Binding Subunit Of Cholera Toxin Research Articles

Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.

HMGB1 released from intestinal epithelia damaged by cholera toxin adjuvant contributes to activation of mucosal dendritic cells and induction of intestinal cytotoxic T lymphocytes and IgA

Cholera toxin (CT) is a potent mucosal adjuvant and oral administration of ovalbumin (OVA) antigens plus CT induces OVA-specific CD8+ cytotoxic T lymphocytes (CTLs) and IgA production in intestinal mucosa. However, the mechanisms of induction of these immune responses remain unknown. Intestinal OVA-specific CD8+ CTLs were not induced by oral administration of the CT active (CTA) or CT binding (CTB) subunit as an adjuvant and CD11c+ DCs were involved in cross-priming of intestinal CTLs. CD8+CD103+CD11c+CD11b−DCs and DCIR2+CD103+CD11c+CD11b+ DCs were distributed in the intestinal lamina propria and mesenteric lymph nodes, both DC subsets expressed DEC-205, and the expression of co-stimulatory molecules such as CD80 and CD86 was enhanced in both DC subsets after oral administration of intact CT but not the CTA or CTB subunit. Intestinal DCs activated by the oral administration of OVA plus CT cross-presented OVA antigens and DCs that captured OVA antigen through DEC-205, but not DCIR2, could cross-present antigen. We found that oral administration of intact CT, but not the CTA or CTB subunit, enhanced cell death, cytoplasmic expression of high-mobility group box 1 protein (HMGB1) in epithelial cell adhesion molecule (EpCAM)+CD45− intestinal epithelial cells (IECs), and HMGB1 levels in fecal extracts. HMGB1 dose-dependently enhanced the expression of CD80 and CD86 on DCs in vitro, and intravenous or oral administration of glycyrrhizin, an HMGB1 inhibitor, significantly suppressed activation of mucosal DCs and induction of intestinal OVA-specific CTLs and IgA by oral CT administration. These results showed that oral administration of intact CT triggers epithelial cell death in the gut and the release of HMGB1 from damaged IECs, and that the released HMGB1 may mediate activation of mucosal DCs and induction of CTLs and IgA in the intestine.

Role of the Antigen Capture Pathway in the Induction of a Neutralizing Antibody Response to Anthrax Protective Antigen.

ABSTRACTToxin neutralizing antibodies represent the major mode of protective immunity against a number of toxin-mediated bacterial diseases, including anthrax; however, the cellular mechanisms that lead to optimal neutralizing antibody responses remain ill defined. Here we show that the cellular binding pathway of anthrax protective antigen (PA), the binding component of anthrax toxin, determines the toxin neutralizing antibody response to this antigen. PA, which binds cellular receptors and efficiently enters antigen-presenting cells by receptor-mediated endocytosis, was found to elicit robust anti-PA IgG and toxin neutralizing antibody responses. In contrast, a receptor binding-deficient mutant of PA, which does not bind receptors and only inefficiently enters antigen-presenting cells by macropinocytosis, elicited very poor antibody responses. A chimeric protein consisting of the receptor binding-deficient PA mutant tethered to the binding subunit of cholera toxin, which efficiently enters cells using the cholera toxin receptor rather than the PA receptor, elicited an anti-PA IgG antibody response similar to that elicited by wild-type PA; however, the chimeric protein elicited a poor toxin neutralizing antibody response. Taken together, our results demonstrate that the antigen capture pathway can dictate the magnitudes of the total IgG and toxin neutralizing antibody responses to PA as well as the ratio of the two responses.

Role of Glycolipid-Binding Bacterial Toxins in the Assembly and Function of Membrane Domains

Cell membranes are characterized by the presence of small regions of specialized protein and lipid composition termed membrane domains. Cholesterol-dependent domains serve as important regulators of several membrane trafficking pathways, including the process of clathrin-independent endocytosis. Some of the best studied examples of proteins that are internalized into cells via clathrin independent endocytic pathways include bacterial toxins in the AB5 family such as Shiga toxin and cholera toxin. Recently, the intrinsic ability of cholera toxin and related glycolipid-binding toxins to initiate domain assembly has been postulated to trigger toxin internalization by generating a novel class of endocytic intermediates. Although the properties of domains induced by toxin binding have been well characterized in model systems, the nature of these structures in intact cells is still unclear, and their exact role in toxin uptake in cells remains to be fully elucidated. We have therefore focused on understanding the properties of toxin-induced domains in living cells, using the membrane binding subunit of cholera toxin (CTxB) as a model. To do so, we are utilizing a variety of methods to study domains induced by CTxB-binding to cell membranes, including FRAP and FCS-based measurements of protein dynamics, as well as live cell imaging and cell biological approaches. These studies are providing new insights into how CTxB-induced domains are assembled, how their structure and dynamics are regulated, the pathways they utilize to enter cells, and the role of both the glycolipid receptor of CTxB and toxin structure itself in these events.

