A detailed structural characterization of ribonuclease B oligosaccharides by 1H NMR spectroscopy and mass spectrometry

Abstract

The structures of ribonuclease B oligosaccharides have previously been shown to be high mannose type by methylation analyses and sequential exoglycosidase digestion. Due to the unique nature of these oligosaccharides, in that all mannosyl residues are attached by α-(1 → 2)-linkages beyond the branch points, methylation analysis fails to solve the exact structures beyond Man5. Therefore, we have undertaken this study using 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. In this study, bovine pancreatic ribonuclease B was first reduced and carboxymethylated, and was then deglycosylated by peptide/ N-glycosidase F (PNGase F). The released oligosaccharides were fractionated by Bio-Gel P-4 chromatography to give five pools, Man5 through Man9. The structures of the oligosaccharide pools were then studied by laser desorption time of flight mass spectrometry and 1H NMR spectroscopy at 300 MHz. For Man5, Man-A and Man-B are attached in α-(1 → 3)- and α-(1 → 6)-linkages to the α-(1 → 6)-linked Man-4′ of the pentasaccharide core structure. For Man6, Man-C is linked α-(1 → 2) to the α-(1 → 3)-linked Man-4. Man7 exists as three structural isomers, and has the additional mannosyl residue (Man-D) linked α-(1 → 2) to Man-A, Man-B, and Man-C is linked α-(1 → 2) to the α-(1 → 3)-linked Man-4. Man-7 exists as three structural isomers, with the additional two mannosyl residues linked α-(1 → 2) to Man-A, Man-B, and Man-C. For each position, Man-A, Man-B, and Man-C, the extent of occupancy by one of the additional α-(→)-linked mannosyl residues was 15, 94, and 91%, respectively. Man9 is a single component, with the three additional mannosyl residues linked α-(1 → 2) to Man-A, Man-B, and Man-C, respectively. The relative molar proportions of Man5 to Man9 are 57, 31, 4, 7, and 1%, respectively. This report presents for the first time the complete structural characterization of the oligosaccharides from ribonuclease B.