Cholera Toxin Peptide Research Articles

A Dipeptide Metalloendoprotease Substrate Completely Blocks the Response of Cells in Culture to Cholera Toxin

Prior exposure (15 min at 37 degrees C) of several cell types (Vero, SH-SY5Y neuroblastoma, human intestinal epithelial T84) to 3 mm N-benzoyloxycarbonyl-Gly-Phe-amide (Cbz-Gly-Phe-NH(2)), a competitive substrate for metalloendoproteases, completely suppressed cholera toxin (CT)-induced intracellular cAMP accumulation. The specificity of the inhibitory effect was demonstrated by the complete lack of effect of the dipeptide Cbz-Gly-Gly-NH(2), an inactive analogue of Cbz-Gly-Phe-NH(2). The effect was reversible and dose- (IC(50) as low as 0.2 mm depending on the cell type) and time-dependent. Adding Cbz-Gly-Phe-NH(2) during the lag phase caused a diminution of its inhibitory effect similar to that observed with brefeldin A (BFA). Whereas the dipeptide completely suppressed the CT-induced adenylate cyclase (AC) activity, a direct effect on AC is unlikely since the elevation of intracellular cAMP by forskolin was only slightly reduced. The A(1) peptide of CT and NAD(+) activated the AC to the same extent in membranes from control and Cbz-Gly-Phe-NH(2)-treated cells or when Cbz-Gly-Phe-NH(2) was added directly to the assay. The inhibitory effects of suboptimal amounts of Cbz-Gly-Phe-NH(2) and BFA were not additive pointing to a similar mode of action of the two substances. However, Madin-Darby canine kidney cells of which the Golgi structure is BFA-resistant were not resistant to the inhibitory action of Cbz-Gly-Phe-NH(2) on CT cytotoxicity. Several lines of evidence indicate that a perturbation of intracellular Ca(2+) homeostasis by Cbz-Gly-Phe-NH(2) is not responsible for the inhibitory effect of the dipeptide. The dipeptide had also no effect on the binding of (125)I-CT to cells and even increased its intracellular internalization. In contrast with BFA, Cbz-Gly-Phe-NH(2) did not completely suppress the formation of the catalytically active A(1) fragment from bound CT. The data are compatible with a role of metalloendoprotease activity in the intracellular trafficking and processing of CT, although other mechanisms of action of Cbz-Gly-Phe-NH(2) cannot be excluded.

Two‐Dimensional nmr studies of the interactions between a peptide of cholera toxin and monoclonal antibodies

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of antipeptide antibodies with native proteins, it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen, and the bound peptide conformation. To extract the information about antibody-peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab-peptide complex (Fab: antibody fragment made of Fv--the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen--the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two-dimensional transferred nuclear Overhauser effect (NOE) difference spectra was developed. Using this methodology the interactions of three monoclonal antibodies with a cholera toxin peptide were studied. The observed interactions were assigned to the antibody protons involved by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies' combining sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional NMR investigations of the interactions of antibodies with peptide antigens.

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of anti-peptide antibodies with native proteins it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen and the bound peptide conformation. We have developed an NMR methodology based on two-dimensional difference spectrum measurements which extract the information concerning antibody-peptide interactions and intramolecular interactions in the bound ligand from the crowded and unresolved spectrum of the Fab complex. These measurements yield restraints on interproton distances in the complex which are used to dock the peptide into calculated models for the antibodies' combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3), which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by antibodies, the structure of their combining sites and relationships between antibodies' primary structure, and their interactions with peptide antigens.

Crystal Structure of an Anticholera Toxin Peptide Complex at 2·3 Å

Cholera toxin peptide 3 (CTP3) is a 15-residue peptide corresponding in sequence to an immunogenic loop on the surface of the B-subnnits of both cholera toxin and the heat-labile toxin from Escherichia coli. TE33 is the Fab fragment of a monoclonal antibody elicited against CTP3. The crystal structure of the TE33-CTP3 complex at 2·3 Å resolution reveals an antigen-binding pocket, 13 Å deep and 13 Å wide, which is lined with many aromatic residues. The N-terminal portion of the peptide antigen CTP3 forms a type II β-turn that fits snugly into this pocket. At gln7 the peptide backbone of CTP3 forms a kink followed by an extended C-terminal chain that seals off the cleft and buries the β-turn underneath it. All six complementarity-determining regions of TE33 contribute to the binding of CTP3. The antibody-peptide contacts include, in addition to van der Waals' interactions and hydrogen bonds, also one salt bridge and one water molecule, which mediates the interaction.

