title: Conformational studies of the Man8 oligosaccharide on native ribonuclease B and on the reduced and denatured protein.
abstract: Site-specific presentation of oligosaccharides in the context of carrier proteins can influence markedly their recognition by carbohydrate-binding proteins. On RNaseB, the Man5-9 N-glycans at Asn-34 are bound by the serum lectin conglutinin when the glycoprotein is reduced and denatured, but there is no binding to the N-glycans on the native form of RNaseB. The RNaseB Man8, which is a glycoform preferentially bound by conglutinin, is the subject of the present study. The conformational behavior of the protein-linked oligosaccharide Man8 is investigated on the native and on the reduced and denatured RNaseB, using a combination of NMR and theoretical calculations. Quantitative data on the NOESY crosspeaks have been obtained, thereby allowing the comparison of mobilities of homologous linkages within the glycan chain. Oligosaccharide conformations compatible with the NMR data have been explored by molecular modeling of the free oligosaccharide, using two different force fields (AMBER and SYBYL). There are some differences between the results produced by the two force fields, the AMBER simulations providing a better agreement with the experimental data. The results indicate that both on the native and on the reduced heat-denatured glycoprotein, the RNase Man8 oligosaccharide exhibits a conformational behavior very similar to that of the free oligosaccharide. However, this conformational freedom of the N-glcyan does not amount to full availability for carbohydrate-recognition proteins and enzymes.
authors.affiliation: Instituto de Química Orgánica General, CSIC, Madrid, Spain.
authors.forename: L
authors.initials: L
authors.lastname: González
authors.forename: M
authors.initials: M
authors.lastname: Bruix
authors.forename: T
authors.initials: T
authors.lastname: Díaz-Mauriño
authors.forename: T
authors.initials: T
authors.lastname: Feizi
authors.forename: M
authors.initials: M
authors.lastname: Rico
authors.forename: D
authors.initials: D
authors.lastname: Solís
authors.forename: J
authors.initials: J
authors.lastname: Jiménez-Barbero
mesh_terms: D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D012260:Ribonucleases / Q000737:chemistry*
chemical_list: D009844:Oligosaccharides; D012260:Ribonucleases; C029600:ribonuclease B