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C Granier

Publications and source records attributed to C Granier.

At least 91 records · Page 5Linked to original sources
PubMed1990

Identification of antigenic residues on apamin recognized by polyclonal antibodies.

The structural requirements for antigenic recognition of apamin--an 18-amino acid, disulfide-bridged peptide--by rabbit antibodies were defined using a set of 18 apamin analogs in a competition liquid-phase radioimmunoassay. Some residues contribute considerably to antigenic recognition, e.g. Ala10, Arg13, and others to a lesser extent, e.g. Arg14, Glu7 and Thr8. The N- and C-terminal moieties of apamin are less antigenically important. These findings suggest that a good part of antibody specificities are directed to the central tightly folded part of the molecule. They are consistent with the observation that in saturating conditions, labeled apamin can, on average, bind one specific Fab fragment.

Amino Acid Sequence↗
PubMed1990

Epitope mapping of apamin by means of monoclonal antibodies raised against free or carrier-coupled peptide.

A panel of 20 monoclonal antibodies raised against the bee-venom peptide apamin (18 residues, 2 disulfide bridges) was prepared. Nine monoclonal antibodies (mAb) were obtained from a mouse immunized with free apamin and 11 from a mouse immunized with a mixture of free and carrier-coupled peptide. Using a panel of 11 synthetic apamin analogs, we examined the fine antigenic specificity of each antibody. The mAb generated against free apamin preferentially bound to the central part of the peptide and less frequently recognized the N- and C-terminal regions. However, monoclonal antibodies obtained by immunization with carrier-bound apamin showed a broader range of specificities, consistent with the possibility of the entire surface of this small antigen becoming immunogenic upon coupling to the carrier.

Amino Acid Sequence↗
PubMed1990

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1. Synthesis, CD analysis and immunoreactivity.

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.

Amino Acid Sequence↗
PubMed1989

Structural analysis of the interaction of apamin with Ia and its recognition by Ad- or Ab-restricted mouse T cells.

Apamin is a single-chain, disulfide-bonded, 18-amino acid peptide that elicits mouse T cell responses when presented by cells expressing syngeneic Ad or Ab class II MHC molecules. We previously showed that both the unfolding of this peptide by APC and the integrity of its N terminus segment were required for efficient apamin T cell recognition. To seek further information on the sites through which this peptide interacts with Ia and/or TCR, we used a panel of Ad- or Ab-restricted, apamin-specific THC to probe the antigenicity of a series of synthetic apamin analogs. These included peptides either truncated at the N terminus, or substituted by Ala at position 2, 4, 6, 7, 8, or 10. Analysis of THC responses to apamin analogs and use of the latter in competition assays for peptide presentation revealed the following: 1) optimal apamin T cell recognition critically involved Lys4, Ala5, Pro6, Glu7, and Leu10. The role of these residues in either "Ia or TCR binding regions" was found to depend upon the restricting Ia molecules at play. Thus, Lys4, Glu7, and Leu10 were TCR-binding residues in both Ad- and Ab-apamin complexes, whereas Lys4 participated in apamin/Ab but not, or to a marginal extent, in apamin/Ad interaction. Furthermore, Pro6 was associated either with an Ia contact region or a TCR interaction site when apamin was presented by Ab or Ad molecules, respectively. Unfolded apamin and the unrelated chicken OVA323-339 peptide were found to bind to the same, or closely related site(s) of Ad, as shown by their ability to compete reciprocally for recognition by appropriate Ad-restricted THC. Four distinct TCR V beta genes (V beta 2, V beta 4, V beta 6, and V beta 8) were found to be used in our panel of 16 apamin-specific THC. These data indicate that apamin interacts with Ad or TCR through a motif resembling other beta-sheeted, Ad-binding sequences; however, based on the spacing of the critical residues (i.e., 4, 7, and 10), the possibility exists that apamin processing permits the folding of this sequence into an alpha-helix.

Amino Acid Sequence↗
PubMed1989

Structure/activity relationships of scorpion alpha-toxins. Multiple residues contribute to the interaction with receptors.

Chemical modifications of tyrosine and tryptophan residues of scorpion alpha-neurotoxins II and III from Androctonus australis Hector were performed as well as modification of the two arginines and the alpha-amino group of toxin I. The pharmacological potencies of each derivative were assessed in vivo by LD50 measurement and in vitro by competition experiments with 125I-toxin for synaptosomal receptors. Arginine residues in positions 2 and 60 and the alpha-amino group of Androctonus toxin I were derivatized by p-hydroxyphenylglyoxal; the corresponding modified toxins exhibit low pharmacological potencies. Tryptophan 38 of toxin II and tryptophan 45 of toxin III were modified by nitrophenylsulfenyl chloride, leading respectively to a poorly and a fully active derivative. The tetranitromethane modification of tyrosine residues in positions 60, 5 and 14 of toxin III induced respectively 60%, 40% and 30% of loss of biological activity. Circular dichroic analysis indicated that for every derivative, except the nitrophenylsulfenyl derivative of Trp-45 of AaH III, the conformation of the toxin was not altered by derivatization. Conformational integrity was also confirmed by full activity of the derivatives in radioimmunoassays. Taken together, the results suggest that aromatic residues belonging to the conserved hydrophobic surface, to the C-terminal and to the loop region 37-44 are involved in the molecular mechanisms by which scorpion alpha-toxins act. Charged residues in the N-terminal and C-terminal also contribute to the high efficacy of the binding process. It appears that all important residues are clustered on one face of the toxin, suggesting a multipoint interaction with the proteins of the sodium channel.