Determination of the conformations of bound peptides using NMR-transferred nOe techniques

NMR-transferred nOe spectroscopy provides a means for determining the structure of a peptide bound to a macromolecular target when the complex is too large to be studied by conventional NMR techniques. Recent examples of structures that have been determined by NMR-transferred nOe techniques include platelet-receptor peptide bound to thrombin, cholera toxin peptide bound to antibodies, troponin I peptide bound to troponin C and the vsv-C peptide bound to chaperones DnaK and GroEL.

A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens

Transferred nuclear Overhauser effect (TRNOE) spectroscopy can be used to study intra- and intermolecular interactions of bound ligands complexed with large proteins. However, the 2D NOE (NOESY) spectra of large proteins are very poorly resolved and it is very difficult to discriminate the TRNOE cross peaks, especially those due to intermolecular interactions, from the numerous cross peaks due to intramolecular interactions in the protein. In previous studies we measured two-dimensional difference spectra that show exclusively TRNOE and exchange cross-peaks (Anglister, J., 1990. Quart. Rev. Biophys. 23:175-203). Here we show that a filtering method based on the difference between the T1rho values of the ligand and the protein protons can be used to directly obtain a two-dimensional transferred NOE spectrum in which the background cross-peaks due to intramolecular interactions in the protein are very effectively removed. The usefulness of this technique to study protein ligand interactions is demonstrated for two different antibodies complexed with a peptide of cholera toxin (CTP3). It is shown that the T1 rho-filtering alleviates t problems encountered in our previous measurements of TRNOE by the difference method. These problems were due to imperfections in the subtraction of two spectra measured for two different samples.

<b>ADP-RIBOSYLATION OF A<sub>1</sub> PEPTIDE OF CHOLERA TOXIN BY CHICKEN ARGININE-SPECIFIC ADP-RIBOSYLTRANSFERASE WITH A CONCOMITANT INCREASE IN ADP-RIBOSYLTRANSFERASE ACTIVITY OF THE </b><b>PEPTIDE </b>

We studied the in vitro ADP-ribosylation of the A 1 peptide of cholera toxin by arginine-specific ADP-ribosyltransferase purified from chicken peripheral polymorphonuclear leukocytes. Chicken ADP-ribosyltransferase modified A 1 peptide, but not the entire toxin. Four moles of ADP-ribose were incorporated into 1 mol of A 1 peptide. Modification of A 1 peptide increased its enzyme activity up to 4-fold, ad demonstrated by zymographic analysis using poly(L-arginine) as an acceptor

Induced peptide conformations in different antibody complexes: molecular modeling of the three-dimensional structure of peptide-antibody complexes using NMR-derived distance restraints.

Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a beta-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a beta-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a beta-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than alpha-helix or beta-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function. (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen. (b) Residues 31, 31c, and 31e of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen. (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.

NMR-derived model for a peptide-antibody complex

The TE34 monoclonal antibody against cholera toxin peptide 3 (CTP3; VEVPGSQHIDSQKKA) was sequenced and investigated by two-dimensional transferred NOE difference spectroscopy and molecular modeling. The VH sequence of TE34, which does not bind cholera toxin, shares remarkable homology to that of TE32 and TE33, which are both anti-CTP3 antibodies that bind the toxin. However, due to a shortened heavy chain CDR3, TE34 assumes a radically different combining site structure. The assignment of the combining site interactions to specific peptide residues was completed by use of AcIDSQRKA, a truncated peptide analogue in which lysine-13 was substituted by arginine, specific deuteration of individual polypeptide chains of the antibody, and a computer model for the Fv fragment of TE34. NMR-derived distance restraints were then applied to the calculated model of the Fv to generate a three-dimensional structure of the TE34/CTP3 complex. The combining site was found to be a very hydrophobic cavity composed of seven aromatic residues. Charged residues are found in the periphery of the combining site. The peptide residues HIDSQKKA form a beta-turn inside the combining site. The contact area between the peptide and the TE34 antibody is 388 A2, about half of the contact area observed in protein-antibody complexes.

