A short framework-III (mini-M-2) conotoxin from the venom of a vermivorous species, Conus archon, inhibits human neuronal nicotinic acetylcholine receptors.

The venoms of Conus snails contain neuroactive peptides named conotoxins (CTXs). Some CTXs are nicotinic acetylcholine receptor (nAChRs) antagonists. nAChRs modulate the release of neurotransmitters and are implicated in several pathophysiologies. One venom peptide from Conus archon, a vermivorous species from the Mexican Pacific, was purified by RP-HPLC and its activity on human α7, α3ß2, and α7ß2 nAChRs was assessed by the two-electrode voltage clamp technique. At 36.3 µM the purified peptide (F27-1, renamed tentatively ArchIIIA) slowly reversibly inhibited the ACh-induced response of the hα7 subtype by 44.52 ± 5.83%, while it had low or no significant effect on the response of the hα3ß2 and hα7ß2 subtypes; the EC50 of the inhibiting effect was 45.7 µM on the hα7 subtype. This peptide has 15 amino acid residues and a monoisotopic mass of 1654.6 Da (CCSALCSRYHCLPCC), with three disulfide bridges and a free C-terminus. This sequence with a CC-C-C-CC arrangement (framework III) belongs to the M superfamily of conotoxins, corresponding to the mini-M´s (M-1-M-3) conotoxins; due to its size and inter-Cys spacings it is an M-2 conotoxin. This toxin is a novel mini-M conotoxin affecting ligand-gated ion channels, like the maxi-M CTX ψ-conotoxins and α-MIIIJ conotoxin (nAChRs blockers). This peptide seems to be homologous to the reg3b conotoxin (from Conus regius) with an identity of 93.3%, differing only in the third residue in the sequence, serine for threonine, both uncharged polar residues. We obtained, in silico, a probable 3D structure, which is consistent with its effect on neuronal subtypes.

A turripeptide from Polystira nobilis venom inhibits human α3ß2 and α7 nicotinic acetylcholine receptors.

Almost all marine snails within superfamily Conoidea produce venoms containing numerous neuroactive peptides. Most toxins characterized from members of this superfamily are produced by species belonging to family Conidae. These toxins (conotoxins) affect diverse membrane proteins, such as voltage- and ligand-gated ion channels, including nicotinic acetylcholine receptors (nAChRs). Family Turridae has been considerably less studied than their Conidae counterpart and, therefore, turrid toxins (turritoxins) have just been barely described. Consequently, in this work the most prominent chromatographic (RP-HPLC) fractions from the East Pacific species Polystira nobilis venom duct extract were isolated. The biological activity of six selected fractions was assayed on human (h) α7 AChRs expressed in Xenopus laevis oocytes. One of these fractions, F21, inhibited the acetylcholine-elicited response by 62 ± 12%. Therefore, this fraction was further purified and the F21-2 peptide was obtained. This peptide (at 5.6 µM) strongly and irreversibly inhibited the acetylcholine-induced response on hα7 and hα3ß2 nAChRs, by 55 ± 4 and 91 ± 1%, respectively. Electrospray mass spectrometry indicates that the average molecular mass of this toxin is 12 358.80 Da. The affinity for hα3ß2 nAChRs is high (IC50 of 566.2 nM). A partial sequence without cysteines was obtained by automated Edman degradation: WFRSFKSYYGHHGSVYRPNEPNFRSFAS…; blastp search revealed that this sequence has low similarity to some non-Cys-containing turripeptides. This is the first report of a turritoxin from a species of the American Pacific and the second description of a turripeptide inhibiting nAChRs.

Effect of pterois volitans (lionfish) venom on cholinergic and dopaminergic systems.

