RESUMO
Low molecular weight fragments of sulfated galactans (Boc-5 and Boc-10) from the red algae Botryocladia occidentalis significantly inhibited Crotalus durissus cascavella sPLA2 enzymatic activity. Equimolar ratios of sPLA2 to Boc-5 or Boc-10 resulted in allosteric inhibition of sPLA2. Under the conditions tested, we observed that both Boc-5 and Boc-10 strongly decreased edema, myonecrosis, and neurotoxicity induced by native sPLA2.
Assuntos
Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/farmacologia , Edema/tratamento farmacológico , Galactanos/química , Galactanos/farmacologia , Mastócitos/efeitos dos fármacos , Fosfolipases A2 Secretórias , Rodófitas/química , Pele/efeitos dos fármacos , Animais , Venenos de Crotalídeos/química , Crotalus/metabolismo , Interações Medicamentosas , Edema/induzido quimicamente , Galactanos/isolamento & purificação , Cinética , Masculino , Fosfolipases A2 Secretórias/química , Fosfolipases A2 Secretórias/metabolismo , Fosfolipases A2 Secretórias/toxicidade , Ratos , Ratos Wistar , Pele/patologia , Relação Estrutura-Atividade , Sulfatos/químicaRESUMO
AIMS: The initial colonization of the tooth by streptococci involves their attachment to adsorbed components of the acquired pellicle. Avoiding this adhesion may be successful in preventing caries at early stages. Salivary mucins are glycoproteins that when absorbed onto hydroxyapatite may provide binding sites for certain bacteria. Algal lectins may be especially interesting for oral antiadhesion trials because of their great stability and high specificity for mucins. This work aimed to evaluate the potential of two algal lectins to inhibit the adherence of five streptococci species to the acquired pellicle in vitro. METHODS AND RESULTS: The lectins used were extracted from Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL). Fluorescence microscopy was applied to visualize the ability of fluorescein isothiocyanate-labelled lectins to attach to the pellicle and revealed a similar capability for both lectins. Streptococcal adherence assays were performed using saliva-coated microtitre plates. BSL inhibited more than 75% of Streptococcus sanguis, Streptococcus mitis, Streptococcus sobrinus and Streptococcus mutans adherence, achieving 92% to the latter. BTL only obtained statistically significant results on S. mitis and S. sobrinus, whose adherence was decreased by 32.5% and 54.4%, respectively. CONCLUSION: Algal lectins are able to inhibit streptococcal adherence. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support the proposed application of lectins in antiadhesion therapeutics.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Película Dentária/microbiologia , Lectinas/farmacologia , Streptococcus/efeitos dos fármacos , Adsorção , Biofilmes/crescimento & desenvolvimento , Durapatita/metabolismo , Eucariotos/química , Humanos , Saliva/metabolismo , Streptococcus/classificação , Streptococcus/crescimento & desenvolvimento , Streptococcus/fisiologiaRESUMO
The biochemical characterization of a new lectin (Hypnea cervicornis agglutinin or HCA) isolated from the Brazilian red alga H. cervicornis is reported. The haemagglutinating activity of the lectin was only inhibited by the glycoprotein porcine stomach mucin at a minimum inhibitory concentration of 19 microg x mL(-1). No haemagglutination inhibition was detected after the addition of simple sugars. The MALDI-TOF molecular masses of native and reduced and carbamidomethylated HCA were, respectively, 9196.6 Da and 9988.2 Da, indicating that the primary structure of the protein is crosslinked by 7 disulfide bonds. This unusual structural feature among lectins, along with its N-terminal sequence and amino-acid composition, clearly shows that HCA belongs to a protein family distinct from the isolectins Hypnin A1 and A2 isolated from the related Japanese alga Hypnea japonica. On the other hand, HCA displayed a high degree of similarity to the agglutinin from the Brazilian species Hypnea musciformis. Our data indicate the occurrence of structural diversity among lectins of closely related species living in distant ecosystems, i.e., the Pacific coast of Japan and the Atlantic coast of Brazil, and support the hypothesis that the lectin content (lectinome) might serve as a biomarker for taxonomical purposes.
