD or buffer (controls) for 1 h. 20 nM CT was bound to control monolayers at 4°C. Some

monolayers were exposed continuously to 20 nM CT at 37°C for the indicated times and then

brought back to 4°C. Surface bound CT was removed by incubation at pH 2.5 (acid stripped)

and total cell associated CT was analyzed by SDS PAGE and Western blot as described in


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methods. Total cell lysates were loaded for equal protein and Western blotted for either the CT

B-subunit, or for actin as control for protein loading.

B. Data collected at the 30 min time point in panel A was analyzed by densitometry.

Endocytosed toxin is expressed as fraction of toxin bound at 4°C (n=2 independent experiments).

C. Treatment with mβ−CD inhibits trafficking of CT to the Golgi. Monolayers were pretreated

with buffer (control) or mβ−CD and loaded with Na2

35SO4. Cells were then continuously exposed

to 20 nM CT containing the sulfation motif SAEDYEYPS for 50 min CT-SAEDYEYPS was

immunoprecipitated and analyzed for sulfation by SDS PAGE gels and autoradiography (upper

panel). The lower panel shows a Western blot of immunoprecipitated CT B-subunit loaded for

equal volume as a “loading control” for the autoradiogram. Data are representative of two

experiments.

Table 1

Relative enrichment for the CT B-subunit, pp60src and caveolin in raft fractions

isolated from cells depleted in cholesterol.

Western blots from Figure 4E were analyzed by densitometry. Relative enrichment for

the proteins per mg protein was determined as the ratio of the mass of protein (measured as

optical density) in raft fractions isolated from cells depleted of cholesterol compared to the mass

of protein in rafts isolated from cells not depleted of cholesterol (+2OHβ-CD/-2OHβ -CD, n=

3). Fractionation of 5’nucleotidase (5’NT) with lipid rafts was assessed by enzyme assay (n=2).


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Anne A Wolf, Yukako Fujinaga and Wayne I Lencermembrane cholesterol

retrograde Golgi trafficking, and downstream signal transduction by depletion of Uncoupling of the cholera toxin GM1 ganglioside-receptor complex from endocytosis,

published online February 21, 2002J. Biol. Chem.

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