Part II: diffraction from two-dimensional cholera toxin crystals bound to their receptors in a lipid monolayer

The structure of cholera toxin (CTAB₅) bound to its putative ganglioside receptor, galactosyl-N-acetylgalactosaminyl (N-acetyl-neuraminyl) galactosylglucosylceramide (GM₁), in a lipid monolayer at the air-water interface has been studied utilizing grazing incidence x-ray diffraction. Cholera toxin is one of very few proteins to be crystallized in two dimensions and characterized in a fully hydrated state. The observed grazing incidence x-ray diffraction Bragg peaks indicated cholera toxin was ordered in a hexagonal lattice and the order extended 600-800 Å. The pentameric binding portion of cholera toxin (CTB₅) improved in-plane ordering over the full toxin (CTAB₅) especially at low pH. Disulfide bond reduction (activation of the full toxin) also increased the protein layer ordering. These findings are consistent with A-subunit flexibility and motion, which cause packing inefficiencies and greater disorder of the protein layer. Corroborative out-of-plane diffraction (Bragg rod) analysis indicated that the scattering units in the cholera layer with CTAB₅ shortened after disulfide bond reduction of the A subunit. These studies, together with Part I results, revealed key changes in the structure of the cholera toxin-lipid system under different pH conditions.     

The cholera toxin receptor ganglioside GM1 remains associated with Triton X-100 cytoskeletons of BALB/c-3T3 cells

A substantial amount of the cholera toxin which binds to the surface of mouse fibroblasts resists solubilization by neutral detergents and remains associated with Triton X-100 cytoskeletons prepared by extraction of monolayer cultures. The observation is surprising given that the receptor for cholera toxin is a ganglioside (GM₁), and that membrane lipids are often assumed to be quantitatively extracted from Triton X-100 cytoskeletons. Indeed such preparations from mouse fibroblasts contain GM₁, and approx. 20% of the total cellular phospholipid and ganglioside. The observations are discussed in terms of the current trend to assume that detergent insolubility implies an association with the cytoskeleton.     

Integration of ganglioside GT1b receptor into DPPE and DPPC phospholipid monolayers: an X-ray reflectivity and grazing-incidence diffraction study

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structures of mixed-ganglioside GT_1b-phospholipid monolayers were investigated at the air-liquid interface and compared with monolayers of the pure components. The receptor GT_1b is involved in the binding of lectins and toxins, including botulinum neurotoxin, to cell membranes. Monolayers composed of 20 mol % ganglioside GT_1b, the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE), and the phospholipid dipalmitoyl phosphatidylcholine (DPPC) were studied in the gel phase at 23°C and at surface pressures of 20 and 40 mN/m, and at pH 7.4 and 5. Under these conditions, the two components did not phase-separate, and no evidence of domain formation was observed. The x-ray scattering measurements revealed that GT_1b was intercalated within the host DPPE/DPPC monolayers, and slightly expanded DPPE but condensed the DPPC matrix. The oligosaccharide headgroups extended normally from the monolayer surfaces into the subphase. This study demonstrated that these monolayers can serve as platforms for investigating toxin membrane binding and penetration.     

Persistence of exogenous, inserted ganglioside GM1 on the cell surface of cultured cells

Ganglioside GM₁, which can insert spontaneously into the membrane of intact cells, has been measured after insertion into transformed fibroblasts by cholera toxin (choleragen) binding, for which ganglioside GM₁ is the natural receptor. Choleragen binding is not altered in starved, quiescent cells over a four-day period. Dividing cells show decreased binding in proportion to cell division. Thus, neither dividing nor quiescent cells appear to metabolize or otherwise degrade this membrane component.     

Binding Cooperativity Matters: A GM1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM₁ and GM₁-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM₁ positive cooperativity. Surprisingly, we demonstrated fucosyl-GM₁ has approximately 7 times higher CTB binding capacity than GM₁. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM₁ caused the increased binding capacity. This was unintuitive, as GM₁ exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM₁ and GM₂, we observed the mild binding avidity fucosyl-GM₁ activated GM₂ receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM₂ receptors have negligible binding avidity in pure GM₂ bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.     

Biomimetic particles for isolation and reconstitution of receptor function

Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB₅ hexameric protein, and its receptor, the monosialoganglioside GM₁, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM₁ molar ratio in perfect agreement with literature. This receptor-lig-and recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM₁ showed that GM₁ incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2–0.3 mM PC and then decreased as a function of PC concentration. At 5 μM GM₁, 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m² silica, which corresponded to the 5 GM₁/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction.     

