RESUMEN
A d-galacturonic acid-specific lectin, named AcL, was purified from the sea hare Aplysia californica by galactose-agarose affinity chromatography. AcL has a molecular mass of 27.5 kDa determined by MALDI-TOF mass spectrometry. This lectin shows a good affinity for d-galacturonic acid and a lower affinity for galactosides: raffinose, melibiose, α and ß-lactose, and d-galactose. We determined the amino acid sequence of AcL by trypsin digestion and subsequent peptide analysis by mass spectrometry, resulting in a 238 amino acid protein with a theoretical molecular mass of 26.4 kDa. The difference between the theoretical and experimental values can be attributed to post-translational modifications. Thiol-disulfide quantification discerned five disulfide bonds and three free cysteines. The structure of Acl is mainly comprised of beta sheets, determined by circular dichroism, and predicted with AlphaFold. Theoretical models depict three nearly identical tandem domains consisting of two beta sheets each. From docking analysis, we identified AcL glycan-binding sites as multiple conserved motifs in each domain. Furthermore, phylogenetic analysis based on its structure and sequence showed that AcL and its closest homologues (GalULs) form a clear monophyletic group, distinct from other glycan-binding proteins with a jelly-roll fold: lectins of types F and H. GalULs possess four conserved sequence regions that distinguish them and are either ligand-binding motifs or stabilizing network hubs. We suggest that this new family should be referred to as GalUL or D-type, following the traditional naming of lectins; D standing for depilans, the epithet for the species (Aplysia depilans) from which a lectin of this family was first isolated and described.
Asunto(s)
Aplysia , Liebres , Animales , Aplysia/química , Aplysia/metabolismo , Liebres/metabolismo , Galectinas/química , Filogenia , Galactosa/metabolismo , Polisacáridos/metabolismoRESUMEN
A new lectin from Aplysia dactylomela eggs (ADEL) was isolated by affinity chromatography on HCl-activated Sepharose™ media. Hemagglutination caused by ADEL was inhibited by several galactosides, mainly galacturonic acid (Ka = 6.05 × 106 M-1). The primary structure of ADEL consists of 217 residues, including 11 half-cystines involved in five intrachain and one interchain disulfide bond, resulting in a molecular mass of 57,228 ± 2 Da, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. ADEL showed high similarity with lectins isolated from Aplysia eggs, but not with other known lectins, indicating that these lectins could be grouped into a new family of animal lectins. Three glycosylation sites were found in its polypeptide backbone. Data from peptide-N-glycosidase F digestion and MS suggest that all oligosaccharides attached to ADEL are high in mannose. The secondary structure of ADEL is predominantly ß-sheet, and its tertiary structure is sensitive to the presence of ligands, as observed by CD. A 3D structure model of ADEL was created and shows two domains connected by a short loop. Domain A is composed of a flat three-stranded and a curved five-stranded ß-sheet, while domain B presents a flat three-stranded and a curved four-stranded ß-sheet. Molecular docking revealed favorable binding energies for interactions between lectin and galacturonic acid, lactose, galactosamine, and galactose. Moreover, ADEL was able to agglutinate and inhibit biofilm formation of Staphylococcus aureus, suggesting that this lectin may be a potential alternative to conventional use of antimicrobial agents in the treatment of infections caused by Staphylococcal biofilms.
Asunto(s)
Antibacterianos/química , Aplysia/química , Biopelículas/efectos de los fármacos , Lectinas/química , Staphylococcus aureus/efectos de los fármacos , Cigoto/química , Secuencia de Aminoácidos , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/metabolismo , Antibacterianos/farmacología , Aplysia/genética , Aplysia/metabolismo , Biopelículas/crecimiento & desarrollo , Escherichia coli/genética , Escherichia coli/metabolismo , Galactósidos/farmacología , Expresión Génica , Pruebas de Inhibición de Hemaglutinación , Ácidos Hexurónicos/farmacología , Lectinas/genética , Lectinas/aislamiento & purificación , Lectinas/farmacología , Simulación del Acoplamiento Molecular , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
This study reports the comparative molecular modeling, docking and dynamic simulations of human alpha9alpha10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and alpha-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary alpha10(-) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal alpha9(+), and Asp114 in the complementary alpha10(-) subunit, might confer the potency and selectivity to alpha-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.