Assuntos
Aglutininas/química , Aglutininas/isolamento & purificação , Rodófitas/química , Sequência de Aminoácidos , Aminoácidos , Animais , Cromatografia por Troca Iônica , Hemaglutinação , Testes de Hemaglutinação , Dados de Sequência MolecularRESUMO
A lectin from the red marine alga Hypnea musciformis (HML) was purified by extraction with 20 mM PBS, precipitation with 70% saturated ammonium sulphate, ion-exchange DEAE-Cellulose chromatography and RP-HPLC. The 9.3 kDa polypeptide agglutinates erythrocytes from various sources and shows oligomerization tendencies under certain MALDI-TOF/MS conditions. Preliminary N-terminal sequencing and biological assays strongly suggest that the HML may belong to a new class of algae lectins.
Assuntos
Lectinas/química , Lectinas/isolamento & purificação , Rodófitas/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Dimerização , Humanos , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Sulfated D-galactans occur on the red algae Botryocladia occidentalis as three fractions that differ in their sulfate content. Fractions F2 and F3 are potent anticoagulants. Like heparin, they enhance thrombin and factor Xa inhibition by antithrombin and/or heparin cofactor II. The inhibition potency increases simultaneously with the sulfate content of the fractions. The antithrombotic activity of these sulfated D-galactans was investigated on an experimental thrombosis model in which thrombus formation was induced by a combination of stasis and hypercoagulability. In contrast with heparin. the sulfated D-galactans showed a dual dose-response curve preventing thrombosis at doses up to approximately 0.5 mg/ kg body weight but losing the effect at higher doses. This unexpected behavior is probably due to a combined action of the sulfated D-galactan as anticoagulant and also as a strong inducer of platelet aggregation. In platelet-depleted animals the antithrombotic activity at higher dose of sulfated D-galactan is restored and almost total inhibition of thrombus formation is achieved. The sulfated D-galactan has no hemorrhagic effect even at high doses, possibly as a consequence of its effect on platelet aggregation. At comparable dose heparin has an intense bleeding effect. These results indicate that new polysaccharides, with well-defined structures, can help to distinguish events, such as antithrombotic and anticoagulant activities, bleeding and platelet-aggregating effects, which are obscure when induced simultaneously by a single compound.
Assuntos
Anticoagulantes/uso terapêutico , Fibrinolíticos/uso terapêutico , Galactanos/uso terapêutico , Fitoterapia , Extratos Vegetais/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Rodófitas/química , Trombose Venosa/prevenção & controle , Animais , Anticoagulantes/isolamento & purificação , Anticoagulantes/farmacologia , Anticoagulantes/toxicidade , Brasil , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Fibrinolíticos/toxicidade , Galactanos/química , Galactanos/isolamento & purificação , Galactanos/farmacologia , Hemorragia/induzido quimicamente , Heparina/farmacologia , Heparina/uso terapêutico , Heparina/toxicidade , Masculino , Tempo de Tromboplastina Parcial , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Ratos , Ratos Wistar , Sulfatos/análise , Tromboplastina/toxicidade , Veia Cava Inferior , Trombose Venosa/tratamento farmacológicoRESUMO
We have characterized the structure of a sulfated d-galactan from the red algae Botryocladia occidentalis. The following repeating structure (-4-alpha-d-Galp-1-->3-beta-d-Galp-1-->) was found for this polysaccharide, but with a variable sulfation pattern. Clearly one-third of the total alpha-units are 2,3-di-O-sulfated and another one-third are 2-O-sulfated. The algal sulfated d-galactan has a potent anticoagulant activity (similar potency as unfractionated heparin) due to enhanced inhibition of thrombin and factor Xa by antithrombin and/or heparin cofactor II. We also extended the experiments to several sulfated polysaccharides from marine invertebrates with simple structures, composed of a single repeating structure. A 2-O- or 3-O-sulfated l-galactan (as well as a 2-O-sulfated l-fucan) has a weak anticoagulant action when compared with the potent action of the algal sulfated d-galactan. Possibly, the addition of two sulfate esters to a single alpha-galactose residue has an "amplifying effect" on the anticoagulant action, which cannot be totally ascribed to the increased charge density of the polymer. These results indicate that the wide diversity of polysaccharides from marine alga and invertebrates is a useful tool to elucidate structure/anticoagulant activity relationships.