Cholera toxin (CT) enters host cells by binding to ganglioside GM1 in the apical plasma membrane (PM). GM1 carries CT retrograde from the PM to the endoplasmic reticulum (ER), where a portion of the ...toxin, the A1-chain, retro-translocates to the cytosol, causing disease. Trafficking in this pathway appears to depend on the association of CT-GM1 complexes with sphingomyelin (SM)- and cholesterol-rich membrane microdomains termed lipid rafts. Here, we find that in polarized intestinal epithelia, the conversion of apical membrane SM to ceramide by bacterial sphingomyelinase attenuates CT toxicity, consistent with the lipid raft hypothesis. The effect is reversible, specific to toxin entry via the apical membrane, and recapitulated by the addition of exogenous long-chain ceramides. Conversion of apical membrane SM to ceramide inhibits the efficiency of toxin endocytosis, but retrograde trafficking from the apical PM to the Golgi and ER is not affected. This result suggests that the cause for toxin resistance occurs at steps required for retro-translocation of the CT A1-chain to the cytosol.
Sphingomyelinases (SMases) hydrolyze membrane sphingomyelin to ceramide and are expressed by diverse host and microbial cell types populating mucosal surfaces. Exogenous bacterial SMase acts on the ...basolateral membrane of polarized human intestinal epithelial cells to repress the cAMP-induced Cl⁻ secretory response, but how this occurs is unknown. We show here that SMase acts by down-regulating a cAMP-gated basolateral membrane K⁺ conductance. Neither phosphocholine, ceramide-1-phosphate, nor sphingosine-1-phosphate recapitulates this effect, indicating that ceramide production is the decisive factor. Basolaterally applied SMase induced the phosphorylation of c-Jun NH₂-terminal kinase (JNK), and inhibition of JNK rescued the effect of SMase on cAMP-dependant secretion. SMase secreted by normal human fibroblasts specifically recapitulated the effect on cAMP-induced Cl⁻ secretion, indicating that cell types inhabiting the subepithelial space can provide such an activity to the basolateral membrane of intestinal enterocytes in trans. Thus, conversion of sphingomyelin to ceramide in basolateral membranes of intestinal cells rapidly activates JNK to inhibit a cAMP-gated K⁺ conductance and thereby attenuates Cl⁻ secretion. These results define a novel lipid-mediated pathway for regulation of salt and water homeostasis at mucosal surfaces.--Saslowsky, D. E., Tanaka, N., Reddy, K. P., Lencer, W. I. Ceramide activates JNK to inhibit a cAMP-gated K⁺ conductance and Cl⁻ secretion in intestinal epithelia.
Abstract only
Cholera toxin (CT) is an AB5‐subunit toxin that enters host cells by binding ganglioside GM1. GM1 carries the toxin retrograde from the apical cell surface through the trans‐Golgi ...network (TGN) into the endoplasmic reticulum, where a portion of the A‐subunit translocates to the cytosol to induce disease. In polarized cells that line mucosal surfaces, a fraction of the toxin enters the transcytotic pathway, moving all the way from apical to basolateral membrane, thus breeching the epithelial barrier. Here, we find that sorting of CT‐GM1 complexes into the transcytotic pathway occurs prior to delivery to the TGN or ER The closely related E. coli toxin LTIIb, which binds a different ganglioside, also enters the endosomal compartment of polarized cells but is not sorted into the retrograde or transcytotic pathways, suggesting that ganglioside structure may dictate the trafficking. To test this, we used fluor‐labeled GM1 species with defined ceramide structures applied to live cells. We found that polarized epithelia preferentially sort GM1 species with unsaturated ceramide acyl chains from apical to basolateral membranes by transcytosis. These results identify the early/common endosome as the site of GM1 sorting in polarized cells and show that trafficking depends on ceramide structure.
Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified ...from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.
Five new alleles of the
Arabidopsis chalcone synthase (CHS) locus,
tt4, have been characterized at the gene, protein, and end product levels as a genetic approach to understanding structure–function ...relationships in a key enzyme of plant secondary metabolism. Together with two previously described mutants, these
tt4 lines represent one of the first allelic series for a central enzyme of the flavonoid pathway and include both null alleles and alleles with leaky, apparently temperature-sensitive, phenotypes. A variety of effects on accumulation of CHS protein and flavonoid glycosides were observed among these lines, including alterations in the apparent stability and activity of the enzyme. Assembly of the CHS homodimer also appeared to be impacted in several cases. A three-dimensional model of the
Arabidopsis CHS protein, based on the recently determined structure for alfalfa CHS, predicts significant effects on protein structure or folding for several of the mutations. This allelic series should provide a useful genetic resource for ongoing studies of flavonoid enzyme structure, function, and subcellular organization.
Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM sub(1). The homopentomeric B-subunit of the toxin ...can bind up to five GM sub(1) molecules at once. Here, we examined the role of polyvalent binding of GM sub(1) in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM sub(1). The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM sub(1) when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM sub(1) molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM sub(1) molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.
ABSTRACT
Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM
1
. The homopentomeric B-subunit of the ...toxin can bind up to five GM
1
molecules at once. Here, we examined the role of polyvalent binding of GM
1
in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM
1
. The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM
1
when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM
1
molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM
1
molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.
Cholera toxin moves from the plasma membrane to the ER of host cells to cause disease. Trafficking in this pathway depends on toxin binding to specific ceramide-based glycolipids that associate with ...lipid rafts at the cell surface. In the ER, a portion of the toxin is unfolded, dissociated from the rest of the toxin and retro-translocated to the cytosol where it activates adenylyl cyclase to initiate the severe secretory diarrhea seen in cholera.
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