Antibodies against a peptide of cholera toxin differing in cross-reactivity with the toxin differ in their specific interactions with the peptide as observed by proton NMR spectroscopy

The interactions between the aromatic residues of the monoclonal antibody TE34, and its peptide antigen CTP3, have been studied by 2D TRNOE difference spectroscopy. The sequence of CTP3 corresponds to residues 50-64 of the B subunit of cholera toxin (VEVPGSQHIDSQKKA). Unlike two previously studied anti-CTP3 antibodies (TE32 and TE33), the TE34 antibody does not bind the toxin. The off-rate of CTP3 from TE34 was found to be too slow to measure strong TRNOE cross-peaks between the antibody and the peptide. Much faster off-rates, resulting in a strong TRNOE, were obtained for two peptide analogues: (a) CTP3 with an amide in the C-terminus (VEVPGSQHIDSQKKA-NH2) and (b) a truncated version of the peptide (N-acetyl-IDSQKKA). These modifications do not interfere significantly either with the interactions of the unmodified part of the peptide with the antibody or with intramolecular interactions occurring in the epitope recognized by the antibody. The combined use of these peptides allows us to study the interactions between the antibody and the whole peptide. Two tyrosine residues and one or more tryptophan and phenylalanine residues have been found to interact with histidine-8, isoleucine-9, aspartate-10, lysine-13 and/or lysine-14, and alanine-15 of the peptide. In the bound peptide, we observe interactions of a lysine residue with aspartate-10 beta protons. While the peptide epitope recognized by TE34 is between histidine-8 and the negatively charged C-terminus, that recognized by TE32 and TE33 is between residues 3 and 10 of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of antibody aromatic residues with a peptide of cholera toxin observed by two-dimensional transferred nuclear Overhauser effect difference spectroscopy.

The interactions between a peptide of cholera toxin and the aromatic amino acids of the TE33 antipeptide antibody, cross-reactive with the toxin, have been studied by NOESY difference spectroscopy. The 2D difference between the NOESY spectrum of the Fab with a 4-fold excess of the peptide and that of the peptide-saturated Fab reveals cross-peaks growing with excess of the peptide. These cross-peaks are due to magnetization transfer between the Fab and neighboring bound peptide protons, and a further transfer to the free peptide protons by exchange between bound and free peptide (transferred NOE). Additional cross-peaks appearing in the difference spectrum are due to a combination of intramolecular interactions between bound peptide protons and exchange between bound and free peptide. Assignment of cross-peaks is attained by specific deuteration of antibody aromatic amino acids using also the resonance assignment of the free peptide, deduced from the COSY spectrum of the peptide solution. The antibody combining site is found to be highly aromatic. We have identified one or two histidine, two tyrosine, and two tryptophan residues and one phenylalanine residue of the antibody interacting with valine-3, proline-4, glycine-5, glutamine-7, histidine-8, and aspartate-10 of the peptide. The 2D TRNOE difference spectroscopy can be used to study protein-ligand interactions, given that the ligand off rate is fast relative to the spin-lattice relaxation time of the protein and ligand protons (about 1 s). The resolution obtained in the difference spectra implies that the technique is equally applicable for studying proteins having a molecular weight larger than 50,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral immunization with a synthetic peptide of cholera toxin B subunit. Obtention of neutralizing antibodies.

The ability of free synthetic fragments of the cholera toxin (CT), administered by parenteral or oral route, without adjuvant, to induce antibodies cross-reacting with CT was tested. Two peptides corresponding to the sequences 30-50 and 50-75 of the CT beta chain were selected and synthesized. Both free peptides, given intraperitoneally or orally, without adjuvant, elicited seric antibodies cross-reacting with CT. The anti-(P50-75) antibodies were able to neutralize the CT activity. Our results show that protection against a toxin at the systemic level can be obtained with a synthetic peptide even when administered by an oral route.

A conjugate of the A1 peptide of chloera toxin and the lectin of Wisteria floribunda that activates the adenylate cyclase of intact cells

The active A1 peptide of cholera toxin was linked by a disulphide bond to the lectin of Wisteria floribunda. The resulting conjugate activated the adenylate cyclase of intact U937 or K562 cells at the same concentrations as native toxin did, but to a greater extent. Activation was inhibited by N-acetyl-D-galactosamine or by antisera to the lectin or peptide. The characteristic lag phase between addition of toxin to cells and activation of cyclase was not found with the conjugate or with free A1 peptide.