Pterois volitans venom induces muscular fibrillation, which results from nerve transmission caused by the presence of acetylcholine (ACh). It also has cardiovascular effects that are due to its actions on muscarinic and nicotinic cholinergic receptors. In this study, we characterized the effects of P. volitans venom on nicotinic acetylcholine receptors (nAChRs) and dopaminergic neurons. After exposure to P. volitans venom, acetylcholinesterase (AChE) mRNA levels and the expression of the α2 subunit of nAChR increased in zebrafish embryos (15-20 somites). In addition, the lionfish venom blocked zebrafish α2 nAChR subunit functional expression and the ACh-induced response of human neuronal α3ß2 receptors. The latter receptor was blocked by a protein fraction named F2, which was isolated from P. volitans venom using reversed phase high performance liquid chromatography (RP-HPLC). This venom causes death in dopaminergic neurons, and affects the cholinergic system. The effect of these two systems may result in retarded embryonic development of zebrafish, since the two systems function in a related manner to control growth hormone secretion.

αD-Conotoxins in Species of the Eastern Pacific: The Case of Conus princeps from Mexico.

Conus snails produce venoms containing numerous peptides such as the α-conotoxins (α-CTXs), which are well-known nicotinic acetylcholine receptor (nAChR) antagonists. Thirty-eight chromatographic fractions from Conus princeps venom extract were isolated by RP-HPLC. The biological activities of 37 fractions (0.07 µg/µL) were assayed by two-electrode voltage clamp on human α7 nAChRs expressed in Xenopus laevis oocytes. Fractions F7 and F16 notably inhibited the response elicited by acetylcholine by 52.7 ± 15.2% and 59.6 ± 2.5%, respectively. Fraction F7 was purified, and an active peptide (F7-3) was isolated. Using a combination of Edman degradation, mass spectrometry, and RNASeq, we determined the sequence of peptide F7-3: AVKKTCIRSTOGSNWGRCCLTKMCHTLCCARSDCTCVYRSGKGHGCSCTS, with one hydroxyproline (O) and a free C-terminus. The average mass of this peptide, 10,735.54 Da, indicates that it is a homodimer of identical subunits, with 10 disulfide bonds in total. This peptide is clearly similar to αD-CTXs from species of the Indo-Pacific. Therefore, we called it αD-PiXXA. This toxin slowly and reversibly inhibited the ACh-induced response of the hα7 nAChR subtype, with an IC50 of 6.2 µM, and it does not affect the hα3ß2 subtype at 6.5 µM.

Actividad enzimática de proteasas de Cyprinus carpio (Cypriniformes: Cyprinidae) extraídas de una laguna contaminada en México / Enzymatic activity of proteases from Cyprinus carpio (Cypriniformes: Cyprinidae) captured in a polluted lagoon in Mexico

ResumenLa carpa común (Cyprinus carpio) es un organismo acuático de valor comercial que puede sobrevivir en ambientes contaminados; la carpa contiene enzimas proteolíticas de importancia fisiológica y potencial aplicación industrial. El objetivo de este estudio fue purificar parcialmente y determinar la actividad proteolítica a diferente pH de proteasas de carpas colonizando un ambiente contaminado. Se capturaron tres carpas en diferentes zonas de la laguna contaminada de Zumpango (México) a 1 m de profundidad máxima. El extracto crudo se obtuvo del músculo dorsal mediante extracción acuosa y se fraccionó con (NH4)2SO4 saturado a 20 %, 50 % y 80 %. Posteriormente, se seleccionaron las fracciones obtenidas con (NH4)2SO4 saturado a 50 % y 80 % por su alta actividad proteolítica, se concentraron por ultrafiltración con membranas de corte de peso molecular de 100 kDa, y se analizaron por electroforesis en geles de poliacrilamida con dodecil sulfato de sodio (SDS-PAGE). La actividad proteolítica del extracto crudo fue significativamente mayor (19.7-20.3 U / mg) a pH 2, 5, y 7 (P < 0.001). Las fracciones obtenidas con (NH4)2SO4 saturado a 20 %, 50 % y 80 % presentaron actividades proteolíticas óptimas a pH 5 (2.8 U / mg) y pH 6 (2.2 U / mg); pH 6 (4.3 U / mg) y pH 3-4 (3.6 - 3.7 U / mg); pH 3 (10.8 U / mg) y pH 10 (10.6 U / mg); respectivamente. Las subfracciones con peso molecular < 100 kDa obtenidas con (NH4)2SO4 saturado a 50 % y 80 % tuvieron máxima actividad proteolítica a pH alcalino. La subfracción < 100 kDa, obtenida con (NH4)2SO4 saturado a 80 % tuvo la mayor actividad proteolítica (37.3-43.7 U / mg) a pH 8-10, factor de purificación de 3 y 19.1 % de recuperación. Trece proteínas entre 9.8 a 104.8 kDa se identificaron en el extracto proteolítico crudo. Las proteínas de 31 - 33 y 39 - 41 kDa tuvieron la concentración más alta en las fracciones estudiadas, sugiriendo la posible predominancia de serín y aspartil proteasas, respectivamente. Nosotros sugerimos que la presencia de proteasas con máxima actividad a pH alcalino está relacionada con la adaptación de C. carpio a aguas contaminadas con pH alto. Aunque estos peces son inadecuados para el consumo humano, pueden ser empleados como materia prima para la producción de proteasas destinadas para varias industrias, incluido el tratamiento de aguas residuales.

