Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis

Abstract

Cholera toxin (CT) is the prototype for the Vibrio cholerae-Escherichia coli family of heat-labile enterotoxins having an AB5 structure. By substituting amino acids in the enzymatic A subunit that are highly conserved in all members of this family, we constructed 23 variants of CT that exhibited decreased or undetectable toxicity and we characterized their biological and biochemical properties. Many variants exhibited previously undescribed temperature-sensitive assembly of holotoxin and/or increased sensitivity to proteolysis, which in all cases correlated with exposure of epitopes of CT-A that are normally hidden in native CT holotoxin. Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT. Several novel variants with wild-type assembly and stability showed significantly decreased toxicity and enzymatic activity (e.g., variants at positions R11, I16, R25, E29, and S68؉V72). In most variants the reduction in toxicity was proportional to the decrease in enzymatic activity. For substitutions or insertions at E29 and Y30 the decrease in toxicity was 10-and 5-fold more than the reduction in enzymatic activity, but for variants with R25G, E110D, or E112D substitutions the decrease in enzymatic activity was 12-to 50-fold more than the reduction in toxicity. These variants may be useful as tools for additional studies on the cell biology of toxin action and/or as attenuated toxins for adjuvant or vaccine use.The massive diarrhea characteristic of the disease cholera is in large part due to the action of cholera toxin (CT), produced by Vibrio cholerae of serogroup O1. A better understanding of the structure and function of CT will provide new insights into the pathogenesis of cholera and may aid in the design of safe and effective vaccines against cholera and related diarrheas.CT is a heterohexameric complex consisting of one A polypeptide and five identical B polypeptides (11). The B pentamer is required for binding to the cell surface receptor ganglioside GM 1 (11). The A subunit can be proteolytically cleaved within the single disulfide-linked loop between C187 and C199 to produce the enzymatically active A1 polypeptide (23) and the smaller A2 polypeptide that links fragment A1 to the B pentamer (32). Upon entry into enterocytes by endocytosis and following reduction and translocation, CT-A1 ADPribosylates a regulatory G-protein (Gs␣), which leads to constitutive activation of adenylate cyclase, increased intracellular concentration of cyclic AMP, and secretion of fluid and electrolytes into the lumen of the small intestine (12). ADP-ribosyl transferase activity of CT is stimulated in vitro by the presence of accessory proteins called ARFs (49), small GTP-binding proteins known to be involved in vesicle trafficking within the eukaryotic cell, but the role of ARFs in the activity of CT in vivo has not yet been determined.CT is the prototype for the V. cholerae-Escherichia coli family of heat-labile enteroto...

Structural and Biochemical Characterization of NarE, an Iron-containing ADP-ribosyltransferase from Neisseria meningitidis

“…For that reason it is very likely that the iron coordination is crucial for the recognition and binding of the target protein. This is further supported by the fact that His-57, which was recently shown to be crucial for the transferase activity (36,37,38), is part of the Cys 2 His 2 iron site. Intriguingly His-57 is part of an ␣-helix which is found in proteins with ADPRT activity but not within structurally similar NAD-binding proteins (Fig.…”

“…This central strand is followed by an ␣-helix and flanked by two antiparallel ␤-strands containing a crucial arginine and glutamate residue (5). A further histidine located within ␣-helix 2, which covers the active site, is considered as crucial for the recognition of, and interaction with, the target protein (36,37,38). By measuring chemical shift perturbations after addition of NAD to the NarE sample we could determine those residues that are affected by the interaction with NAD (Fig.…”

Cholera toxin activates nonconventional adjuvant pathways that induce protective CD8 T-cell responses after epicutaneous vaccination

“…CT induces apoptosis in some epithelial cell lines (33,34), a trait that may be relevant for adjuvanticity of the toxin and thus account for such mechanistic similarities. However, we observed residual priming activity of enzymatically inactive CT-OVAp, despite the fact that this form of CT has lost its ability to induce cAMP and is correspondingly less toxic (35). Other properties of enzymatically inactive CT, such as its continued ability to direct transfer of the (inactive) A1 subunit from the ER lumen to the cytosol, may be relevant in antigen processing and presentation.…”

“…GalnT1/2 (GalT −/− ) DKO mice, lacking expression of GalNAcT (N-acetylgalactosaminyltransferase) 1 and 2, were obtained from the Mouse Model Core of the Consortium for Functional Glycomics (National Institute of General Medical Sciences) and were maintained by breeding mice deficient for GalnT2 (44) and heterozygous for GalnT1 (45). Typing of double KO mice was confirmed by peripheral blood cells failing to stain with FITC-CTB (B subunit of CT) (Sigma Aldrich) (35,46). MyD88/TRIF DKO mice (47) and ASC KO mice (20) were obtained from Shizuo Akira (Osaka University, Osaka, Japan) via Marc Jenkins (University of Minnesota).…”

Vesicular Transport Is Not Required for the Cytoplasmic Pool of Cholera Toxin To Interact with the Stimulatory Alpha Subunit of the Heterotrimeric G Protein

“…Cell culture reagents were from Gibco BRL (Grand Island, N.Y.), while Lipofectamine was from Invitrogen (Carlsbad, Calif.). Monoclonal and polyclonal antibodies to CTA were produced in this lab (10,11), the anti-␣1-antitrypsin (␣1AT) antibody was from DAKO (Carpinteria, Calif.), the anti-PDI antibody was from Stressgen (San Diego, Calif.), and the secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, Pa.). Immobilized antibodies were generated by a 4°C overnight incubation with 50 mg of protein G-agarose (anti-CTA antibody) or 100 mg of protein A-Sepharose (anti-␣1AT antibody) in 1 ml of phosphatebuffered saline supplemented with 0.1% bovine serum albumin.…”