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Cholera Toxin As a Probe for Membrane Biology toxins Review Cholera Toxin as a Probe for Membrane Biology Anne K. Kenworthy 1,*, Stefanie S. Schmieder 2,3,4, Krishnan Raghunathan 5 , Ajit Tiwari 1 , Ting Wang 1, Christopher V. Kelly 6,* and Wayne I. Lencer 2,3,4,* 1 Center for Membrane and Cell Physiology and Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22903, USA; [email protected] (A.T.); [email protected] (T.W.) 2 Division of Gastroenterology, Boston Children’s Hospital, Boston, MA 02115, USA; [email protected] 3 Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA 4 Harvard Digestive Diseases Center, Boston, MA 02115, USA 5 Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA; [email protected] 6 Department of Physics and Astronomy, Wayne State University, Detroit, MI 48201, USA * Correspondence: [email protected] (A.K.K.); [email protected] (C.V.K.); [email protected] (W.I.L.) Abstract: Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1. Keywords: cholera toxin B-subunit; membrane nanodomains; endocytosis; retrograde trafficking; membrane curvature; membrane rafts; glycolipids; GM1; GL-Lect hypothesis Citation: Kenworthy, A.K.; Key Contribution: Recent advances in our understanding of membrane biology emerging from Schmieder, S.S.; Raghunathan, K.; studies using cholera toxin B-subunit and synthetic derivatives of its receptor GM1 are highlighted. Tiwari, A.; Wang, T.; Kelly, C.V.; Lencer, W.I. Cholera Toxin as a Probe for Membrane Biology. Toxins 2021, 13, 543. https://doi.org/10.3390/ 1. Introduction toxins13080543 Cholera toxin (CTx) typifies the AB5 bacterial toxins, and it is the essential pathogenic Received: 20 June 2021 factor that causes the massive secretory diarrhea seen in humans infected with V. cholerae [1,2]. Accepted: 29 July 2021 The Vibrio pathogen first secretes the toxin into the intestinal lumen after colonization of Published: 3 August 2021 the mucosal surface, but CTx is not active in this space. Remarkably, the toxin encodes within its protein structure everything necessary to breach the intestinal epithelial barrier Publisher’s Note: MDPI stays neutral and enter the cytosol of host cells. Here, in the cytosol, a portion of the toxin induces disease with regard to jurisdictional claims in by activation of adenylyl cyclase. This alters the physiology of the intestinal epithelium by published maps and institutional affil- activating the Cl− channel CFTR and inhibiting the Na+/H+ exchanger NHE3 to cause Cl iations. secretion and Na malabsorption leading to a severe form of secretory watery diarrhea [3]. The toxin accomplishes cytosolic entry by co-opting normal aspects of host cell mem- brane and organelle biology. It does not induce pathogenic membrane pores, or penetrate cell membranes, or damage the integrity of the mucosal surface in any way. Rather, it Copyright: © 2021 by the authors. traffics into the cell and across the mucosal barrier by riding along endogenous pathways Licensee MDPI, Basel, Switzerland. of membrane lipid and protein trafficking, and by engaging different aspects of normal sub- This article is an open access article cellular organelle biology. The evolutionarily driven adaptations enabling these processes distributed under the terms and have rendered the toxin one of the most potent and informative probes of cell and mem- conditions of the Creative Commons brane structure and function and mucosal tissue biology. This is the topic of the current Attribution (CC BY) license (https:// review: how CTx has illuminated our understanding of basic membrane and subcellular creativecommons.org/licenses/by/ processes fundamental to cell biology. 4.0/). Toxins 2021, 13, 543. https://doi.org/10.3390/toxins13080543 https://www.mdpi.com/journal/toxins Toxins 2021, 13, x FOR PEER REVIEW 2 of 18 Toxins 2021, 13, 543 membrane structure and function and mucosal tissue biology. This is the topic of the2 cur- of 18 rent review: how CTx has illuminated our understanding of basic membrane and subcel- lular processes fundamental to cell biology. To enter the cytosol of host cells, CTx has evolved to bind with high affinity affinity to the oligosaccharide moiety moiety of a raft-associated glycosphingolipid, ganglioside GM1. This is mediated entirely byby thethe toxin’stoxin’s binding binding B-subunit, B-subunit, CTxB CTxB (Figure (Figure1[ 14 ]).