ABSTRACT
A new neurotoxin RTX-VI that modulates the voltage-gated sodium channels (NaV) was isolated from the ethanolic extract of the sea anemone Heteractis crispa. Its amino acid sequence was determined using the combination of Edman degradation and tandem mass spectrometry. RTX-VI turned out to be an unusual natural analogue of the previously described sea anemone toxin RTX-III. The RTX-VI molecule consists of two disulfide-linked peptide chains and is devoid of Arg13, which is important for the selectivity and affinity of such peptides for the NaV channels. Electrophysiological screening of RTV-VI on NaV channel subtypes showed its selective interaction with the central nervous system (NaV1.2, NaV1.6) and insect (BgNaV1, VdNaV1) sodium channels.
Subject(s)
Cnidarian Venoms/pharmacology , Insect Proteins/metabolism , NAV1.2 Voltage-Gated Sodium Channel/metabolism , NAV1.6 Voltage-Gated Sodium Channel/metabolism , Sea Anemones/chemistry , Amino Acid Sequence , Animals , Cnidarian Venoms/chemistry , Ion Channel Gating/drug effects , Sequence Homology , Structure-Activity RelationshipABSTRACT
The ion channel TRPV1, which is one of the most important integrators of pain and inflammatory stimuli, is considered a promising therapeutic target in the treatment of pain conditions. In this work, we performed a comparative study of the analgesic effect in the "hot plate" test of recombinant analogues of Kunitz-type peptides from the sea anemone Heteractis crispa venom: APHC1-modulator of TRPV1 and HCRG21-a full blocker of TRPV1. As a result of biological tests, it was shown that the full blocker HCRG21, despite the higher value of 50% effective concentration of TRPV1 inhibition, had an equal analgesic ability with the APHC1 upon intramuscular administration and retained it for 13 h of observation. The analgesic effect of APHC1 at a dose of 0.1 mg/kg when administered intramuscularly developed very quickly in 5 min but lasted 3 h. The differences in the pharmacodynamic profile of the peptides are in good agreement with different mechanisms of binding to TRPV1.
Subject(s)
Analgesics/pharmacology , Cnidarian Venoms/pharmacology , Pain/drug therapy , Peptides/pharmacology , TRPV Cation Channels/antagonists & inhibitors , Amino Acid Sequence , Analgesics/administration & dosage , Animals , Cnidarian Venoms/administration & dosage , Disease Models, Animal , Hot Temperature , Injections, Intramuscular , Mice , Mice, Inbred ICR , Pain/metabolism , Peptides/administration & dosage , Sea Anemones , Sequence HomologyABSTRACT
Recombinant analogue of the sea anemone Heteractismagnifica peptide was obtained, and the kinetic parameters of its interaction with mammalian α-amylases were determined. Magnificamide inhibits α-amylases significantly stronger than the medical drug acarbose (PrecoseTM or GlucobayTM). Magnificamide is assumed to find application as a drug for prevention and treatment of metabolic disorders and type 2 diabetes mellitus.
Subject(s)
Enzyme Inhibitors/pharmacology , Peptides/pharmacology , alpha-Amylases/antagonists & inhibitors , Amino Acid Sequence , Animals , Enzyme Inhibitors/chemistry , Peptides/chemistry , Sea Anemones/chemistrySubject(s)
Analgesics/pharmacology , Peptides/pharmacology , Sea Anemones , Amino Acid Sequence , Animals , Animals, Outbred Strains , Disease Models, Animal , Hot Temperature , Injections, Intraperitoneal , Mice , Molecular Sequence Data , Pain/drug therapy , Peptides/genetics , Recombinant Proteins/pharmacology , Sea Anemones/genetics , Sequence Homology, Amino AcidABSTRACT
The anti-inflammatory effect of the recombinant polypeptide HCGS 1.20, a Kunitz-type serine protease inhibitor of the sea anemone Heteractis crispa, was investigated. It was shown that the polypeptide inhibits the increase of the concentration of calcium ions in mouse bone marrow derived macrophages elicited by histamine, and reduces the content of NO in lipopolysaccharide stimulated macrophages. A presumable mechanism of anti-inflammatory action of the polypeptide was being discussed.
Subject(s)
Anti-Inflammatory Agents , Macrophages/metabolism , Peptides , Sea Anemones , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Cell Line , Histamine/metabolism , Lipopolysaccharides/pharmacology , Macrophages/pathology , Mice , Nitric Oxide/metabolism , Peptides/chemistry , Peptides/genetics , Peptides/pharmacology , Sea Anemones/chemistry , Sea Anemones/geneticsABSTRACT
A new actinoporin Hct-S4 (molecular mass 19,414 ± 10 Da) belonging to the sphingomyelin-inhibited α-pore forming toxin (α-PFT) family was isolated from the tropical sea anemone Heteractis crispa (also called Radianthus macrodactylus) and purified by methods of protein chemistry. The N-terminal nucleotide sequence (encoding 20 amino acid residues) of actinoporin Hct-S4 was determined. Genes encoding 18 new isoforms of H. crispa actinoporins were cloned and sequenced. These genes form a multigene Hct-S family characterized by presence of N-terminal serine in the mature proteins. Highly conserved residues comprising the aromatic phosphorylcholine-binding site and significant structure-function changes in the N-terminal segment (10-27 amino acid residues) of actinoporins were established. Two expressed recombinant actinoporins (rHct-S5 and rHct-S6) were one order less hemolytically active than native actinoporins.