Effect of HFE Variants on Sphingolipid Expression by SH-SY5Y Human Neuroblastoma Cells

C282Y and H63D are two common variants of the hemochromatosis protein HFE. SH-SY5Y human neuroblastoma cells stably transfected to express either wild type HFE (WT-HFE), or the C282Y or H63D allele were analyzed for effect of expression of the mutant proteins on transcription of 14 enzymes involved in sphingolipid metabolism. Cells expressing the C282Y variant showed significant increases (>2-fold) in transcription of five genes and decreases in two compared to that seen for cells expressing WT-HFE, while cells expressing the H63D variant showed an elevation in transcription of one gene and a decrease in two. These changes were seen as alterations in ganglioside composition, cell surface binding by the binding subunit of cholera toxin, expression of sphingosine-kinase-1 and synthesis of sphingosine-1-phosphate. These changes may explain why C282Y-HFE is a risk factor for colon and breast cancer and possibly protective against Alzheimer's disease while H63D-HFE is a risk factor for neurodegenerative diseases.

Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles

Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1null (Cav1−/−) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1−/− MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveolae and to identify noncaveolar endocytic vehicles. In WT MEFs, a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2% per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.

A biologically active lectin of enteroaggregative Escherichia coli

The pathogenesis of enteroaggregative Escherichia coli, a major contributor to paediatric diarrhoea, is still not clearly understood. A complex carbohydrate specific lectin was identified from the culture supernatant of an enteroaggregative E. coli strain. The lectin was purified to 660-fold by a combination of sequential saturated ammonium sulphate precipitation and gel filtration chromatography in the FPLC system. The homogeneity of the purified lectin was established by analytical isoelectrofocusing [pI 6.75]. Hemagglutination of rabbit erythrocytes by the purified lectin was best inhibited by fetuin. The N-terminal sequence of the 41.7 kDa subunit showed homology to the outermembrane porins and the 23.4 kDa subunit showed homology to a hypothetical protein of Yersinia pestis and secreted Hcp protein. This protein could induce extensive morphological changes in HEp-2 cells and significant amount of fluid accumulation in rabbit ileal loop. GM1 showed maximum binding to the lectin among all other gangliosides. This purified protein showed cross-reactivity to the binding subunit of cholera toxin in western immunoblot. The presence of this toxin in some of the clinical isolates of enteroaggregative E. coli was also observed. The structural and functional characteristics of the toxin revealed that it is a novel virulence determinant of aggregative E. coli.

Inhibition of the adherence of cholera toxin and the heat-labile enterotoxin of Escherichia coli to cell-surface GM1 by oligosaccharide-derivatized dendrimers

The adherence of either cholera toxin or the heat-labile enterotoxin of Escherichia coli to monosialoganglioside gal(β1–3)galNAc(β1–4)[sialic acid (α2–3)]gal(β1–4)glcβ1–ceramide (GM1) present on the surface of epithelial cells lining the intestine is the first step of a series that results in the induction of a watery diarrhea. While cholera is more severe, both can lead to death as a result of dehydration. To determine the potential of defined multivalent oligosaccharides, synthesized by the covalent attachment of multiple phenylisothiocyanate (PITC) derivatives of gal(β1–3)galNAc(β1–4)[sialic acid(α2–3)]gal(β1–4)glc (oligo-GM1) to the arms of a poly(propylene imine) dendrimer, as therapeutic agents for these diseases, their ability to inhibit adherence of the toxins to cell surface-associated GM1 was determined. They not only inhibited choleragenoid (binding subunit of cholera toxin) binding to GM1-treated NCTC-2071 cells (chemically transformed murine fibroblasts) at 5°, but also inhibited adherence of the choleragenoid, cholera toxin, and heat-labile enterotoxin of E. coli to GM1-treated NCTC-2071 cells at 37°. Inhibition was observed whether the toxin was preincubated with the oligo-GM1-PITC-derivatized dendrimer prior to addition to cells or given just after the addition of the derivatized dendrimer to cells. The derivatized dendrimer had no effect on cell viability, as monitored by trypan blue exclusion. Blue-shifts in tryptophan fluorescence emission spectra maxima induced by adherence of either choleragenoid, cholera holotoxin, or the heat-labile enterotoxin of E. coli to oligo-GM1-PITC-derivatized dendrimers were similar to those induced by adherence to GM1 or oligo-GM1. Comparable shifts were not observed when the toxins were incubated with gangliosides that fail to function as receptors.

Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting.

Transepithelial transport of antigens and pathogens across the epithelial barrier by M cells may be a prerequisite for induction of mucosal immunity in the intestine. Efficient transport of antigens and pathogens requires adherence to M cell apical surfaces. Coupling of antigen-containing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed as a means for increasing antigen uptake because the CTB receptor, ganglioside GM1, is a glycolipid present in apical membranes of all intestinal epithelial cells. To test the accessibility of enterocyte and M cell membrane glycolipids to ligands in the size ranges of viruses, bacteria, and particulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, 28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent microparticles (final diameter, 1.13 microns) failed to adhere to enterocytes or M cells in vivo or to well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound specifically to GM1 on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that lacked brush borders and glycocalyx. Measurements of glycocalyx thickness by electron microscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of 1-micron microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithelial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system.

Interleukin-1-induced IL-8 and IL-6 gene expression and production in human mesangial cells is differentially regulated by cAMP

We have previously proposed that activated mesangial cells (MC) have a direct role in the initiation and propagation of inflammatory events within the glomerulus via the generation of the mesangioproliferative cytokine IL-6 and the chemokines IL-8 and MCP-1. The objective of this study was to investigate the role of cAMP in the regulation of IL-6 and IL-8 gene expression and peptide production in IL-1 stimulated human MC. Agents known to elevate cAMP, including dibutyryl cAMP (db-cAMP), forskolin or isobutyl-methylxanthine (IBMX) were alone unable to induce IL-6 or IL-8 expression or production above media control levels, indicating activation of the cAMP pathway could not mimic IL-1 signaling events. In the presence of IL-1, all three agents produced a marked potentiation of IL-6 mRNA expression and dose-dependent increase in IL-6 peptide production (twofold), but had little or no effect on IL-8 mRNA expression or peptide generation. In marked contrast cholera toxin (CT) caused a dose-dependent potentiation of both IL-1-induced IL-6 (approximately fourfold) and IL-8 peptide (approximately twofold) generation. The control agent, the purified binding subunit of cholera toxin (CT-B) which is devoid of ADP-ribosylating activity also enhanced IL-6 and IL-8 (approximately twofold) peptide generation indicating cAMP-independent mechanisms may be involved in the CT up-regulation of these cytokines. Treatment of MC with the cycloxygenase inhibitor indomethacin resulted in partial inhibition (37%) of IL-6 production but had no effect on IL-8 generation. Thus our data show that cAMP can potentiate IL-1 induced IL-6 production, while having no effect on IL-8 induction, and PGE2 may operate via a positive feedback loop to up-regulate IL-1 induced IL-6. Taken together, our results demonstrate that cAMP differentially regulates IL-6 and IL-8 production in IL-1-stimulated human MC.

Synthesis of a photoreactive, radiolabelled derivative of the oligosaccharide of GM1 ganglioside.

A photoreactive, radioiodinatable derivative of the oligosaccharide (GM1OS) of ganglioside GM1 was synthesized as follows: GM1OS was generated from GM1 by ozonolysis and alkaline fragmentation, and reductively aminated to GM1OSNH2 (1-amino-1-deoxymonosialogangliotetraitol). The latter compound was then reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) to form GM1OSNH-ASA [1-(4-azidosalicoylamido)-1-deoxymonosialogangliotetraitol], which was radioiodinated and further purified. To test the [125I]GM1OSNH-IASA [1-(4-iodoazidosalicoylamido)-1-deoxymonosialogangliotetraitol+ ++] as a probe for ganglioside-binding proteins, the derivative was incubated with cholera toxin, which specifically binds GM1, followed by photolysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The probe only labelled the B or binding subunit of cholera toxin, but not the A or adenylyl cyclase activating subunit. Labelling was inhibited by excess GM1OS, but not by the oligosaccharides from gangliosides GD1a and GD1b. [125I]GM1OSNH-IASA and analogous oligosaccharide derivatives may be valuable probes for detecting ganglioside-binding proteins.

