The arrangement of subunits in cholera toxin.

Cholera toxin consists of five similar B subunits of apparent molecular weight about 10 600 and one A subunit (29 000) consisting of two peptides (A1 23 000-24 000 and A2 about 5500) linked by a single disulfide bond. Each B subunit also contains one internal disulfide bond which is readily reduced but is protected from carboxymethylation unless the reduced subunits are heated in urea. Tyrosine residues in A1 and in B subunits are readily iodinated, but the intact B assembly does not react with iodine. Upon reaction with the cross-linking reagent dimethyl suberimidate, B subunits may be covalently connected to each other, to A1 and to A2. A1 and A2 may also be cross-linked. The B subunits are probably arranged in a ring with A on the axis. A2 is required for the re-assembly of toxin from its subunits and may serve to hold A1 on the B ring. The maximum activity of cholera toxin in vitro is obtained only when the active peptide, A1, is separated from the rest of the molecule. Such separation, and the insertion of A1 into the cytosol, must follow the binding of the complete toxin, through component B, to the exterior of intact cells. This binding increases the effective concentration of the toxin in the vicinity of the plasma membrane. Possible ways in which A1 then crosses the membrane are considered in the Discussion.     

Reduction of adenylyl cyclase activity by cholera toxin in myeloid cells. Long-term down-regulation of Gs alpha subunits by cholera toxin treatment

In IPC-81 cells, the adenylyl-cyclase activation by cholera toxin produces an elevation of cAMP that causes a rapid cytolysis. A resistant clone with deficient cholera toxin-induced cyclase activity (yet sensitive to cAMP) showed a rapid decrease in the amount of membrane-bound Gs alpha (42-47 kDa) detectable soon after ADP-ribosylation of these proteins; pertussis toxin-sensitive G proteins (41 kDa) were not affected. Resistant cells showed a rapid decrease of Gs alpha that is consistent with the finding that cAMP did not accumulate in these cells. Cholera toxin treatment of resistant cells had long-lasting effects (several weeks) on the level of Gs alpha in the cell membrane. The duration of Gs alpha decrease does not correspond to the probable life of catalytically active cholera toxin in the cells, and suggests a regulated process more complex than a proteolytic degradation targeted on ADP-ribosylated molecules.     

The amino acid sequence of toxin V from Anemonia sulcata

Preparations of the β-galactoside-binding lectin of bovine heart have been shown to stimulate $\text{in vitro}$ the sialylation of the oligosaccharide Ga1β1→4G1cNAc and asialo-α₁-acid glycoprotein by bovine colostrum β-D-galactoside α2→6 sialyltransferase. Kinetic data revealed that in the presence of lectin the K_m values for Ga1β1→4G1cNAc and CMP-NeuAc were reduced from 25.0 to 11.6 mM and from 0.42 to 0.19 mM respectively, but the K_m for asialo-α₁-acid glycoprotein and the V_max values for all three substrates were little affected. Stimulation by the lectin was partially inhibited by Fucα1→2Ga1β1→4G1cNAc. This, together with the effects of certain plant lectins, suggests that the stimulation of sialytransferase may be mediated through the carbohydrate-binding properties of the lectin.     

High performance liquid chromatography purification and amino terminal sequence analysis of the subunits of bovine heart mitochondrial F1-ATPase

All five subunits of bovine heart mitochondrial F1-ATPase have been isolated by reverse-phase HPLC and NH2-terminal sequences determined by gas phase Edman degradations. Bovine gamma exhibits 16 identities in the first 30 residues compared with the NH2-terminus of gamma from E.coli F1. Bovine delta exhibit about 27% identity with residues 28-59 of precursor delta from N.crassa and in the first six residues is identical with delta from S.cerevisiae. Approximately half of bovine epsilon has been sequenced. Possibly significant sequence similarities exist between bovine gamma and epsilon and kinase-related gene and oncogene products. The bovine alpha subunit has a blocked NH2-terminus.     