AbstractCommon carp (Cyprinus carpio) is an aquatic organism of commercial value able to survive in polluted environments; carps contain proteolytic enzymes of physiological importance and potential industrial application. The objective of this work was partially purify and study the proteolytic activity at different pH of carp proteases living in a polluted environment. Three carps were captured in different zones of Zumpango polluted lagoon (Mexico) at 1 m of maximum deep. Protease crude extracts were obtained from dorsal muscle by aqueous extraction and fractionated by 20 %, 50 %, 80 %-saturated (NH4)2SO4. Fractions extracted with 50 % and 80 %-saturated (NH4)2SO4 were selected for their high proteolytic activity and concentrated by ultrafiltration through 100 kDa molecular weight cutoff membranes and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The crude proteolytic extract had significantly higher activity (19.7 - 20.3 U / mg) at pH 2, 5, and 7 (P < 0.001). Fractions obtained with 20 %, 50 % and 80 % - saturated (NH4)2SO4 showed peak activity at pH 5 (2.8 U / mg) and pH 6 (2.2 U / mg); pH 6 (4.3 U / mg) and pH 3 - 4 (3.6 - 3.7 U / mg); pH 3 (10.8 U / mg) and pH 10 (10.6 U / mg); respectively. Subfractions of < 100 kDa, obtained with 50 % and 80 %-saturated (NH4)2SO4, had peak proteolytic activity at alkaline pH. A < 100 kDa fraction, obtained with 80 %-saturated (NH4)2SO4, had the highest proteolytic activity (37.3 - 43.7 U / mg) at pH 8 - 10, purification factor of 3 and 19.1 % recovery. Thirteen proteins between 9.8 to 104.8 kDa were identified in the crude extract. Peak protein concentration was observed for 31 - 33 and 39 - 41 kDa, suggesting the possibility predominance of serine- and aspartyl- proteases, respectively. We suggest this protease with maximum activity at alkaline pH is related to the adaptation of C. carpio to polluted waters with high pH. Although unsuitable for human consumption, these organisms can be a source of protease production aimed to several uses as in the industry and waste water treatment among others.

Release of Antioxidant Peptides from the Body Wall Proteins of the Sea Cucumber Isostichopus fuscus.

Proteases from the tentacles of the sea cucumber Isostichopusfuscus were partially purified and used to produce antioxidant peptides from body wall proteins of this marine species. Three proteins (105, 68, and 39 kDa) were identified by SDS-PAGE in the proteolytic extract of the tentacles. Protein hydrolyzates were generated with gelatin and crude protein substrates from body wall, and peptidic fractions lower and higher than 3 kDa were obtained to evaluate their oxygen radical scavenging capacity (ORAC). The 3 kDa-fraction obtained from the crude protein hydrolyzate showed the highest ORAC value (0.92 +/- 0.04 micromol Trolox equivalent/mg protein). This fraction was selected to purify peptides potentially responsible for the activity that might be used as ingredients for development of functional foods.