A monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short‐chain variants

Trémeau, O.; Boulain, Jean‐Claude; Couderc, Jacques; Fromageot, P.; Ménèz, Andre

doi:10.1016/0014-5793(86)81024-9

Cited by 41 publications

(39 citation statements)

References 19 publications

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“…Radioimmunoassays were carried out as previously described [3] using 3H-labelled toxin oe from Naja nigricollis [4] and Moe2-3 monoclonal antibody [5]. Specific binding and affinity of fused and cleaved erabutoxins for acetylcholine receptor-rich membranes were determined from competition experiments [6] using 3H-labelled toxin oe.…”

Section: Biological Assaysmentioning

confidence: 99%

“…Treatment with CNBr was therefore expected to release the toxin moiety. Chromatography on the RP-column of the cleaved hybrid led to three main resolved components which were investigated for their ability to compete with a radioactive toxin in binding with a toxin-specific monoclonal antibody named M~2-3 [5]. Components 1 and 3 were recognized by the antibody and coeluted respectively with native erabutoxin and the hybrid.…”

Section: Purification and Characterizationmentioning

confidence: 99%

“…Firstly, we examined the antigenicity of Ear using M~2-3, a monoclonal antibody which is highly specific for correctly folded neurotoxins and which recognizes a large domain encompassing the three adjacent loops on the concave side of neurotoxins [5]. As shown on Fig.…”

Section: Structurementioning

confidence: 99%

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A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties

Boyot¹,

Pillet²,

Ducancel³

et al. 1990

FEBS Letters

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We previously reported the production of a fused snake neurotoxin composed of protein A and erabutoxin a in E. coli [1]. The hybrid had much lower toxicity and affinity for the acetylcholine nicotinic receptor than natural erabutoxin. By treating the hybrid with cyanogen bromide we generated a toxin which was purified in a single step by RP-HPLC. This compound, produced in a good yield, recovered all properties of native erabutoxin a, implying that the lower toxic activities of the hybrid were due to the bulky protein A and not to an incorrect folding of the toxin.This work serves as a basis for future studies of toxin-receptor interactions using engineered toxin mutants.

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Section: Biological Assaysmentioning

confidence: 99%

Section: Purification and Characterizationmentioning

confidence: 99%

See 1 more Smart Citation

A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties

Boyot¹,

Pillet²,

Ducancel³

et al. 1990

FEBS Letters

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“…Two monoclonal antibodies were also used as controls: M␣ 2-3 , as positive control, which is an antibody raised against the native toxin and is able to neutralize its toxic activity (48) and M 14-4-4, which recognizes the class II major histocompatibility complex molecule (49), used as negative control. Fig.…”

Section: Engineering Of a Curaremimetic Snake-scorpion Chimeramentioning

confidence: 99%

“…Microtiter plates were coated with solubilized AchoR as described above. Serial dilutions of 50 l of either the affinity-purified anti-chimera antibodies or the monoclonal antibodies M␣ [2][3] (48) or M 14-1-1 (49) were added to the wells. The starting concentration for all three antibodies was 5 ϫ 10 Ϫ7 M. A constant dilution of HRP-toxin ␣ in 50 l was then added to the wells, and the plate was incubated for 3 h at room temperature.…”

Section: ϫ3mentioning

confidence: 99%

Changing the Structural Context of a Functional β-Hairpin

Drakopoulou

Zinn‐Justin

Guenneugues³

et al. 1996

Journal of Biological Chemistry

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An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure acting as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26 -37, sandwiched between two hairpins, onto the structurally similar ␤-hairpin of the scorpion toxin charybdotoxin, stabilized by a short helix. The resulting chimeric molecule, only 31 amino acids long, was produced by solid phase synthesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected ␣/␤ fold characteristic of scorpion toxins and presented a remarkable structural stability. The chimera competitively displaces the snake curaremimetic toxin ␣ from the acetylcholine receptor at 10 ؊5 M concentrations. Antibodies, elicited in rabbits against the chimera, recognize the parent snake toxin and prevent its binding to the acetylcholine receptor, thus neutralizing its toxic function. All these data demonstrate that the strategy of active site transfer to the charybdotoxin scaffold has general applications in the engineering of novel ligands for membrane receptors and in vaccine design.

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Structural model of the anti-snake-toxin antibody, Mα2,3

1996

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We present results of structural modeling of the variable fragment of M alpha 2,3, an antibody capable of neutralizing all short snake toxins. Three different methods were used to model the hypervariable loops: the conformational search algorithm CONGEN (Bruccoleri and Karplus, Biopolymers 26:137-168, 1987), high-temperature molecular dynamics (Bruccoleri and Karplus, Biopolymers 29:1847-1862, 1990), and a combined knowledge-based and energy-based algorithm (Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272, 1989). Ninety plausible conformations were generated and were clustered into 13 classes. The clustering results indicate that there was little overlap of the conformational space explored by the different methods. Canonical loop structures were found by all methods for two of the loops, in agreement with previously established empirical modeling criteria. Nine of the 13 classes of structure were rejected on the ground of their lacking common features of antibody combining-site structure. The remaining four models were refined using restrained molecular dynamics. It was found that interconversion between the four resulting structures is possible with no significant energy barriers, suggesting that they are in thermodynamic equilibrium at 300 K. Features of the combining-site structure likely to be particularly important for antigen binding are discussed.

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A monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short‐chain variants

Cited by 41 publications

References 19 publications

A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties

A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties

Changing the Structural Context of a Functional β-Hairpin

Structural model of the anti-snake-toxin antibody, Mα2,3

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