Asunto(s)
Modelos Moleculares , Receptores Nicotínicos/química , Acetilcolina/química , Aminoácidos , Animales , Aplysia/química , Sitios de Unión , Simulación por Computador , Conotoxinas/química , Humanos , Lymnaea/química , Nicotina/química , Unión ProteicaRESUMEN
The first, total synthesis of (+)-brasilenyne (1) has been achieved in 19 steps from l-(S)-malic acid. The key elements of this approach are a highly diastereoselective ring-opening of a 1,3-dioxolanone with bis(trimethylsilyl)acetylene) promoted by TiCl4 to set a propargylic stereocenter and the successful application of the sequential ring closing metathesis/silicon-assisted intramolecular cross-coupling reaction for construction of the oxonin core structure of 1.
Asunto(s)
Éteres Cíclicos/síntesis química , Silicio/química , Animales , Aplysia/química , EstereoisomerismoRESUMEN
Trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana, negatively modulates vagal response, indicating a probable ability to inhibit cholinergic responses. In the present study, the pharmacological profile of trimethylsulfonium was characterized on muscarinic and nicotinic acetylcholine receptors. In rat jejunum the contractile response induced by trimethylsulfonium (pD2 = 2.46 +/- 0.12 and maximal response = 2.14 +/- 0.32 g) was not antagonized competitively by atropine. The maximal response (Emax) to trimethylsulfonium was diminished in the presence of increasing doses of atropine (P<0.05), suggesting that trimethylsulfonium-induced contraction was not related to muscarinic stimulation, but might be caused by acetylcholine release due to presynaptic stimulation. Trimethylsulfonium displaced [3H]-quinuclidinyl benzilate from rat cortex membranes with a low affinity (Ki = 0.5 mM). Furthermore, it caused contraction of frog rectus abdominis muscles (pD2 = 2.70 +/- 0.06 and Emax = 4.16 +/- 0.9 g), which was competitively antagonized by d-tubocurarine (1, 3 or 10 microM) with a pA2 of 5.79, suggesting a positive interaction with nicotinic receptors. In fact, trimethylsulfonium displaced [3H]-nicotine from rat diaphragm muscle membranes with a Ki of 27.1 microM. These results suggest that trimethylsulfonium acts as an agonist on nicotinic receptors, and thus contracts frog skeletal rectus abdominis muscle and rat jejunum smooth muscle via stimulation of postjunctional and neuronal prejunctional nicotinic cholinoreceptors, respectively.
Asunto(s)
Aplysia/química , Colinérgicos/farmacología , Agonistas Nicotínicos/farmacología , Receptores Muscarínicos/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Compuestos de Sulfonio/farmacología , Animales , Atropina/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Wistar , Compuestos de Sulfonio/antagonistas & inhibidoresRESUMEN
The structures and absolute stereochemistries of two chamigrene-type metabolites (spiro[5.5]undecane derivatives) isolated from the red algae Laurencia scoparia are described. One, a non-sesquiterpene named mailione (8-bromo-9-hydroxy-7,7-dimethyl-11-methylenespiro[5.5]undec-1-en-3-one), C(14)H(19)BrO(2), was detected previously in Laurencia cartilaginea, while the other, the sesquiterpene isorigidol (8-bromo-3,7,7-trimethyl-11-methylenespiro[5.5]-undec-1-ene-3,9-diol), C(15)H(23)BrO(2), is a new isomer of rigidol, first isolated from Laurencia rigida. The A rings of these spirocyclic compounds show the same carbon skeleton. However, the relative stereochemistry of the 8-Br and 9-OH substituents is different. While mailione displays the usual syn (or cis) relative stereochemistry of the bromohydroxy vicinal group, isorigidol shows an anti (or trans) arrangement. The 8-Br and 9-OH groups are both in equatorial positions in isorigidol, while the 9-OH group is axial in mailione, as in most chamigrenes. The absolute configurations of the chiral centers were determined as 6S, 8S and 9R in mailione, and 3R, 6S, 8S and 9S in isorigidol.