OLIGOMANNOSIDES OR OLIGOSACCHARIDE-LIPIDS AS POTENTIAL SUBSTRATES FORRAT-LIVER CYTOSOLIC ALPHA-D-MANNOSIDASE

We have previously reported the substrate specificity of the cytosolic alpha-D-mannosidase purified from rat liver using Man(9)GlcNAc, i.e. Man alpha 1-2Man alpha 1-3(Man alpha 1-2Man alpha 1-6)Man alpha 1-6(Ma n alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc, as substrate [Grard, Saint-Pol, Haeuw, Alonso, Wieruszeski, Strecker and Michalski (1994) Eur. J. Biochem. 223, 99-106]. Man(9)GlcNAc is hydrolysed givi ng Man(5)GlcNAc, i.e. Man alpha 1-2Man alpha 1-2Man alpha 1-3(Man alph a 1-6)Man beta 1-4GlcNAcAc, possessing the same structure as the oligo saccharide of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. We study here t he activity of the purified cytosolic alpha-D-mannosidase towards the oligosaccharide-diphosphodolichol intermediates formed during the bios ynthesis of N-glycans, and also towards soluble oligosaccharides relea sed from the endoplasmic reticulum which are glucosylated or not and p ossessing at their reducing end either a single N-acetylglucosamine re sidue or a di-N-acetylchitobiose sequence. We demonstrate that (1) dol ichol pyrophosphate oligosaccharide substrates are poorly hydrolysed b y the cytosolic alpha-D-mannosidase; (2) oligosaccharides with a termi nal reducing di-N-acetylchitobiose sequence are not hydrolysed at all; (3) soluble oligosaccharides bearing a single reducing N-acetylglucos amine are the real substrates for the enzyme. These results suggest a role for alpha-D-mannosidase in the catabolism of glycans released fro m the endoplasmic reticulum rather than in the regulation of the biosy nthesis of asparagine-linked oligosaccharides.