Amino Acid Sequence↗
PubMed1989

Specificity of antibodies to sea anemone toxin III and immunogenicity of the pharmacological site of anemone and scorpion toxins.

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues. It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion alpha-toxins. The results obtained suggest that a region of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti-(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.

Action Potentials↗
PubMed1989

The antigenic structure of a scorpion toxin.

Scorpion toxins constitute a family of homologous proteins that exert potent pharmacological effects on ion channels. These proteins are immunogenic and constitute a good model for investigation of the molecular basis of antigenicity. In the first part of this article we summarize the results we have obtained in recent years concerning the location of the main antigenic regions of a model toxin, toxin II of the North African scorpion Androctonus australis Hector. Then, thanks to the recently available atomic coordinates of this toxin, we analyzed the relationships between the structural features of the protein and the location of the antigenic regions: we found that antigenic regions are located at exposed parts of the molecular surface, i.e. in reverse turns and the alpha-helix. These surface parts also correspond to segments of the polypeptide chain which are most accessible to a large spherical probe modelizing an antibody molecule. Finally, we obtained a general idea of what could be the main discontinuous antigenic determinants by looking for the neighboring relationships between the most exposed residues of the protein.

Animals↗
PubMed1989

Accessibility of the highly conserved amino- and carboxy-terminal regions from HIV-1 external envelope glycoproteins.

Amino- and carboxy-terminal extremities of the envelope external glycoproteins are regions that have remained highly conserved between human immunodeficiency viruses HIV-1 and HIV-2. The corresponding peptides have been synthesized and their structure and function analyzed. Circular dichroism spectra showed evidence of alpha helical conformation when the peptides were dissolved in the nonpolar solvent trifuoroethanol. These two regions are indeed exposed on the molecule because they were accessible to their respective specific antibodies on the native gp160 precursor or processed gp120 glycoproteins of HIV-1. Neither the peptides nor rabbit or human antibodies directed against the N- and C-terminal peptides interfered with the interaction between HIV-1 external glycoprotein gp120 and its CD4 cellular receptor. Taken together, these results indicate that N- and C-terminal regions of gp120 are accessible on the quaternary structure of the virion as well as on the soluble form of gp120 and that these regions are not directly or indirectly involved in the binding of gp120 to CD4.

Animals↗
PubMed1989

Use of synthetic peptides for the detection of antibodies against the nef regulating protein in sera of HIV-infected patients.

Human sera were tested for the presence of anti-nef antibodies by radioimmunoassay (RIA), with recombinant radiolabelled nef expressed in E. coli. Of the 300 HIV-positive sera tested by RIA, 70 +/- 5.3% were found to be anti-nef positive. Anti-nef antibodies bound to nef with a high affinity (K 0.5 = 2.2 x 10(-9) M). In 31 of the sera, the specificity of anti-nef antibodies was further analysed by enzyme-linked immunosorbent assay (ELISA) with large synthetic peptides ranging from 31 to 66 amino acid residues and spanning the total sequence of nef from HIV-1. The results obtained showed that the immunodominant antigenic sites of nef were located close to the N- and C-terminal regions of the molecule.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Monoclonal antibodies to scorpion toxins. Characterization and molecular mechanisms of neutralization.

Two mAb specific for the potent toxin II of the scorpion Androctonus australis Hector have been produced. One of them shows both high affinity binding to the toxin (Kd) = 0.8 nM) and in vivo and in vitro neutralizing properties. The mechanism by which the antibody neutralizes toxin binding to its receptor was shown to be of the competitive type, the epitope overlapping or being close to the receptor-binding region of the toxin. Several residues of the toxin clustered in the C-terminal region were shown likely to be part of the discontinuous epitope recognized by the antibody. The positive charge of the N epsilon-Lys-58 seems to play a pivotal role in the binding of the toxin to both the mAb and the sodium channel receptor.

Acetylation↗
PubMed1988

Processing by accessory cells for presentation to murine T cells of apamin, a disulfide-bonded 18 amino acid peptide.

