RESUMO
The catalytic domains of murine Golgi alpha1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted proteins in P.pastoris . Recombinant mannosidases IA and IB both required divalent cations for activity, were inhibited by deoxymannojirimycin and kifunensine, and exhibited similar catalytic constants using Manalpha1,2Manalpha-O-CH3as substrate. Mannosidase IA was purified as a 50 kDa catalytically active soluble fragment and shown to be an inverting glycosidase. Recombinant mannosidases IA and IB were used to cleave Man9GlcNAc and the isomers produced were identified by high performance liquid chromatography and proton-nuclear magnetic resonance spectroscopy. Man9GlcNAc was rapidly cleaved by both enzymes to Man6GlcNAc, followed by a much slower conversion to Man5GlcNAc. The same isomers of Man7GlcNAc and Man6GlcNAc were produced by both enzymes but different isomers of Man8GlcNAc were formed. When Man8GlcNAc (Man8B isomer) was used as substrate, rapid conversion to Man5GlcNAc was observed, and the same oligosaccharide isomer intermediates were formed by both enzymes. These results combined with proton-nuclear magnetic resonance spectroscopy data demonstrate that it is the terminal alpha1, 2-mannose residue missing in the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate. When rat liver endoplasmic reticulum membrane extracts were incubated with Man9GlcNAc2, Man8GlcNAc2was the major product and Man8B was the major isomer. In contrast, rat liver Golgi membranes rapidly cleaved Man9GlcNAc2to Man6GlcNAc2and more slowly to Man5GlcNAc2. In this case all three isomers of Man8GlcNAc2were formed as intermediates, but a distinctive isomer, Man8A, was predominant. Antiserum to recombinant mannosidase IA immunoprecipitated an enzyme from Golgi extracts with the same specificity as recombinant mannosidase IA. These immunodepleted membranes were enriched in a Man9GlcNAc2to Man8GlcNAc2-cleaving activity forming predominantly the Man8B isomer. These results suggest that mannosidases IA and IB in Golgi membranes prefer the Man8B isomer generated by a complementary mannosidase that removes a single mannose from Man9GlcNAc2.
Assuntos
Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Isoenzimas/metabolismo , Manosidases/metabolismo , Animais , Sequência de Carboidratos , Hidrólise , Isoenzimas/genética , Fígado/enzimologia , Manosidases/genética , Camundongos , Dados de Sequência Molecular , Pichia/genética , Testes de Precipitina , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The complete primary structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5, an anaerobic bacterium implicated in food poisoning, was determined. The polysaccharide was isolated from C. perfringens Hobbs 5 cells, after deproteination, by ethanol precipitation and by ion-exchange chromatography. The polysaccharide was comprised of glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, and glucuronic acid, in equimolar ratios. Sequence and linkage assignments of the glycosyl residues were obtained by NMR spectroscopy, specifically by the combination of two-dimensional homonuclear TOCSY and NOESY experiments and heteronuclear (1H, 13C) multiple-quantum coherence (HMQC, HMQC-COSY, HMQC-TOCSY and HMBC) experiments. Thus, the envelope polysaccharide of C. perfringens Hobbs 5 was found to be a polymer composed of a hexasaccharide repeating unit with the following structure: [formula: see text] This structure is novel among bacterial cell-surface polysaccharides, and it is the first of many serotypically distinct capsular polysaccharides of C. perfringens to be described.