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Screening and cDNA cloning of Kv1 potassium channel toxins in sea anemones.

Yamaguchi Y, Hasegawa Y, Honma T, Nagashima Y, Shiomi K - Mar Drugs (2010)

Bottom Line: When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive.The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions.As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo, Japan.

ABSTRACT
When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3'RACE and 5'RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.

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Nucleotide sequence of the cDNA encoding κ1.3-SHTX-Sg1a. Deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown on the right. In-frame stop codons (TAA) are shown by asterisks. A poly(A) signal is boxed. Putative signal peptide, propart and mature peptide are shaded with light blue, yellow and light green, respectively.
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f2-marinedrugs-08-02893: Nucleotide sequence of the cDNA encoding κ1.3-SHTX-Sg1a. Deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown on the right. In-frame stop codons (TAA) are shown by asterisks. A poly(A) signal is boxed. Putative signal peptide, propart and mature peptide are shaded with light blue, yellow and light green, respectively.

Mentions: After subcloning each PCR product into the pT7Blue T-vector, at least three clones were analyzed for nucleotide sequence. For each PCR product, there was no difference in nucleotide sequence among the clones analyzed, suggesting that isoforms of the cloned toxin, if present, are trace in the six species. The determined nucleotide sequences of the full-length cDNAs coding for six type 1 potassium channel toxins (named κ1.3-SHTX-Sg1a, κ1.3-SHTX-Sha1a, κ1.3-SHTX-Sm1a, κ1.3-TLTX-Ca1a, κ1.3-TLTX-Hh1a and κ1.3-TLTX-Ta1a for the toxins of S. gigantea, S. haddoni, S. mertensii, C. adhaesivum, H. hemprichii and T. aster, respectively) have been deposited in the DDBJ/EMBL/GenBank databases under the following accession numbers: AB595204 for κ1.3-SHTX-Sg1a (501 bp), AB595205 for κ1.3-SHTX-Sha1a (461 bp), AB595206 for κ1.3-SHTX-Sm1a (461 bp), AB595207 for κ1.3-TLTX-Ca1a (464 bp), AB595208 for κ1.3-TLTX-Hh1a (403 bp) and AB595209 for κ1.3-TLTX-Ta1a (469 bp). The six cDNAs had the following common features; a stop codon (TAA or TGA) is contained in the 5′-untranslated region upstream of the initiating Met and a poly(A) signal (AATAAA) and a poly(A) tail in the 3′-terminal region. In addition, the cDNAs encoding the toxins of the Stichodactyla species and Thalassianthidae species contain open reading frames composed of 222 bp (corresponding to 74 amino acid residues) and 225 bp (corresponding to 75 amino acid residues), respectively. As a typical example, the nucleotide sequence of the κ1.3-SHTX-Sg1a cDNA is illustrated in Figure 2 (refer to Supplementary Data for the nucleotide sequences of the cDNAs encoding the remaining five toxins).


Screening and cDNA cloning of Kv1 potassium channel toxins in sea anemones.

Yamaguchi Y, Hasegawa Y, Honma T, Nagashima Y, Shiomi K - Mar Drugs (2010)

Nucleotide sequence of the cDNA encoding κ1.3-SHTX-Sg1a. Deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown on the right. In-frame stop codons (TAA) are shown by asterisks. A poly(A) signal is boxed. Putative signal peptide, propart and mature peptide are shaded with light blue, yellow and light green, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3039155&req=5

f2-marinedrugs-08-02893: Nucleotide sequence of the cDNA encoding κ1.3-SHTX-Sg1a. Deduced amino acid sequence is aligned below the nucleotide sequence. Nucleotide and amino acid numbers are shown on the right. In-frame stop codons (TAA) are shown by asterisks. A poly(A) signal is boxed. Putative signal peptide, propart and mature peptide are shaded with light blue, yellow and light green, respectively.
Mentions: After subcloning each PCR product into the pT7Blue T-vector, at least three clones were analyzed for nucleotide sequence. For each PCR product, there was no difference in nucleotide sequence among the clones analyzed, suggesting that isoforms of the cloned toxin, if present, are trace in the six species. The determined nucleotide sequences of the full-length cDNAs coding for six type 1 potassium channel toxins (named κ1.3-SHTX-Sg1a, κ1.3-SHTX-Sha1a, κ1.3-SHTX-Sm1a, κ1.3-TLTX-Ca1a, κ1.3-TLTX-Hh1a and κ1.3-TLTX-Ta1a for the toxins of S. gigantea, S. haddoni, S. mertensii, C. adhaesivum, H. hemprichii and T. aster, respectively) have been deposited in the DDBJ/EMBL/GenBank databases under the following accession numbers: AB595204 for κ1.3-SHTX-Sg1a (501 bp), AB595205 for κ1.3-SHTX-Sha1a (461 bp), AB595206 for κ1.3-SHTX-Sm1a (461 bp), AB595207 for κ1.3-TLTX-Ca1a (464 bp), AB595208 for κ1.3-TLTX-Hh1a (403 bp) and AB595209 for κ1.3-TLTX-Ta1a (469 bp). The six cDNAs had the following common features; a stop codon (TAA or TGA) is contained in the 5′-untranslated region upstream of the initiating Met and a poly(A) signal (AATAAA) and a poly(A) tail in the 3′-terminal region. In addition, the cDNAs encoding the toxins of the Stichodactyla species and Thalassianthidae species contain open reading frames composed of 222 bp (corresponding to 74 amino acid residues) and 225 bp (corresponding to 75 amino acid residues), respectively. As a typical example, the nucleotide sequence of the κ1.3-SHTX-Sg1a cDNA is illustrated in Figure 2 (refer to Supplementary Data for the nucleotide sequences of the cDNAs encoding the remaining five toxins).

Bottom Line: When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive.The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions.As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo, Japan.

ABSTRACT
When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3'RACE and 5'RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.

Show MeSH