Anterograde horseradish peroxidase tracing and immunohistochemistry of trigeminal ganglion tooth pulp neurons after dental nerve lesions in the rat

The peripheral reorganization of pulpal nerves after tooth injury was studied, in the rat, with anterograde horseradish peroxidase tracing techniques, and combined retrograde Fluorogold tracing and immunohistochemistry was employed to examine the effects of inferior alveolar nerve lesions or tooth injury on some cytochemical characteristics of pulpal trigeminal ganglion nerve cells, namely content of substance P, calcitonin gene-related peptide and the ganglioside GM1 (binding subunit of cholera toxin), as well as affinity to RT 97 (antibody to neurofilament protein) and the lectin Griffonia simplicifolia isolectin I-B4. Anterograde horseradish peroxidase tracing demonstrated that pulpal nerves either disappear or reinnervate novel targets after loss of pulpal tissue. There were no obvious signs of neuroma formation. Retrograde Fluorogold labelling with immunohistochemistry showed that after inferior alveolar nerve lesions with subsequent regeneration, a much higher proportion of Fluorogold cells (15%) were substance P-positive compared to normal (2%). In addition, 3% of the cells were Griffonia simplicifolia isolectin I-B4-positive. Such cells were very rare in controls. Proportions of calcitonin gene-related peptide-, GM1- and RT-97-positive cells were normal. After tooth lesions, the proportions of Fluorogold-positive substance P-, Griffonia simplicifolia isolectin I-B4-, GM1- and RT 97-labelled cells were similar to controls, while the proportion of calcitonin gene-related peptide-positive neurons was reduced. The results show that pulpal deafferentation may change the long-term cytochemical characteristics of affected trigeminal ganglion neurons.

Lumbar dorsal root projections to spinocerebellar cell groups in the rat spinal cord: a double labeling study

The aim of this study has been to investigate projections to spinocerebellar cell groups from lumbar dorsal root ganglia (DRGs) in the rat. The binding subunit of cholera toxin conjugated to horseradish peroxidase (B-HRP) was used to label primary afferent fibers. Spinocerebellar neurons were labeled retrogradely by Fluoro-Gold (FG). To determine the orientation of dendrites, retrogradely labeled spinocerebellar neurons were studied, following injections of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into cerebellum. FG or WGA-HRP labeled neurons were found mainly in laminae V and VII, in the lateral group of lamina IX, in Clarke's column (CC) and in the dorsal funiculus. B-HRP labeled primary afferent fibers overlapping with FG labeled cells were observed at all these locations after injections of B-HRP into different DRGs. The overlap in lamina V was found mainly medially and dorsolaterally. CC was found to receive dense projections from DRGs L1-6. In the lumbar part of CC, labeling from DRGs L4-5 overlapped and was distributed over the entire mediolateral extent of the CC, whereas labeling from DRGs L1-3 was somatotopically organized and projected to successively more dorsomedial areas. The central area of lamina VII showed moderate labeling from DRGs L3-5. The lateral group of lamina IX received only smaller amounts of labeled fibers from DRGs L3-5.

Immunocytochemical evidence for the localization of the GM1 ganglioside in carbonic anhydrase-containing and RT 97-immunoreactive rat primary sensory neurons

Localization of GM1 ganglioside, the receptor for cholera toxin, and choleragenoid, which is the binding subunit of cholera toxin, was studied in the rat L5 dorsal root ganglion. Sections were incubated with choleragenoid and treated immunocytochemically. Choleragenoid-like immunoreactive cells were then examined for possible co-localization with carbonic anhydrase-like, RT 97 (antibody to neurofilament proteins), substance P-like, somatostatin-like and calcitonin gene-related peptide-like immunoreactivity and fluoride-resistant acid phosphatase (FRAP) activity, using adjacent sections. A subpopulation of dorsal root ganglion neurons exhibited choleragenoid-like immunoreactivity. The majority of these were medium-sized and large neurons. The strongest immunoreactivity was found in the area of the plasma membrane, but strong reactivity was also seen in the cytoplasm. The majority of the choleragenoid-like immunoreactive cells showed carbonic anhydrase-like and RT 97 immunoreactivity. Cells showing co-localization of choleragenoid-like and neuropeptide-like immunoreactivity or activity for FRAP were rarely observed. Our results suggest that the GM1 receptor is localized primarily on carbonic anhydrase-containing and RT 97-immunoreactive primary sensory neurons.

