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Purification and sequence determination of a new neutral mammalian neurotoxin from the scorpion Buthus martensii
1State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Academia Sinica, P.R. China.
Abstract:
A new neutral mammalian neurotoxin, designated BmK M4, with an isoelectric point (pI) of 7.6 and a relatively low toxicity (LD50 = 4.0 +/- 0.25 microgram/g mice, i.v.) was purified from the venom of scorpion Buthus martensii Karsch (BmK). The complete amino acid sequence of the toxin composed of 64 amino acid residues was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidomethylated protein (up to 30 amino acid residues) and its peptide fragments degraded by lysylendopeptidase or Staphylococcus aureus Va protease. The calculated mol. wt based on the amino acid composition was 7001. By comparison with the sequences of other basic BmK mammalian neurotoxins, it was concluded that the weaker toxicity and lower pI value of BmK M4 might be the result of mutations H10E, R18G and K28D. The sequence comparison of BmK M4 with an acidic toxin, BmK M8, showed that the weakest toxicity and acidic property of BmK M8 may be the consequence of mutations K8D, D53A, V55E and V59E. The substitution of 21 Gly in BmK M4 for Tyrin other BmK toxins may also be of importance. In their tertiary structures, these mutated charged residues are mainly distributed in the surface (face B) that is roughly opposite to the "conserved hydrophobic surface" (face A) proposed by Fontecilla-Camps et al. in 1982. Therefore the toxin-receptor interaction may take a multiposition mode.
Insights
Researchers purified a new scorpion neurotoxin, BmK M4, from Buthus martensii Karsch venom. Its lower toxicity and neutral charge are linked to specific amino acid mutations compared to other toxins.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Scorpion venoms contain diverse neurotoxins affecting ion channels.
- Buthus martensii Karsch (BmK) venom is a source of potent mammalian neurotoxins.
- Understanding structure-activity relationships is crucial for neurotoxin research.
Purpose of the Study:
- To purify and characterize a novel neutral neurotoxin from BmK venom.
- To determine the complete amino acid sequence of the new toxin, BmK M4.
- To investigate the structural basis for BmK M4's reduced toxicity and neutral isoelectric point.
Main Methods:
- Purification of BmK M4 from BmK venom.
- Automated Edman degradation for N-terminal sequencing.
- Enzymatic digestion (lysylendopeptidase, Staphylococcus aureus Va protease) and fragment analysis.
- Amino acid composition analysis to calculate molecular weight.
- Sequence comparison with known BmK neurotoxins.
Main Results:
- A new neutral neurotoxin, BmK M4, was isolated with a pI of 7.6 and low toxicity (LD50 = 4.0 μg/g).
- The complete 64-amino acid sequence of BmK M4 was elucidated.
- Sequence comparisons identified specific mutations (H10E, R18G, K28D) potentially responsible for BmK M4's properties.
- Further comparisons with an acidic toxin (BmK M8) suggested other mutations contributing to toxicity and charge.
- Mutated charged residues are primarily located on the toxin surface, distinct from the conserved hydrophobic face.
Conclusions:
- BmK M4 represents a novel, less toxic, neutral neurotoxin from BmK venom.
- Specific amino acid substitutions significantly influence neurotoxin charge and toxicity.
- The distribution of mutated residues on the toxin surface suggests a multiposition mode for toxin-receptor interactions.