Oligomannosides or oligosaccharide-lipids as potential substrates for rat liver cytosolic alpha-D-mannosidase.
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.
Grard
T
T
Laboratoire de Chimie Biologique (Unité mixte du Centre National de la Recherche Scientifique no, 111), Université des Sciences et Technologies de Lille, Villieneuve d'Ascq, France.
Herman
V
V
Saint-Pol
A
A
Kmiecik
D
D
Labiau
O
O
Mir
A M
AM
Alonso
C
C
Verbert
A
A
Cacan
R
R
Michalski
J C
JC
D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008361:Mannosidases / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D043323:alpha-Mannosidase
D009844:Oligosaccharides; D011104:Polyisoprenyl Phosphate Oligosaccharides; D011994:Recombinant Proteins; C024962:dolichyl diphosphate oligosaccharides; C028569:oligomannoside; D008361:Mannosidases; D043323:alpha-Mannosidase