| Abstract
| - Normal human fibroblasts and fibroblasts from a patient with α-mannosidosis were grown in the presence or absence of 100 μM swainsonine for 7 days. Accumulated oligosaccharides were isolated and analysed by high performance liquid chromatography (HPLC) and methylation analysis. Manα1→3Manβ1→4GlcNAc and Manα1→2Manα1→3Manβ1→4GlcNAc (where Man is D-mannose and GlcNAc is N-acetyl-D-glucosamine) comprised >80% of the total oligosaccharides in untreated mannosidosis cells. However, Manα1→6[Manα1→3]Manβ1→4GlcNAc was the major Man3 GlcNAc 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. Manα1→6[Manα1→3] Manα1→6[Manα1→3]-Manβ1→4GlcNac and Manα1→6-[Manα1→3]Manβ1→4GlcNac were the major components (67%). Surprisingly, Manα1→3Manβ1→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 α-mannosidase activity which is unaffected in genetic α-mannosidosis, but is inhibited by swainsonine. This enzyme would cleave the α(1→6)-linked mannose residue from branched Man3 GlcNAc to form Manα1→3Manβ1β4GlcNAc. To confirm this hypothesis, fractions from α-mannosidosis and control fibroblasts that bound to concanavalin A (ConA)-Sepharose and were eluted with 0.5 M α-methyl mannoside were incubated at pH 4.0 with Manα1→6[Manα1→3]Manβ1→4-GlcNac. As anticipated,Manαl→3Manβ1→4GlcNac was the sole product using enzyme from mannosidosis fibroblasts, while the major product from control fibroblasts was Manαl→6Manβ1-→4GlcNAc. This confirmed the presence of a swainsonine-inhibitable α(1→6)-mannosidase activity unaffected by the disease. The differing substrate specificities of the α(1→6)-mannosidase and the major lysosomal α-mannosidase indicate that the α(1→6)-mannosidase plays an important role in the generation of the oligosaccharides accumulated in α-mannosidosis patients.
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