Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.

Wen Hua Liu, Lei Wang, Yi Liang Wang, Li Sheng Peng, Wen Yan Wu, Wen Lie Peng, Xiaoyu Jiang, Hong Bin Tu, Hui Ping Chen, Ping Ou-Yang, An Long Xu

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3′,5′-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum-Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C. The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (Benzinger et al., 1998), having 61.7% β-sheet and no α-helix. Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD50 of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5±160.5%. The increase in contractile amplitude reached a plateau value within 3-5 min after addition of this toxin.

Original languageEnglish (US)
Pages (from-to)793-801
Number of pages9
JournalToxicon
Volume41
Issue number7
DOIs
StatePublished - Jun 1 2003
Externally publishedYes

Fingerprint

Sea Anemones
Cloning
Neurotoxins
Organism Cloning
Peptides
Amino Acids
Amino Acid Sequence
Fusion reactions
Complementary DNA
Affinity chromatography
Thioredoxins
DNA Primers
Lethal Dose 50
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Molecular mass
Protein Sorting Signals
Circular Dichroism
Affinity Chromatography
Endoplasmic Reticulum
Escherichia coli

Keywords

  • Fusion expression
  • Gene cloning
  • Increasing contracting
  • LD
  • Neurotoxins from sea anemone
  • rHk2a

ASJC Scopus subject areas

  • Toxicology

Cite this

Liu, W. H., Wang, L., Wang, Y. L., Peng, L. S., Wu, W. Y., Peng, W. L., ... Xu, A. L. (2003). Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp. Toxicon, 41(7), 793-801. https://doi.org/10.1016/S0041-0101(03)00033-3

Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp. / Liu, Wen Hua; Wang, Lei; Wang, Yi Liang; Peng, Li Sheng; Wu, Wen Yan; Peng, Wen Lie; Jiang, Xiaoyu; Tu, Hong Bin; Chen, Hui Ping; Ou-Yang, Ping; Xu, An Long.

In: Toxicon, Vol. 41, No. 7, 01.06.2003, p. 793-801.

Research output: Contribution to journalArticle

Liu, WH, Wang, L, Wang, YL, Peng, LS, Wu, WY, Peng, WL, Jiang, X, Tu, HB, Chen, HP, Ou-Yang, P & Xu, AL 2003, 'Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.', Toxicon, vol. 41, no. 7, pp. 793-801. https://doi.org/10.1016/S0041-0101(03)00033-3
Liu, Wen Hua ; Wang, Lei ; Wang, Yi Liang ; Peng, Li Sheng ; Wu, Wen Yan ; Peng, Wen Lie ; Jiang, Xiaoyu ; Tu, Hong Bin ; Chen, Hui Ping ; Ou-Yang, Ping ; Xu, An Long. / Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp. In: Toxicon. 2003 ; Vol. 41, No. 7. pp. 793-801.
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abstract = "A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3′,5′-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum-Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95{\%} amino acid sequence similarity to Ap-C. The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B (Benzinger et al., 1998), having 61.7{\%} β-sheet and no α-helix. Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD50 of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5±160.5{\%}. The increase in contractile amplitude reached a plateau value within 3-5 min after addition of this toxin.",
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AU - Peng, Wen Lie

AU - Jiang, Xiaoyu

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AU - Xu, An Long

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