Apamin, an 18 amino acid peptide with two disulfide bonds, elicits specific T cell proliferative responses in H-2d and H-2b mouse strains. We evaluated the processing requirement of this compact peptide by accessory cells for presentation to apamin-reactive T hybridoma cells (THC) by analyzing the IL-2 responses of 16 THC from apamin-primed BALB/c or C57BL/6 mice, to various forms of either native or chemically synthesized apamin analogs. These included: unfolded peptides (whose four sulfhydryl groups were blocked by acetamidomethyl residues), N-and/or C-truncated peptides, and an analog with a single amino acid substitution at position 10. Assessment of the Ag-specific THC responses in the presence of either live or formaldehyde-prefixed APC indicated the following: 1) all THC stringently required Ag processing; 2) in 8 of 16 cases, the simple unfolding of apamin was sufficient to eliminate the need for Ag processing, or even induced increased THC IL-2 responses (other cells required further antigenic alterations in addition to unfolding, or rare processing steps dependent on the integrity of the two disulfide bonds); and 3) for most THC, the Leu10 and the N terminus arm of apamin were shown to be critical for expression of the epitopes involved in T cell recognition. These data indicate that apamin, a natural peptide having an appropriate size for T cell triggering, acquires its antigenic conformation after a processing by APC which primarily involves an alteration of a disulfide bond-dependent peptide folding.

Amino Acid Sequence↗
PubMed1988

Immunochemistry of scorpion toxins. Synthesis and antigenic properties of a model of a loop region specific to alpha-toxins.

A region of the toxin II of the scorpion Androctonus australis Hector, possessing a loop structure, is shown to be antigenic. Some clear hints for the probable antigenic character of this region were obtained by the protruding properties of the loop region, as assessed by accessibility computations using atomic coordinates of the toxin and Lee-Richards algorithm. A synthetic replica of the loop region was obtained in a linear and cyclised form. Within the total anti-toxin antibody population, we have found and isolated those that recognize the model peptides. A high affinity binding of these specific antibodies to the parent toxin was demonstrated, affording experimental evidence for the antigenic properties of the loop region.

Amino Acid Sequence↗
PubMed1987

Isolation of two 67 kDa calcium-binding proteins from pig lung differing in affinity for phospholipids and in anti-phospholipase A2 activity.

Two 67 kDa proteins adsorbed to membranes in the presence of Ca2+ have been purified to homogeneity from pig lung using conventional procedures, followed by calcium-dependent affinity chromatography on polyacrylamide-immobilized phosphatidylserine. The two proteins were, respectively, excluded (67E) and retained (67R) on the column in the presence of Ca2+. On the basis of amino acid composition and isoelectric point, 67R was identified as 67 kDa calelectrin/calcimedin, whereas 67E could be differentiated from albumin, calregulin, 67 kDa fragment of protein kinase C and surfactant-associated proteins. Only 67R was slightly phosphorylated by protein kinase C, reacted with an antibody raised against 32.5 kDa endonexin and inhibited pig pancreas phospholipase A2 in a way similar to that of lipocortin or endonexin. These data bring further support to the view that inhibition of phospholipase A2 by lipocortin or other related proteins involves interaction with the lipid/water interface. They also provide evidence for a new kind of Ca2+-binding protein (67E), whose role still remains to be determined.

Amino Acids↗
PubMed1987

Immunochemistry of scorpion toxins. Immunogenicity of peptide 19-28 a model of an accessible and relatively rigid region.

Peptide 19-28, a model of an antigenic region of Androctonus australis Hector toxin II, has a rigid alpha-helix structure in the native protein. It was used as immunogen either in the free form, or bound to bovine serum albumin (BSA) or linked to its synthesis support (macroporous polyacrylamide resin). Only the anti-(peptide-BSA) and the anti-(peptide-resin) antibodies recognized the native toxin. The use of short synthetic analogues of peptide 19-28 suggests a specificity difference in the two antipeptides. Anti-(peptide-BSA) recognizes probably two determinants localized at the C and N terminals of the peptide chain. Anti-(peptide-resin) preferentially recognizes the N-terminal extremity. Finally we showed that the alpha-helix region remains accessible to antipeptide 19-28 when the toxin is bound to its receptor.

Amino Acid Sequence↗
PubMed1987

Antigenicity of peptide 19-28 of toxin II from the scorpion Androctonus australis as measured by different solid-phase tests and characterization of specific antibodies purified by immunoaffinity on the peptide or the toxin.

The antigenicity of peptide 19-28, a model of one of the major antigenic regions of toxin II of the scorpion Androctonus australis Hector was tested in different solid phase radioimmunoassay systems. The type of the solid phase and the mode of binding the synthetic peptide to the phase had a considerable effect on the resulting antigenicity. Two subpopulations of anti-toxin II antibodies were purified by affinity chromatography, one on Sepharose-peptide 19-28, the other on sepharose-toxin. The native or chemically modified toxin bound in the same way to these subpopulations. Denatured toxin was only poorly recognized by these antibodies. This suggests that the antibodies purified on peptide 19-28 recognize the same conformation dependent antigenic surface as do total anti-toxin antibodies.

Antibodies↗
PubMed1986

Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic region.

Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp-9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and alpha-NH2 of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.

Amino Acid Sequence↗