Cholera and Shiga toxin B-subunits: thermodynamic and structural considerations for function and biomedical applications

Pina, David G.; Johannes, Ludger

doi:10.1016/j.toxicon.2004.12.014

Cited by 45 publications

(36 citation statements)

References 28 publications

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“…Bacterial toxins in the AB 5 family, including Shiga toxin and cholera toxin, are an example of a class of proteins with the intrinsic capacity to crosslink glycolipids via their multivalent membrane binding B-subunits [4]–[11]. The ability of cholera toxin B-subunit (CTxB) and related molecules such as Shiga toxin B-subunit to cluster glycolipids and organize membrane domains has been linked to their functional uptake into cells by clathrin-independent, cholesterol-dependent endocytic pathways [3], [7], [12], [13].…”

Section: Introductionmentioning

confidence: 99%

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

Day

Kenworthy

2012

PLoS ONE

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Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM1 in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM1 complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes.

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Section: Introductionmentioning

confidence: 99%

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

Day

Kenworthy

2012

PLoS ONE

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“…The strategy of this assay method may be applicable for other binding assays, such as Cholera toxin B-subunit for ganglioside GM1. 25) Although the ELISA method using verotoxin B subunit for the determination of Gb3 content was already established, 14) the assay procedure in our method (only two-step binding procedure) was simpler than the ELISA method (three steps). We believe minimum steps in a procedure have benefits not only for the quick assay but also for the specific assay.…”

Section: Discussionmentioning

confidence: 98%

Histidine-Tagged Shiga Toxin B Subunit Binding Assay: Simple and Specific Determination of Gb3 Content in Mammalian Cells

Shin

Nishikawa

Maruyama

et al. 2006

CHEMICAL & PHARMACEUTICAL BULLETIN

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The recombinant 1B-His, in which 6 histidine residues were added at the carboxy termini of the B subunits, was prepared as described previously.15) The BL21DE cells expressing 1B-His were cultured in 300 ml of LB broth (Difco laboratories, Detroit, MI, U.S.A.) supplemented with 50 mg/ml kanamycin (Nacalai Tesque, Inc., Kyoto, Japan) at 37°C for 2 h. The cells were subsequently treated with 1.0 mmol/l isopropyl b-D(Ϫ)thiogalactopyranoside (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 37°C for 4 h. Cells were pooled by centrifugation at 6000 rpm for 15 min at 4°C. The 1B-His was extracted from cell pellets with the Bugbuster protein extraction reagent (Novagen, Madison, WI, U.S.A.) and purified by using the His-bind purification kit (Novagen) according to manufacturer's recommendations. Purified 1B-His fractions were applied to an NAP10 column (Amersham Biosciences, Uppsala, Sweden) equilibrated with phosphate-buffered saline (PBS). The purified 1B-His was revealed as a single band on SDS-PAGE (data not shown) and its aliquots were stored at Ϫ20°C.Materials HisProbe-HRP was purchased from Pierce Biotechnology, A two-step binding assay for globotriaosylceramide (Gb3) content was developed by histidine-tagging strategy, which is a well-established method for the purification of recombinant proteins. The complete binding of the recombinant His-tagged Shiga toxin 1B subunit (1B-His) (1 m mg/ml) to the standard Gb3 adsorbed on a multi-well H type plate was observed within 30 min at 37°C; and its binding could be visualized by the following applications of HisProbe-HRP (8 m mg/ml) and tetramethylbenzidine (TMB) peroxidase substrate. The 1B-His binding assay was linear over the range of 1 to 100 ng of Gb3 per well. The binding of 1B-His was specific to Gb3 separated from HeLa cells, and no major cross-reactivity of other glycolipids in Folch's lower fractions extracted from HeLa cells was detected. The glycolipids in Folch's lower fractions from HeLa cells, human fibroblasts and mouse heart were suitable for this assay, but the further purification was needed for glycolipids from human plasma, thus sample preparation is critical factor for the reliable determination of Gb3 content. The 1B-His binding to Gb3 was inhibited by the addition of galactose, but not mannose. This 1B-His binding assay will be useful not only for the determination of Gb3 content, but also for screening for the compounds which inhibit the toxin-binding to Gb3. The strategy of our present method may be applicable for other binding assay, such as Cholera toxin B-subunit for ganglioside GM1.

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“…The toxin belongs to the AB 5 -class of bacterial toxins [65]. The design and molecular conformation of Shiga toxin as a holotoxin is composed of the monomeric catalytic A-subunit (StxA), providing cell toxicity, which is non-covalently linked to the homopentameric B-subunit (StxB), promoting receptor binding, internalization and trafficking [66][67][68][69][70][71]. Each B-subunit monomer contains three binding sites, illustrating a total number of 15 binding pockets for the entire Shiga toxin molecule [72,73].…”

Section: Endocytosis Of Shiga Toxinmentioning

confidence: 99%

How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis

Schubert

Römer

2015

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Synthetic membrane systems, such as giant unilamellar vesicles and solid supported lipid bilayers, have widened our understanding of biological processes occurring at or through membranes. Artificial systems are particularly suited to study the inherent properties of membranes with regard to their components and characteristics. This review critically reflects the emerging molecular mechanism of lipid-driven endocytosis and the impact of model membrane systems in elucidating the complex interplay of biomolecules within this process. Lipid receptor clustering induced by binding of several toxins, viruses and bacteria to the plasma membrane leads to local membrane bending and formation of tubular membrane invaginations. Here, lipid shape, and protein structure and valency are the essential parameters in membrane deformation. Combining observations of complex cellular processes and their reconstitution on minimal systems seems to be a promising future approach to resolve basic underlying mechanisms. This article is part of a Special Issue entitled: Mechanobiology.

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Cholera and Shiga toxin B-subunits: thermodynamic and structural considerations for function and biomedical applications

Cited by 45 publications

References 28 publications

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

Histidine-Tagged Shiga Toxin B Subunit Binding Assay: Simple and Specific Determination of Gb3 Content in Mammalian Cells

How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis

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