Subject(s)
Cnidarian Venoms/chemistry , Cytotoxins/chemistry , Sea Anemones/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Cnidarian Venoms/genetics , Cnidarian Venoms/isolation & purification , Cytotoxins/genetics , Cytotoxins/isolation & purification , Molecular Sequence Data , Protein Isoforms/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sea Anemones/genetics , Sequence Alignment , Structure-Activity RelationshipABSTRACT
Two new serine proteinase inhibitors (RmIn I and RmIn II) from the tropical sea anemone Radianthus macrodactylus have been isolated and characterized. The purification procedure includes polychrome-1 hydrophobic chromatography, Superdex Peptide 10/30 FPLC, and Nucleosil C(18) reverse-phase HPLC. The molecular masses of RmIn I, RmIn II, and the complexes RmIn II/trypsin and RmIn I,II/alpha-chymotrypsin have been determined. The K(i) values of RmIn I and RmIn II for trypsin and alpha-chymotrypsin have been determined. The polypeptides RmIn I and RmIn II are shown to be nontoxic and to exhibit antihistamine activity. The N-terminal amino acid sequences of RmIn I (GICSEPIVVGPCKAG-) and RmIn II (GSTCLEPKVVGPCKA-) have been determined. A high homology of the amino acid sequences is demonstrated for the proteinase inhibitors produced by such evolutionarily distant species as coelenterates, reptiles, and mammals.
Subject(s)
Protease Inhibitors/chemistry , Protease Inhibitors/isolation & purification , Sea Anemones/chemistry , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Chymotrypsin/antagonists & inhibitors , Chymotrypsin/metabolism , Kinetics , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Protease Inhibitors/pharmacology , Protein Isoforms/chemistry , Protein Isoforms/isolation & purification , Protein Isoforms/pharmacology , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Trypsin/metabolismABSTRACT
The spatial organization of actinoporin RTX-SII from the sea anemone Radianthus macrodactylus on the level of tertiary and secondary structures was studied by UV and CD spectroscopy and intrinsic protein fluorescence. The specific and molar extinction coefficients of RTX-SII were determined. The percentages of canonical secondary structures of actinoporin were calculated. The tertiary structure of the polypeptide is well developed and its secondary structure is highly ordered and contains about 50% antiparallel folded beta-sheets. The irreversible thermal denaturation of RTX-SII was studied by CD spectroscopy; a conformational transition occurs at 53 degrees C. Above this temperature irreversible conformational changes are observed in the secondary and tertiary structures. This is accompanied by redistribution of the content of regular and distorted forms of beta-sheet and also by increase in the content of an unordered form. It is suggested that an intermediate is formed in the process of thermal denaturation. Acid-base titration of RTX-SII results in irreversible conformational changes at pH below 2.0 and above 12.0. As shown by intrinsic protein fluorescence, tyrosine residues of RTX-SII make a fundamental contribution to emission, and the total fluorescence depends more on temperature and ionic strength of the solution than tryptophan fluorescence. The data on conformational stability of actinoporin are correlated with data on its hemolytic activity. Activity of RTX-SII significantly decreases at increased temperature and slightly decreases at low pH. Hemolytic activity drastically increases at high pH. Increase in the actinoporin activity at pH above 10 seems to be caused by ionization of the molecule.
Subject(s)
Cnidarian Venoms/chemistry , Hemolysin Proteins/chemistry , Sea Anemones/chemistry , Animals , Circular Dichroism , Cnidarian Venoms/isolation & purification , Fluorescence , Hemolysin Proteins/isolation & purification , Hemolysis , Hydrogen-Ion Concentration , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Spectrophotometry, Ultraviolet , Temperature , Time FactorsABSTRACT
Differential scanning microcalorimetry, intrinsic protein fluorescence and SDS-electrophoresis have been applied for the study of the hemolytic toxin from sea anemone, Radianthus macrodactylus, when it is incorporated into pure sphingomyelin/water systems and upon its effect on the human and dog erythrocyte membranes. The results obtained by using these techniques showed that one molecule of toxin withdraws six sphingomyelin molecules from the cooperative transition. Effect of hemolytic toxin on human erythrocyte ghosts causes an appearance of a new heat sorption peak in their differential scanning calorimetric curve with a maximum of 36 degrees C and, moreover, it leads to a loss of one of cytoskeleton proteins (actin). These effects are essentially weaker in the case of dog erythrocyte ghosts. This suggests differences in the structural organization of human and dog erythrocyte membranes.