Location and amino acid sequence around the ADP-ribosylation site in the cholera toxin active subunit A1

Renatured, S-carboxymethylated subunit A₁ of cholera toxin possess the ADP-ribose transferase activity (Lai, et.al., Biochem. Biophys. Res. Commun. 1981, $\text{102}$, 1021). In the absence of acceptor self ADP-ribosylation of A₁ subunit was observed. Stoicheometric incorporation of ADP-ribose moiety was achieved in 20 min at room temperature in a 0.1 – 0.2M PO₄(Na) buffer, pH 6.6. On incubation of the complex with polyarginine, 75% of the enzyme-bound ADP-ribose moiety was transferred to the acceptor in 25 min. The ADP-ribosylated A₁ was stable at low pH, and on cleavage with BrCN, the ADP-ribose moiety was found associated with peptide Cn I, the COOH-terminal fragment of A₁ subunit. On further fragmentation with cathepsin D, a dodecapeptide containing ADP-ribose moiety was isolated whose structure was determined as: Asp-Glu-Glu-Leu-His-Arg-Gly-Tyr-Arg¹-Asp-Arg-Tyr. The Arg¹ in the peptide was indicated to be the site of ADP-ribosylation.     

Biological and biochemical characterization of variant A subunits of cholera toxin constructed by site-directed mutagenesis

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.     

Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin

Transducin was [32P]ADP-ribosylated by cholera toxin in bovine retinal rod outer segments and then partially purified on omega-amino octyl agarose to remove other ADP-ribosylated proteins. Trypsin digestion of the ADP-ribosylated transducin and further purification using boronate-polyacrylamide beads and high performance liquid chromatography yielded a single radiolabeled tetrapeptide, Ser-Arg-Val-Lys. The ADP-ribose is linked to the guanidinium group of arginine.     

Chloroquine inhibition of cholera toxin

Cholera toxin (CT) stimulated adenylate cyclase and a phospholipase which elevated cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and arachidonic acid (AA). The AA was quickly converted to prostaglandins (PGs) via the cyclo-oxygenase pathway. Chloroquine exerted minimal inhibition of cAMP levels in CT-treated cells, although CT-induced release of [3H]AA and PGs was blocked completely when the drug was added in concentrations as low as 0.1 mM (50 micrograms/ml). Inhibition of [3H]AA release was complete when chloroquine was added before or within 30 min after CT. The capacity of chloroquine to inhibit either phospholipase C (PLC) or phospholipase A2 (PLA2) could explain the antisecretory activity of this drug.     

Stimulation of adenylate cyclase in washed pigeon erythrocyte membrane with cholera toxin and its subunits.

Cholera toxin was found to stimulate adenylate cyclase activity in washed membrane of pigeon erythrocytes in the presence of dithiothreitol and NAD. When tested with isolated cholera toxin components, the stimulatory activity was found with subunit A or polypeptide A1 derived from this subunit, but not with A2 or subunit B. On a molar basis, polypeptide A1 was approximately four times more active than cholera toxin. Dithiothreitol was not required in the action of polypeptide A1, suggesting that the reagent was needed only to release A1 from subunit A or the holotoxin for their action on adenylate cyclase. The single SH group in polypeptide A1 was not involved in the activity of the peptide, since chemical modification of the thiol group did not alter the stimulatory activity of the peptide. The presence of NAD was, however, essential for the activation of adenylate cyclase with cholera toxin, subunit A, or polypeptide A1. Elevation of the adenylate cyclase activity was also observed when the intact pigeon erythrocytes were incubated with polypeptide A1, although a 30-fold molar excess of A1 over that of holotoxin was required for the same level of activation.     

