Structures of Oligomannoside Chains of α-Mannosidase from Porcine Kidney1

Abstract

Lysosomal acid α-mannosidase from porcine kidney was found to contain mannose (4.8%), galactose (0.9%), fucose (0.5%), N-acetylglucosamine (3.1%), and mannose 6-phosphate (0.1%). Approximately 50% of the total hexose of the oligosaccharide chains could be released by endo-β-N-acetylglucosaminidase-H (endo-H). They were predominantly neutral, oligomannoside-type oligosaccharides containing 5, 6, and 9 mannose residues, respectively, in the centesimal ratio of 36:25:34. 500-MHz ¹H-NMR spectroscopy in conjunction with sequential exoglycosidase digestion of the reduced compounds revealed that each of the three fractions consisted of a single isomer only; the Man₉ compound has the following structure: The Man₆ lacks Man residues D₁ D₂ and D₃ while the Man₅-compound lacks Man-C as well. In addition to the neutral ones, some (5%) phosphorylated oligomannoside-type oligosaccharides were obtained. The endo-H resistant glycopeptides were subjected to hydrazinolysis. Approximately 60% of the oligosaccharides released by hydrazine were found to be of rather small size; their composition can be represented as Man_2–3GlcNAc[Fuc]_0–1GlcNAcol. The remaining 40% consist of larger-size, galactose-containing, N-acetyllactosamine-type oligosaccharides. Studies involving sequential exoglycosidase digestion and 500-MHz ²H-NMR spectroscopy performed on the highly purified small-sized compounds revealed the following four structures for the endo-H-resistant oligosaccharides: