Cholera toxin (CTx) is an AB^sub 5^ cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B^sub 5^) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B^sub 5^ pentamer (SLTx B), the CTx B^sub 5^ pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional α-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.

Cholera toxin (CTx), produced by toxinogenic strains of Vibrio cholerae, is a heterohexameric AB^sub 5^ protein belonging to the family of related toxins that are associated with human disease. Infection with V. cholerae can lead to an acute gastrointestinal disease resulting in toxemia and chronic diarrhea causing the loss of over 10-20 liters of body fluids per day in some cases (1) and frequently resulting in a mortality of 20-50% in untreated cases (2,3)- Indeed, there are over 200,000 reported new cases of cholera each year (4). CTx, the main toxic product produced during infection, has been characterized, along with the producing bacterium, as a potential 'biowarfare' agent by the US and UK defense agencies (5). The structure of CTx holotoxin and the CTx B pentameric subunits have been determined by means of x-ray crystallography (6-8). These studies revealed that the five B subunits (~11.6 kDa/subunit) form a doughnut-shaped ring and that the A subunit (~27.5 kDa) is composed of two distinct domains, A1 and A2. The A1-domain is responsible for the enzymatic activity of the toxin, whereas the A2-domain assists in the tethering of the A1-domian to the B subunit pentamer by inserting noncovalently into and through the central pore of the B5 complex (Fig. 1). The CTx A1 subunit is an ADP-ribosyltransferase and NAD-glycohydroIase that can modify G proteins by ADP ribosylation (9). The main intracellular target of CTx directly associated with the induction of fluid secretion is Gs, a G protein involved in the activation of the adenylate cyclase complex (10-12), although the toxin can affect numerous metabolic processes through the modification of other G proteins. Modification of Gs by CTx leads to constitutive activation of the adenylate cyclase complex and elevated intracellular cAMP levels (13-15), which in turn stimulates active chloride secretion (16-18) resulting in a severe form of secretory diarrhea.

To accomplish these intracellular changes, CTx must reach the cytosol of its target cells. CTx binds, via its B pentamer, to the glycosphingolipid GM1 ganglioside (19-21), which is present in the plasma membrane of virtually all cell types, including those cells that line the intestinal tract and certain cells associated with the control and function of the immune system (22). Binding of CTx to GM1 at the plasma membrane triggers toxin uptake and subsequent retrograde delivery of the toxin to the endoplasmic reticulum (ER) (23-28) where the A1-domain is reduced from the A2-B5 complex and then retrotranslocated across the ER membrane into the target cell cytosol where it can modify its targets (27,29-32).

In addition to the well-characterized cytotonic properties of CTx, interaction between the CTx holotoxin or the isolated BS pentamer and specific target cells can also promote signaling pathways not linked to cAMP induction or indeed to other second messenger molecules. This can lead to different immunomodulatory functions, such as mucosal adjuvant effects, depending on the target cell type (22). A key element in the diverse signaling activities of this toxin is the initial oligomerization of GM1 receptors upon binding the B-chain pentamer. This is believed to promote the recruitment of both signaling and accessory molecules into cholesterol-enriched membrane microdomains (lipid rafts) to establish and enhance signaling cascades (22,33). As such, analysis of the noncovalent interactions that promote and maintain the quaternary structure of this toxin is of fundamental importance since such interactions have significant biochemical and medical implications.

There have been relatively few reports of the analysis of CTx by mass spectrometry (MS) (34,35). An in-depth study by liquid chromatography MS (LC-MS) and LC tandem MS (LC-MS/MS) provided sequence information derived from tryptic digestion and showed that the toxin was well suited to mass spectrometric detection (35). During this previous investigation, the AB^sub 5^ holotoxin was not detected. Here, we demonstrate that the complete noncovalent assembled AB^sub 5^ holotoxin remains intact during the electrospray ionization (ESI) process and can be observed in the mass spectrum.