RESUMO
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.
Assuntos
Toxinas Marinhas/farmacologia , Neurotoxinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Anêmonas-do-Mar/química , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Superfamília Shaker de Canais de Potássio/metabolismo , Sinaptossomos/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Avaliação Pré-Clínica de Medicamentos , Venenos Elapídicos/metabolismo , Toxinas Marinhas/química , Toxinas Marinhas/genética , Toxinas Marinhas/metabolismo , Dados de Sequência Molecular , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/metabolismo , Oceanos e Mares , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/metabolismo , Ratos , Anêmonas-do-Mar/genética , Sinaptossomos/efeitos dos fármacosRESUMO
Four peptide toxins, SHTX I-III with crab-paralyzing activity and SHTX IV with crab lethality, were isolated from the sea anemone Stichodactyla haddoni and their primary structures elucidated by protein sequencing and cDNA cloning. SHTX I (new toxin, 28 residues), II (analogue of SHTX I, 28 residues) and III (Kunitz-type protease inhibitor, 62 residues) are potassium channel toxins and SHTX IV (48 residues) is a member of the type 2 sea anemone sodium channel toxins. The precursor protein of SHTX IV is composed of a signal peptide, propart and mature peptide, while the propart is missing in that of SHTX III. In addition to these four toxins, an epidermal growth factor-like peptide was detected in S. haddoni by RT-PCR.
Assuntos
Toxinas Marinhas/química , Toxinas Marinhas/toxicidade , Peptídeos/química , Peptídeos/toxicidade , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/metabolismo , Toxinas Marinhas/isolamento & purificação , Dados de Sequência Molecular , Canais de Potássio/metabolismo , Alinhamento de SequênciaRESUMO
A potassium channel peptide toxin (AETX K) was isolated from the sea anemone Anemonia erythraea by gel filtration on Sephadex G-50, reverse-phase HPLC on TSKgel ODS-120T and anion-exchange HPLC on Mono Q. AETX K inhibited the binding of (125)I-alpha-dendrotoxin to rat synaptosomal membranes, although much less potently than alpha-dendrotoxin. Based on the determined N-terminal amino acid sequence, the nucleotide sequence of the full-length cDNA (609bp) encoding AETX K was elucidated by a combination of degenerate RT-PCR, 3'RACE and 5'RACE. The precursor protein of AETX K is composed of a signal peptide (22 residues), a propart (27 residues) ended with a pair of basic residues (Lys-Arg) and a mature peptide (34 residues). AETX K is the sixth member of the type 1 potassium channel toxins from sea anemones, showing especially high sequence identities with HmK from Heteractis magnifica and ShK from Stichodactyla helianthus. It has six Cys residues at the same position as the known type 1 toxins. In addition, the dyad comprising Lys and Tyr, which is considered to be essential for the binding of the known type 1 toxins to potassium channels, is also conserved in AETX K.