Peptide fingerprinting of the neurotoxic fractions isolated from the secretions of sea anemones Stichodactyla helianthus and Bunodosoma granulifera. New members of the APETx-like family identified by a 454 pyrosequencing approach

Abstract

Sea anemones are known to contain a wide diversity of biologically active peptides, mostly unexplored according to recent peptidomic and transcriptomic studies. In the present work, the neurotoxic fractions from the exudates of Stichodactyla helianthus and Bunodosoma granulifera were analyzed by reversed-phase chromatography and mass spectrometry. The first peptide fingerprints of these sea anemones were assessed, revealing the largest number of peptide components (156) so far found in sea anemone species, as well as the richer peptide diversity of B. granulifera in relation to S. helianthus. The transcriptomic analysis of B. granulifera, performed by massive cDNA sequencing with 454 pyrosequencing approach allowed the discovery of five new APETx-like peptides (U-AITX-Bg1a-e - including the full sequences of their precursors for four of them), which together with type 1 sea anemone sodium channel toxins constitute a very distinguishable feature of studied sea anemone species belonging to genus Bunodosoma. The molecular modeling of these new APETx-like peptides showed a distribution of positively charged and aromatic residues in putative contact surfaces as observed in other animal toxins. On the other hand, they also showed variable electrostatic potentials, thus suggesting a docking onto their targeted channels in different spatial orientations. Moreover several crab paralyzing toxins (other than U-AITX-Bg1a-e), which induce a variety of symptoms in crabs, were isolated. Some of them presumably belong to new classes of crab-paralyzing peptide toxins, especially those with molecular masses below 2kDa, which represent the smallest peptide toxins found in sea anemones.

Estudio bioquímico del veneno de la anémona de mar Phymactis papillosa (Actiniidae)

“…En algunos trabajos realizados con Stichodactyla helianthus, Bunodosoma granulifera y Phymanthus crucifer, en donde también se utilizó el shock hipotónico para obtener el veneno, se ha encontrado que cada ejemplar expele, respectivamente, 20,3, 39 y 89 mg de proteína (Rodríguez et al 2012a, Rodríguez et al 2012b. Esta amplia diferencia en la cantidad de proteína obtenida, podría deberse a particularidades de cada especie de anémona de mar, al estado nutricional o al método de cuantificación utilizado.…”

“…Por estas características el Sephadex G-50 es ampliamente utilizado para separar, preliminarmente, los componentes de los venenos de anemonas de mar, tal como se ha reportado en los casos de Bunodosoma caissarum, Actinia equina, Anthopleura xanthogrammica, Anthopleura fuscoviridis, Urticina crassicornis, Phymanthus crucifer, Bunodosoma granulifera y Stichodactyla helianthus (Malpezzi et al 1993, Minagawa et al 2008, Razpotnik et al 2009, Rodríguez et al 2012a, Rodríguez et al 2012b). Sin embargo, la purificación total de cualquier componente de estos venenos, normalmente requiere del uso adicional de otras técnicas cromatográficas como la cromatografía de intercambio iónico, la cromatografía de afinidad y la cromatografía líquida de alta presión (HPLC).…”

Combining multidimensional liquid chromatography and MALDI–TOF-MS for the fingerprint analysis of secreted peptides from the unexplored sea anemone species Phymanthus crucifer

“…However, the application of these approaches to other venomous animals such as sea anemones still remains limited, even though these organisms are well known sources of peptide toxins [4][5][6][7][8][9][10][11] (ion channel toxins, protease inhibitors, peptide cytolysins) and protein toxins [12,13] (protein cytolysins and phospholipases A2), of pharmacological and therapeutic interest. The applications of genomic/transcriptomic approaches [14][15][16][17] to the study of sea anemone toxins have been accelerated since 2006, from major studies of Nematostella vectensis [18,19] and Anemonia viridis [20], and very recently from Bunodosoma granulifera [17] using for the first time the high-throughput 454 pyrosequencing technology in the study of sea anemones. Besides the genomic/transcriptomic analyses, a very limited number of peptidomic studies focused on sea anemones toxins has been performed, in Bunodosoma cangicum [21], B. granulifera and Stichodactyla helianthus [17].…”

“…The applications of genomic/transcriptomic approaches [14][15][16][17] to the study of sea anemone toxins have been accelerated since 2006, from major studies of Nematostella vectensis [18,19] and Anemonia viridis [20], and very recently from Bunodosoma granulifera [17] using for the first time the high-throughput 454 pyrosequencing technology in the study of sea anemones. Besides the genomic/transcriptomic analyses, a very limited number of peptidomic studies focused on sea anemones toxins has been performed, in Bunodosoma cangicum [21], B. granulifera and Stichodactyla helianthus [17].…”

“…As mentioned above, few peptidomic studies of sea anemones venoms have been reported [17,21], comprising profiling and structural characterization of novel peptides by Edman sequencing and transcriptomic analyses. The separation and detection strategy employed in these studies allowed finding a large number of peptides; however several factors may have led to an underestimation of this overall peptide complexity contained in sea anemone secretions.…”