[4]). CTxB CTxB assembles assembles as asa homopentamer a homopentamer of 11of kDa11 kDa peptide peptide chains chains and and it functions it functions as a as lectin a lectin with with five bindingfive binding sites sitesfor the for oligosaccharide the oligosaccharide head head group group of GM1 of (andGM1 other(and closelyother closely related related gangliosides gangliosides [5–7]). [5–7].)It is thus It is capable thus capable of clustering of clustering up to up five to glycosphingolipidsfive glycosphingolipids together together [8]. [8]. GM1 GM1 acts acts as asthe the vehicle vehicle for for endocytic endocytic uptake uptake and and retrograde retrograde trafficking trafficking of CTx of CTx all the all way the backwardsway back- wardsin the in secretory the secretory pathway pathway into theinto endoplasmic the endoplasmic reticulum reticulum (ER) (ER) [9–11 [9–11].]. The The structure structure of ofthe the ceramide ceramide moiety moiety of of GM1 GM1 dictates dictates the the trafficking trafficking of of the the toxin-GSLtoxin-GSL complexescomplexes in this pathway [12,13]. [12,13]. Once Once in in the the ER, ER, the the CTx CTx A-subunit A-subunit co-opts co-opts the mechanics the mechanics of protein of protein fold- ingfolding quality quality control control to separate to separate from the from B subunit the B subunit and retrotranslocate and retrotranslocate across the across ER-lim- the itingER-limiting membrane membrane into theinto cytosol the cytosolwhere it where induces itinduces toxicity toxicityby enzymatically by enzymatically ADP-ribosyl- ADP- atingribosylating Gαs and G activatingαs and activating adenylyl adenylyl cyclase (Figure cyclase 2) (Figure [14,15].2 )[Separate14,15]. secondary Separate secondaryand lower affinityand lower binding affinity sites binding for glycoproteins sites for glycoproteins also exist on also CTxB exist that on modify CTxB that toxin modify action toxin [16– 27].action [16–27]. Figure 1. The molecular structures structures of of CTx and GM1. ( (AA)) A A side side view view of of CTx CTx is shown with the A subunitsubunit in in grey grey and the five five B subunits shown in color. ( (BB)) A A view view from from the bottom, membrane- membrane- binding surface of CTxB. The five G33 amino acids of the GM1 binding pockets are shown as space- binding surface of CTxB. The five G33 amino acids of the GM1 binding pockets are shown as filling spheres. (C,D) Structure of the CTx receptor ganglioside GM1. GM1 structures with space-filling spheres. (C,D) Structure of the CTx receptor ganglioside GM1. GM1 structures with ceramides containing acyl chains of C16:0 (C) and C16:1 (D) are shown. The crystal structure for CTxceramides was downloaded containing acyl from chains the ofProtein C16:0 Data (C) and Bank C16:1 1S5E (D )[4]. are CTx shown. structure The crystal is from structure O’Neal, for C.J.; CTx Amaya,was downloaded E.I.; Jobling, from M.G.; the Holmes, Protein DataR.K.; BankHol, W.G. 1S5E Crystal [4]. CTx structures structure of is an from intrinsically O’Neal, C.J.; active Amaya, chol- eraE.I.; toxin Jobling, mutant M.G.; yield Holmes, insight R.K.; into Hol, the W.G.toxin Crystalactivation structures mechanism of an. Biochemistry intrinsically 2004 active, 43 cholera, 3772–3782. toxin [4].mutant yield insight into the toxin activation mechanism. Biochemistry 2004, 43, 3772–3782. [4]. Here, we focus in this review on how the CTxB subunit can be used as a non-toxic reporter to probe basic aspects of membrane structure, mechanisms of endocytosis, nan- odomain assembly, and membrane trafficking enabled by glycosphingolipid biology. Toxins 2021, 13, 543 3 of 18 Toxins 2021, 13, x FOR PEER REVIEW 3 of 18 Figure 2. Intracellular itinerary of CTx trafficking and mechanism of intoxification. The CTx holotoxin bindsFigure the 2. plasmaIntracellular membrane itinerary via of its CTx pentameric trafficking membrane and mechanism binding of B intoxification. subunit. It is The subsequently CTx holo- internalizedtoxin binds andthe plasma delivered membrane to endosomes. via its From pentameric there the membrane toxin enters binding into the B retrograde subunit. It trafficking is subse- pathway,quently internalized leading to itsand delivery delivered to theto endosomes. endoplasmic From reticulum there (ER).the toxin In the enters lumen into of the the retrograde ER, the A subunittrafficking isreleased pathway, from leading the B to subunit its delivery and unfolded to the endoplasmic by protein disulfidereticulum isomerase (ER). In the (PDI), lumen enabling of the itsER, translocation the A subunit across is released the ER from membrane the B subunit into the and
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