RESUMEN
Complex glycans are ubiquitous in nature and essential to life. Despite their diverse roles, however, only a fraction of their potential chemical space has been explored. New regions of this chemical space can, nevertheless, be accessed by generating structures that do not occur in nature or by modifying naturally-occurring polysaccharide structures - collectively, termed new polysaccharides (NPs). Two synthetic routes to NPs are described; the de novo route, directly from monosaccharide starting materials and the functionalization route, involving glycosylation of existing polysaccharides. The reaction involves a simple condensation step under microwave heating, catalysed by environmentally benign organic acids and is illustrated by the generation of structures with biological activities ranging from cell signalling and inhibition of bacterial growth, to mimicking carbohydrate antigens of pathogenic microorganisms. The method is as applicable to fine chemicals as it is to industrial waste, for example, biotechnologically-derived d-allulose (d-psicose), or the waste products of biofermentation. Accessing this chemical space unlocks new functionalities, generating complex glycans with applications in the biological, medical, biotechnological and materials science arenas.
RESUMEN
3-O-Sulfotransferase enzyme (sHS) from Litopenaeus vannamei was cloned and its substrate specificity was investigated against a number of GAG structures, including modified heparin polysaccharides and model oligosaccharides. For the heparin polysaccharides, derived from porcine intestinal mucosa heparin, sulfate groups were incorporated into glucosamine residues containing both N-sulfated and N-acetylated substitution within the regions of the predominant repeating disaccharide, either I-ANS or I-ANAc. However, the resulting polysaccharides did not stabilize antithrombin, which is correlated with anticoagulant activity. It was also shown that the enzyme was able to sulfate disaccharides, I2S-ANS and G-ANAc. The results further illustrate that 3-O-sulfation can be induced outside of the classical heparin-binding pentasaccharide sequence, show that 3-O-sulfation of glucosamine is not a sufficient condition for antithrombin stabilization and suggest that the use of this enzyme during HS biosynthesis may not occur as the final enzymatic step.
Asunto(s)
Heparitina Sulfato/biosíntesis , Sulfotransferasas/metabolismo , Animales , Estabilidad de Enzimas , Heparitina Sulfato/química , Modelos Moleculares , Penaeidae/enzimología , TemperaturaRESUMEN
The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100 mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.
Asunto(s)
Adhesión Bacteriana , Bacteroides fragilis/crecimiento & desarrollo , Proteínas Portadoras/metabolismo , Laminina/metabolismo , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteroides fragilis/metabolismo , Bacteroides fragilis/ultraestructura , Matriz Extracelular/metabolismo , Immunoblotting , Microscopía Electrónica de Transmisión , Oxidación-Reducción , Polisacáridos Bacterianos/metabolismo , Factores de TiempoRESUMEN
The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.