Purification and biochemica characterisation of endoplasmic reticulum á 1-2-mannosidase from Sporothrix schenckiil

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, á1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one á1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound á-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-á1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This á1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised á1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi á1,2-mannosidases and therefore, the processing of N-glycans by á1,2-mannosidases is similar to that present in lower eukaryotes.

Heterologous expression and biochemical characterization of an alpha 1, 2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

Identificação de princípios ativos presentes na Ipomoea carnea brasileira / Identification of Brazilian Ipomoea carnea toxic compounds

Dentre as espécies pertencentes à família das Convolvulceae destacam-se as Ipomoeas, amplamente distribuídas por todo o mundo, bastante conhecidas e cultivadas devido ao aspecto ornamental que suas flores campanuladas e de cores vibrantes oferecem. É sabido porém que espécies de Ipomoeas são tóxicas. A Ipomoea carnea, espécie de nosso estudo, provoca emagrecimento, apatia, incoordenção motora, fraqueza progressiva e até mesmo a morte em animais de produção, se ingerida por período prolongado. Os alcalóides suainsonina e calisteginas presentes nesta planta são certamente responsáveis por tais efeitos tóxicos, já que inibem a ação das manosidases e glicosidases, enzimas fundamentais para um adequado metabolismo de carboidratos pelo organismo...

Alfa-manosidasa: su acción sobre antitrombina III, fibrinógeno y plasminógeno / Alfa-manosidase: its action on antithrombin III, fibrinogen and plasminogen

La alfa-manosiadasa (alfa m) es una hidrolasa ácida que se encuentra en los gránulos azurófilos de los leucocitos polimorfonucleares y en menor concentración en los linfocitos. En leucemias agudas no linfoides sus niveles se hallan muy aumentados respecto de los controles normales (p < 0,001). En estos pacientes, se encuentran alteraciones funcionales e inmunológicas de algunas glicoproteínas del sistema plasmático de coagulación y de fibrinólisis, tales como la antitrombina III (AT III), el fibrinógeno o factor I de coagulación (I) y el plasminógeno (Plg). Debido a esto, investigamos la acción de alfa m sobre estas proteínas purificadas, a partir de plasma humano normal, utilizando métodos inmunoelectroforéticos y electroforesis en gel de poliacrilamida con SDS. Se hallan modificaciones importantes en AT III y I y menores en Plg, similares a las obtenidas en los plasmas de los pacientes. Luego, parece que la acción de hidrolasas ácidas es posible in vivo bajo ciertas condiciones patológicas