Analysis of transmembrane dynamics of cholera toxin using photoreactive probes

Using sodium dodecyl sulfate--polyacrylamide gel electrophoresis and autoradiography, we have shown that ¹²⁵I-labeled cholera toxin binds to Newcastle disease virus. Pretreatment of Newcastle disease virus with “cold” cholera toxin (at 37°C for 30 minutes) inhibits the binding of ¹²⁵I-labeled toxin in a subsequent incubation (at 37°C for 30 minutes). These results suggest that cholera toxin binds to Newcastle disease virus in a specific manner. The precise receptor for toxin is unknown in Newcastle disease virus but it is presumed to be the ganglioside GM₁. We have previously shown that the photoreactive probe 12-(4-azido-2-nitrophenoxy)stearoylgucosamine[1-¹⁴C] labels the membrane proteins of Newcastle disease virus. Since the reactive group of the probe, ie, N₃, resides within the membrane bilayer, studies were initiated to determine which, if any, of the subunits of cholera toxin cross the membrane of Newcastle disease virus and become radioactively labeled upon photoactivation of the probe at 360 nm. After a 15-minute incubation of cholera toxin with Newcastle disease virus containing the photoreactive probe, irradiation effected the ¹⁴C-labeling of the active A₁ subunit of cholera toxin. Irradiation of cholera toxin in solution with an equivalent amount of probe but without virus resulted in no labeling of toxin subunits.     

Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L_o) and liquid-disordered (L_d) phases. Here, we show μm-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the plasma membrane were predominantly labelled with L_d markers 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and weakly stained by L_o marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM₁ enriched plasma membrane rafts. Cholesterol depletion with methyl-β-cyclodextrin enhanced DiI vesiculation, GM₁/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total ‘L_o/L_d’ boundary line tension arising from the coalescence of smaller L_d-like domains makes it energetically favourable for L_d-like domains to bend from flat μm-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events.     

Role of membrane gangliosides in the binding and action of bacterial toxins

Summary Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside G_M1. Toxin-resistant cells which lack G_M1 can be sensitized to cholera toxin by treating them with G_M1. Cholera toxin specifically protects G_M1 from cell surface labeling procedures and only G_M1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that G_M1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to G_M1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. G_M1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin ofE. coli is very similar to cholera toxin in both form and function and can also use G_M1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides G_D1b and G_T1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established G_M1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.     

Cholera toxin and membrane gangliosides: Binding and adenylate cyclase activation in normal and transformed cells

Summary A virally transformed, ganglioside GM₁-deficient cell line binds 2% of the cholera toxin (choleragen) bound by the parent, line and is less responsive to choleragen with respect to adenylate cyclase stimulation. This biological response is maximal when 10% of choleragen-binding sites in the transformed line, or 0.5% in the parent line, are occupied. In contrast, in isolated fat cells saturation of binding and adenylate cyclase stimulation are seen at very similar concentrations.Incubation of ganglioside GM₁ with intact cells increases choleragen binding (defined here as ganglioside incorporation) in the transformed cell line but does not enhance the biological response to choleragen. Stimulation of adenylate cyclase is enhanced in isolated fat cells, however, by exogenous ganglioside GM₁. The binding and cyclase response in fat cells can be reduced by the addition of the inactive analog and competitive antagonist, choleragenoid, and there is recovery of the enzyme response and binding upon subsequent addition of exogenous GM₁. Failure of enhancement in the transformed cell line is explained by the presence of a five- to tenfold excess of binding sites over the number required for the full biological effect of choleragen. Cells with a large excess of toxin receptors are relatively refractory to the blocking effects of choleragenoid on biological responses. Notably, untransformed cells, which contain large quantities of toxin receptor, cannot incorporate exogenously added ganglioside GM₁. These findings suggest the possible existence in the cytoplasmic membrane of specific molecular structures, present in finite and limited number, for recognizing and accepting ganglioside molecules exposed to the external medium.     

Action of cholera toxin on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of ⁴⁵Ca or cellular cyclic AMP. Binding of ¹²⁵I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of bindind of ¹²⁵I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in ⁴⁵Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretion or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Polarized distribution of GM1-ganglioside in human duodenal absorptive enterocytes as visualized with cholera toxin-gold complex

Cholera toxin bound to particles of colloidal gold was used to investigate by electron microscopy the binding of the toxin in human duodenum. Cholera toxin binding was detected only in the apical (brush border) plasma membrane domain suggesting that the ganglioside G_M1 is absent from the basolateral plasma membrane domain. Intracellularly, toxin binding became detectable in thetrans side of the Golgi apparatus. Labeling of endosomes may indicate that the non toxin-occupied G_M1-ganglioside becomes internalized.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Sticholysin I–membrane interaction: An interplay between the presence of sphingomyelin and membrane fluidity

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.     

