Delineation of the functional site of a snake venom cardiotoxin: preparation, structure, and function of monoacetylated derivatives

E Gatineau, M Takechi, F Bouet, P Mansuelle, H Rochat, A L Harvey, T Montenay-Garestier, A Ménez

Research output: Contribution to journalArticle

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Abstract

Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.
LanguageEnglish
Pages6480-6489
Number of pages10
JournalBMC Biochemistry
Volume29
Issue number27
DOIs
Publication statusPublished - 10 Jul 1990

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Cardiotoxins
Cobra Venoms
Snake Venoms
Cobra Cardiotoxin Proteins
Fluorescence
Derivatives
Snakes
Neurotoxins
Reverse-Phase Chromatography
Cytotoxicity
Muscle Cells
Cations
Skeletal Muscle
Cell Culture Techniques
Binding Sites
Chromatography
Muscle
Substitution reactions
Cells

Keywords

  • acetylation
  • amino acid sequence
  • animals
  • binding sites
  • cultured cells
  • circular dichroism
  • cobra cardiotoxin proteins
  • female
  • membrane potentials
  • mice
  • inbred BALB C mice
  • molecular models
  • molecular sequence data
  • muscles
  • protein conformation
  • snake venoms
  • fluorescence spectrometry
  • structure-activity relationship

Cite this

Gatineau, E ; Takechi, M ; Bouet, F ; Mansuelle, P ; Rochat, H ; Harvey, A L ; Montenay-Garestier, T ; Ménez, A. / Delineation of the functional site of a snake venom cardiotoxin : preparation, structure, and function of monoacetylated derivatives. In: BMC Biochemistry. 1990 ; Vol. 29, No. 27. pp. 6480-6489.
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abstract = "Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.",
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Gatineau, E, Takechi, M, Bouet, F, Mansuelle, P, Rochat, H, Harvey, AL, Montenay-Garestier, T & Ménez, A 1990, 'Delineation of the functional site of a snake venom cardiotoxin: preparation, structure, and function of monoacetylated derivatives' BMC Biochemistry, vol. 29, no. 27, pp. 6480-6489. https://doi.org/10.1021/bi00479a021

Delineation of the functional site of a snake venom cardiotoxin : preparation, structure, and function of monoacetylated derivatives. / Gatineau, E; Takechi, M; Bouet, F; Mansuelle, P; Rochat, H; Harvey, A L; Montenay-Garestier, T; Ménez, A.

In: BMC Biochemistry, Vol. 29, No. 27, 10.07.1990, p. 6480-6489.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Delineation of the functional site of a snake venom cardiotoxin

T2 - BMC Biochemistry

AU - Gatineau, E

AU - Takechi, M

AU - Bouet, F

AU - Mansuelle, P

AU - Rochat, H

AU - Harvey, A L

AU - Montenay-Garestier, T

AU - Ménez, A

PY - 1990/7/10

Y1 - 1990/7/10

N2 - Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.

AB - Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.

KW - acetylation

KW - amino acid sequence

KW - animals

KW - binding sites

KW - cultured cells

KW - circular dichroism

KW - cobra cardiotoxin proteins

KW - female

KW - membrane potentials

KW - mice

KW - inbred BALB C mice

KW - molecular models

KW - molecular sequence data

KW - muscles

KW - protein conformation

KW - snake venoms

KW - fluorescence spectrometry

KW - structure-activity relationship

U2 - 10.1021/bi00479a021

DO - 10.1021/bi00479a021

M3 - Article

VL - 29

SP - 6480

EP - 6489

JO - BMC Biochemistry

JF - BMC Biochemistry

SN - 1471-2091

IS - 27

ER -