Modification of the cholera toxin B subunit coding sequence to enhance expression in plants

The cholera toxin B subunit (CTB) contains five identical polypeptides and targets glycosphingolipid receptors on eukaryotic cell surfaces. Increased expression of CTB in plants is critical for the development of edible vaccines. In this study, the coding sequence of the CTB gene was optimized, based on the modification of codon usage to that of tobacco plant genes and the removal of mRNA-destabilizing sequences. The synthetic CTB gene was cloned into a plant expression vector and expressed in tobacco plants under the control of the CaMV 35S promoter. The recombinant CTB protein constituted approximately 1.5% of the total soluble protein in transgenic tobacco leaves. This level of CTB production was approximately 15-fold higher than that in tobacco plants that were transformed with the bacterial CTB gene. The recombinant CTB produced by tobacco plants demonstrated strong affinity for GM1-ganglioside, which indicates that the sites required for binding and proper folding of the pentameric CTB structure were conserved. This is the first report on the optimization of the CTB-coding sequence to give a dramatic increase in CTB expression in plants.     

The amino acid sequence of toxin RpIII from the sea anemone, Radianthus paumotensis

The amino acid sequence of the sodium channel toxin RpIII from the sea anemone Radianthus paumotensis has been determined. The protein is homologous with five analogous toxins from three anemone species, and is most similar to a less toxic protein, RpII, from the same organism. Twelve residues are conserved in all six toxins, one of which is an arginine residue thought to be essential for toxicity. The others (Cys, Gly, Pro and Trp) tend to be conserved in other sets of homologous proteins to maintain functional folds. Comparisons of the sequences suggest the existence of two separate but related classes of toxins cumon the three species of anemone.     

N-terminal amino acid sequence analysis of the subunits of pea photosystem I

Six 'core' subunits of pea photosystem I have been isolated and their N-terminal amino acid sequences determined by gas-phase or solid-phase sequencing. On average more than thirty residues were determined from the N-terminus of each polypeptide. This sequence analysis has revealed three polypeptides with charged N-terminal regions (21, 17 and 11 kDa subunits), one polypeptide with a predominantly hydrophobic N-terminal region (9 kDa subunit), one polypeptide which is cysteine-rich (8 kDa subunit) and one which is alanine-rich (13 kDa subunit).     

Homologous sequences in cholera toxin A and B subunits to peptide domains in myelin basic protein

Recent reports that myelin basic protein (MBP) can be ADP-ribosylated and contains specific sites that bind GTP and GM1 ganglioside, have suggested an analogy to the properties of cholera toxin. Comparisons of pairs of sequences between these two proteins yielded two regions of homology between MBP and the cholera toxin B (chol B) subunit, and one region of homology with the cholera toxin A (chol A) subunit. The matching sites within chol B consisted of a 17 amino acid residue sequence (residues 30-46 in chol B and residues 102-118 in human-MBP, hMBP, p less than 0.0007) and an 11 residue span (residues 31-41 in chol B and sequence 29-39 in hMBP, p less than 0.0004). The homologous site within chol A corresponded to an 11 residue span (residues 130-140 in chol A and 67-77 in hMBP sequence, p less than 0.00007). Since portions of the cholera toxin sequence are virtually identical to sections of the sequence in E. coli toxin, the homology is also valid for the same sequences in this toxin. The highly antigenic behavior of MBP that is related to the induction of experimental allergic encephalomyelitis may be paralleled by comparable neural pathology from the homologous regions of cholera toxin.     

The intracellular voyage of cholera toxin: going retro

Cholera toxin (CT) and related AB₅-subunit toxins move from the plasma membrane through the trans-Golgi and endoplasmic reticulum (ER) to the cytosol of host cells. The toxins exploit a specific glycolipid pathway rather than a protein pathway. They bind glycolipids that associate with lipid rafts at the cell surface, which carry the toxins retrograde to the Golgi and ER. In the ER, the A1-chain of the CT unfolds and enters the cytosol by hijacking the cellular machinery that enables misfolded proteins to cross the membrane for degradation by the proteasome, a process termed retro-translocation. Upon entering the cytosol, the A1-chain rapidly refolds, avoids the proteasome and induces toxicity.