Gravimetric antigen detection utilizing antibody-modified lipid bilayers

Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO₂ have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA-Ni²⁺-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing G_M1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750 pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum.     

On the role of polysialoglycosphingolipids as tetanus toxin receptors. A study with lipid monolayers

Lipid monolayers of different compositions were used to study the interaction of tetanus toxin with membrane lipids and to evaluate the role of polysialoglycosphingolipids as membrane receptors. At neutral pH, the toxin binds to dioleoylglycerophosphocholine monolayers and inserts into the phospholipid layer. This effect is potentiated by acidic phospholipids without an apparent preference for a single class of phospholipids. Polysialoglycosphingolipids further increase the fixation and penetration of tetanus toxin in lipid monolayers, but no specific requirement for a particular ganglioside was identified. The ganglioside effect is abolished in the presence of other nervous tissue lipids: cerebrosides and glycosphingolipid sulfates are partially responsible for this effect. The penetration of tetanus toxin in the lipid monolayer is pH dependent. It increases with lowering pH, it is facilitated by acidic phospholipids and by glycosphingolipid sulfates and it is mediated both by hydrophobic and electrostatic interactions as deduced from an analysis of the effect of ionic strength. Fragment B of tetanus toxin the low-pH-driven lipid interaction of the toxin. On the basis of the present findings, the possible role of polysialoglycosphingolipids in the neurospecific binding of tetanus toxin is discussed.     

Ganglioside GM1-mediated Transcytosis of Cholera Toxin Bypasses the Retrograde Pathway and Depends on the Structure of the Ceramide Domain

Cholera toxin causes diarrheal disease by binding ganglioside GM1 on the apical membrane of polarized intestinal epithelial cells and trafficking retrograde through sorting endosomes, the trans-Golgi network (TGN), and into the endoplasmic reticulum. A fraction of toxin also moves from endosomes across the cell to the basolateral plasma membrane by transcytosis, thus breeching the intestinal barrier. Here we find that sorting of cholera toxin into this transcytotic pathway bypasses retrograde transport to the TGN. We also find that GM1 sphingolipids can traffic from apical to basolateral membranes by transcytosis in the absence of toxin binding but only if the GM1 species contain cis-unsaturated or short acyl chains in the ceramide domain. We found previously that the same GM1 species are needed to efficiently traffic retrograde into the TGN and endoplasmic reticulum and into the recycling endosome, implicating a shared mechanism of action for sorting by lipid shape among these pathways.     

Focal junctions retard lateral movement and disrupt fluid phase connectivity in the plasma membrane

In cultured keratinocytes, focal junctions are enriched in major constituents of lipid rafts, such as GM₁ ganglioside, phosphoinositides, caveolins and flotillins. We have therefore speculated that focal junctions represent superrafts formed by coalescence of microdomains into large areas containing liquid-ordered (L_o) lipids. Indeed, values of maximal fluorescence recovery after photobleaching revealed that the long-range mobility of cholera toxin B subunit (CTB, marker of L_o) was ∼1.5-fold retarded within the focal junctions compared to the surrounding membrane. However, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI-C_18:0), which specifically partitions to the liquid-disordered (L_d), non-raft phase, was also enriched in focal junctions and its mobility was slightly retarded. Cross-linking of GM₁ by CTB or raft aggregation by methyl-β-cyclodextrin further decreased the recovery of DiI-C_18:0. We propose a model in which focal junctions impose lateral heterogeneity in the plasma membrane by entrapment of lipid microdomains between dense arrays of immobilized transmembrane molecules which can enmesh otherwise freely percolating L_d phase lipids.     

Cholera toxin assault on lipid monolayers containing ganglioside GM1

Many bacterial toxins bind to and gain entrance to target cells through specific interactions with membrane components. Using neutron reflectivity, we have characterized the structure of mixed DPPE:GM(1) lipid monolayers before and during the binding of cholera toxin (CTAB(5)) or its B-subunit (CTB(5)). Structural parameters such as the density and thickness of the lipid layer, extension of the GM(1) oligosaccharide headgroup, and orientation and position of the protein upon binding are reported. The density of the lipid layer was found to decrease slightly upon protein binding. However, the A-subunit of the whole toxin is clearly located below the B-pentameric ring, away from the monolayer, and does not penetrate into the lipid layer before enzymatic cleavage. Using Monte Carlo simulations, the observed monolayer expansion was found to be consistent with geometrical constraints imposed on DPPE by multivalent binding of GM(1) by the toxin. Our findings suggest that the mechanism of membrane translocation by the protein may be aided by alterations in lipid packing.     

Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3’s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3’s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment’s impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.