In vitro hydrolysis of oligomannosyl oligosaccharides by the lysosomal alpha-D-mannosidases.
In vitro incubation of the oligomannosyl oligosaccharides Man9GlcNAc and Man5GlcNAc with isolated disrupted lysosomes yields different oligosaccharide isomers resulting from mannosidase hydrolysis. These isomers were isolated by HPLC and characterized by 1H-NMR spectroscopy. The first steps of the degradation involve an (alpha 1-2)mannosidase activity and lead to the formation of one Man8GlcNAc, one Man7GlcNAc, two Man6GlcNAc and two Man5GlcNAc isomers. These reactions do not require Zn2+ as activator. On the other hand, the following steps, which lead to the formation of Man3GlcNAc and Man2GlcNAc, are Zn2(+)-dependent. This process is characterized by the preferential action of an (alpha 1-3)mannosidase activity, and the formation of Man(alpha 1-6)Man(alpha 1-6)Man(beta 1-4)GlcNAc and Man(alpha 1-6)Man(beta 1-4)GlcNAc. Therefore, the digestion of Man9GlcNAc inside the lysosome appears to follow a very specific pathway, since only nine intermediate compounds can be identified instead of the 38 possible isomers. Our results are consistent both with the existence of several specific enzymes for alpha 1-2, alpha 1-3 and alpha 1-6 linkages, and with the presence of a unique enzyme whose specificity would be dependent either on Zn2+ or on the spatial conformation of the glycan. Nevertheless, previous work on the structural analysis of oligosaccharides excreted in the urine of patients suffering from mannosidosis, demonstrates the absence of the core alpha 1-6-linked mannosyl residue in the major storage product derived from oligomannosyl oligosaccharides. This observation indicates the presence of a specific (alpha 1-6)mannosidase form, unaffected in mannosidosis.
Michalski
J C
JC
Laboratoire de Chimie Biologique, Université des Sciences et Techniques de Lille Flandres-Artois, France.
Haeuw
J F
JF
Wieruszeski
J M
JM
Montreuil
J
J
Strecker
G
G
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002458:Cell Fractionation; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology* / Q000648:ultrastructure; D008361:Mannosidases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D009844:Oligosaccharides; D008361:Mannosidases