Oligomannosides or oligosaccharide-lipids as potential substrates for rat liver cytosolic alpha-D-mannosidase

• Published in: Biochemical journal 1996-06, Vol.316 ( Pt 3) (3), p.787-792
• Main Authors: Grard, T, Herman, V, Saint-Pol, A, Kmiecik, D, Labiau, O, Mir, A M, Alonso, C, Verbert, A, Cacan, R, Michalski, J C
• Format: Article
• Language: English
• Subjects: alpha-Mannosidase; Animals; Biochemistry; Biochemistry, Molecular Biology; Carbohydrate Conformation; Carbohydrate Sequence; CHO Cells; Chromatography, High Pressure Liquid; Cricetinae; Cytosol - enzymology; Endoplasmic Reticulum - metabolism; Life Sciences; Liver - enzymology; Mannosidases - metabolism; Models, Biological; Molecular Sequence Data; Oligosaccharides - chemical synthesis; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Polyisoprenyl Phosphate Oligosaccharides - metabolism; Rats; Recombinant Proteins - metabolism; Substrate Specificity; Transfection
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3160787

We have previously reported the substrate specificity of the cytosolic alpha-D-mannosidase purified from rat liver using Man9GlcNAc, i.e. Man alpha 1-2Man alpha 1-3(Man alpha 1-2Man alpha 1-6)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3) Man beta 1-4G1cNAc, as substrate [Grard, Saint-Pol, Haeuw, Alonso, Wieruszeski, Strecker and Michalski (1994) Eur. J. Biochem. 223, 99-106]. Man9 G1cNAc is hydrolysed giving Man5GlcNAc, i.e. Man alpha 1-2 Man alpha 1-2Man alpha 1-3(Man alpha 1-6)Man beta 1-4GlcNAc, possessing the same structure as the oligosaccharide of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. We study here the activity of the purified cytosolic alpha-D-mannosidase towards the oligosaccharide-diphosphodolichol intermediates formed during the biosynthesis of N-glycans, and also towards soluble oligosaccharides released from the endoplasmic reticulum which are glucosylated or not and possessing at their reducing end either a single N-acetylglucosamine residue or a di-N-acetylchitobiose sequence. We demonstrate that (1) dolichol pyrophosphate oligosaccharide substrates are poorly hydrolysed by the cytosolic alpha-D-mannosidase; (2) oligosaccharides with a terminal reducing di-N-acetylchitobiose sequence are not hydrolysed at all; (3) soluble oligosaccharides bearing a single reducing N-acetylglucosamine are the real substrates for the enzyme. These results suggest a role for alpha-D-mannosidase in the catabolism of glycans released from the endoplasmic reticulum rather than in the regulation of the biosynthesis of asparagine-linked oligosaccharides.