Recent advances in identifying the functions of gangliosides

The recent development of several new approaches has proven extremely useful in identifying functions for gangliosides, the sialic-acid containing glycosphingolipids. The first is the incorporation of exogenous gangliosides into the plasma membrane of ganglioside-deficient cells. Using this approach, specific gangliosides have been identified as the receptors for certain bacterial toxins and viruses and as important factors in the organization of fibronectin into an extracellular matrix. The second approach has been a ligand blotting technique which allows detection of ganglioside-binding proteins such as toxins and antibodies. Gangliosides are separated by thin-layer chromatography and overlain with the protein of interest. Specific binding of the ligand to gangliosides can then be detected by either direct or indirect methods. The third approach is the use of the B or binding subunit of cholera toxin as a specific probe for endogenous plasma membrane ganglioside function. The ability of the B subunit to alter the growth of cells directly demonstrates a role for gangliosides as biotransducers of signals for the regulation of cell growth.

A comparison between wheat germ agglutinin- and choleragenoid-horseradish peroxidase as anterogradely transported markers in central branches of primary sensory neurones in the rat with some observations in the cat

Horseradish peroxidase conjugates of either the lectin wheat germ agglutinin or choleragenoid, the binding subunit of cholera toxin, were injected into the L5 spinal ganglion of adult rats. This enabled comparison of these two conjugates as anterograde tracers in the primary sensory system. After a postoperative survival of 4 h to 30 days, the rats were perfused and frozen sections from spinal and medullary regions receiving primary afferents were processed for horseradish peroxidase histochemistry with tetramethylbenzidine as the chromogen. Additional observations were made in two adult cats. Following injection of wheat germ agglutinin-horseradish peroxidase the labelling appeared mostly as small-sized granules. The concentration of labelled primary afferents in the grey matter of the spinal cord was greatest in the marginal zone and the substantia gelatinosa and less pronounced in the deep parts of the dorsal horn. Labelling was also found in a region lateral to the central canal and in the ventral horn. Following injection of choleragenoid-horseradish peroxidase the labelling appeared mainly as larger-sized granular profiles. The concentration of labelled primary afferents was greatest in the deep part of the dorsal horn and pronounced in a region lateral to the central canal and in the ventral horn. All these regions are known to receive large calibre fibres. The marginal zone and the substantia gelatinosa, known to receive fine calibre fibres, showed almost no labelling in the rat. In the cat, however, there was somewhat more labelling in the substantia gelatinosa. Labelling of neuronal cell bodies indicating transneuronal transport was seen after injection of wheat germ agglutinin-horseradish peroxidase. Transneuronal labelling did not seem to occur after injection of choleragenoid-horseradish peroxidase. The present findings show that wheat germ agglutinin- and choleragenoid-horseradish peroxidase give rise to markedly different labelling patterns. A possible explanation for the different labelling in the marginal zone and substantia gelatinosa could be that certain primary sensory neurones lack either receptors for choleragenoid on their neuronal plasma membrane or the ability to transport the choleragenoid-horseradish peroxidase complex.

Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response.

Horseradish peroxidase (HRP) was covalently coupled to the binding subunit of cholera toxin (CTB) via a two-step glutaraldehyde procedure. The HRP-CTB conjugate was characterized by physiochemical as well as immunochemical methods. Mice were immunized intraduodenally with the HRP-CTB conjugate, with HRP alone, or with a mixture of uncoupled CTB and HRP. The functionally active dose of CTB was 50 micrograms and the HRP dose was in the 30- to 90-micrograms range. Both IgA and IgG antibody responses were measured in serum, intestinal washes, and bile by using a solid phase immunoradiometric assay. Mice immunized with the HRP-CTB conjugate showed a significantly higher level of IgA anti-HRP in intestinal washes and bile, as well as increased levels of serum IgG anti-HRP. Animals that received only HRP or the mixture of CTB and HRP had reduced levels of HRP-specific antibody of either class in both gut washes and bile. The IgA anti-HRP responses in the gut washes were 33- to 120-fold higher when the conjugate was used as the immunogen in comparison with immunization with the CTB + HRP or the HRP alone. Vaccines to stimulate mucosal immunity to any antigenic determinant might thus be prepared by covalent conjugation to effective mucosal immunogens such as CTB.

β Sheet to α helix transition in the binding subunit of cholera toxin

Circular dichroism spectra have been measured for the binding subunit of cholera toxin in water and in the presence of dodecyl sulfate. In water the protein has an appreciable amount of β structure and almost no α helix. In the presence of dodecyl sulfate the spectrum undergoes drastic change over a time period of approximately four hours, and at equilibrium resembles that expected for a chain with an appreciable amount of α helix but no β structure. The change in helicity is in good agreement with that expected from our formulation of the configuration partition function for the binding subunit. The conformational change may have an important relationship to the means by which the binding subunit permits penetration of the active subunit into the cell.