A HUMAN LYSOSOMAL ALPHA(1-]6)-MANNOSIDASE ACTIVE ON THE BRANCHED TRIMANNOSYL CORE OF COMPLEX GLYCANS

Normal human fibroblasts and fibroblasts from a patient with alpha-mannosidosis were grown in the presence or absence of 100-mu-M swainsonine for 7 days. Accumulated oligosaccharides were isolated and analysed by high performance liquid chromatography (HPLC) and methylation analysis. Man-alpha-1 --> 3Man-beta-1 --> 4GlcNAc and Man-alpha-1 --> 2Man-alpha-1 --> 3Man-beta-1 --> 4GlcNAc (where Man is D-mannose and GlcNAc is N-acetyl-D-glucosamine) comprised > 80% of the total oligosaccharides in untreated mannosidosis cells. However, Man-alpha-1 --> 6[Man-alpha-1 --> 3]Man-beta-1 --> 4GlcNAc was the major Man3GlcNAc isomer present after 7 days of swainsonine treatment. No mannose-containing oligosaccharides were detected in control fibroblasts in the absence of swainsonine but, in its presence, oligosaccharides containing 2-9 mannose residues accumulated. Man-alpha-1 --> 6[Man-alpha-1 --> 3]Man-alpha-1 --> 6[Man-alpha-1 --> 3]Man-beta-1 --> 4GlcNAc and Man-alpha-1--> 6[Man-alpha-1 --> 3]Man-beta-1 --> 4GlcNAc were the major components (67%). Surprisingly, Man-alpha-1 --> 3Man-beta-1 --> 4GlcNAc was only observed in swainsonine-treated control cells during the recovery period after removal of swainsonine. These studies suggest the presence of a second lysosomal alpha-mannosidase activity which is unaffected in genetic alpha-mannosidosis, but is inhibited by swainsonine. This enzyme would cleave the alpha(1 --> 6)-linked mannose residue from branched Man3GlcNAc to form Man-alpha-1 --> 3Man-beta-1 --> 4GlcNAc. To confirm this hypothesis, fractions from ce-mannosidosis and control fibroblasts that bound to concanavalin A (ConA)-Sepharose and were eluted with 0.5 M alpha-methyl mannoside were incubated at pH 4.0 with Man-alpha-1 --> 6[Man-alpha-1 --> 3]Man-beta-1 --> 4GlcNAc. As anticipated, Man-alpha-1 --> 3Man-beta-1 --> 4GlcNAc was the sole product using enzyme from mannosidosis fibroblasts, while the major product from control fibroblasts was Man-alpha-1 --> 6Man-beta-1 --> 4GlcNAc. This confirmed the presence of a swainsonine-inhibitable alpha(1 --> 6)-mannosidase activity unaffected by the disease. The differing substrate specificities of the alpha(1 --> 6)-mannosidase and the major lysosomal alpha-mannosidase indicate that the alpha(1 --> 6)-mannosidase plays an important role in the generation of the oligosaccharides accumulated in alpha-mannosidosis patients.