Detection of Man₅GlcNAc and Related Free Oligomannosides in the Cytosol Fraction of Hen Oviduct

Abstract

The presence of free oligomannosides in cytosol has been demonstrated by metabolic radiolabeling, and Manα1-2Manα1-2Manα1-3(Manα1-6)Manβ1-4GlcNAc was detected as the main free oligomannoside in Chinese hamster ovary cells [Kmiécik, D., Herman, V., Stroop, C. J. M., Michalski, J. C., Mir, A. M., Labiau, O., Verbert, A., and Cacan, R. (1995) Glycobiology, 5, 483-494]. In the present paper, the isomeric structures and amounts of oligomannosides in the cytosol fraction of hen oviduct were analyzed by pyridylamination and exoglycosidase digestion. Hen oviduct was used since our group has already characterized the cytosolic neutral α-mannosidase and endo-β-N-acetylglucosaminidase obtained from the same source. The amounts of Man₂GlcNAc, Man₃GlcNAc, Man₄GlcNAc, and Man₅GlcNAc were 0.6, 0.6, 0.5, and 0.8 nmol/g tissue, respectively, but Man₆GlcNAc-Man₉GlcNAc were not detected. The isomeric structures of the Man₃GlcNAc-Man₅GlcNAc found were (Manα1-2)_0-2Manα1-3(Manα1-6)Manβ1-4GlcNAc, which were compatible with the substrate specificities of cytosolic endo-β-N-acetylglucosaminidase and neutral α-mannosidase, indicating that these enzymes participate in the formation of the oligomannosides present in the cytosol.