Daniel P F, Evans J E, De Gasperi R, Winchester B, Warren C D
Department of Neurology, Massachusetts General Hospital, Charlestown 02129.
Glycobiology. 1992 Aug;2(4):327-36. doi: 10.1093/glycob/2.4.327.
Normal human fibroblasts and fibroblasts from a patient with alpha-mannosidosis were grown in the presence or absence of 100 microM 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----3-Man beta 1----4GlcNAc (where Man is D-mannose and GlcNAc is N-acetyl-D-glucosamine) comprised greater than 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 alpha-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----4-GlcNAc. 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.
将正常人成纤维细胞和来自一名α-甘露糖苷贮积症患者的成纤维细胞，在有或无100微摩尔苦马豆素的情况下培养7天。分离出积累的寡糖，并通过高效液相色谱法（HPLC）和甲基化分析进行分析。Manα1----3Manβ1----4GlcNAc和Manα1----2Manα1----3-Manβ1----4GlcNAc（其中Man为D-甘露糖，GlcNAc为N-乙酰-D-葡糖胺）占未处理的甘露糖苷贮积症细胞中总寡糖的80%以上。然而，Manα1----6[Manα1----3]Manβ1----4GlcNAc是苦马豆素处理7天后存在的主要Man3GlcNAc异构体。在没有苦马豆素的情况下，对照成纤维细胞中未检测到含甘露糖的寡糖，但在有苦马豆素的情况下，积累了含有2-9个甘露糖残基的寡糖。Manα1----6[Manα1----3]-Manα1----6[Manα1----3]Manβ1----4GlcNAc和Manα1----6-[Manα1----3]Manβ1----4GlcNAc是主要成分（67%）。令人惊讶的是，仅在去除苦马豆素后的恢复期，在经苦马豆素处理的对照细胞中观察到Manα1----3Manβ1----4GlcNAc。这些研究表明存在第二种溶酶体α-甘露糖苷酶活性，其在遗传性α-甘露糖苷贮积症中不受影响，但被苦马豆素抑制。这种酶会从分支的Man3GlcNAc上切割α(1----6)-连接的甘露糖残基，形成Manα1----3Manβ1----4GlcNAc。为了证实这一假设，将与伴刀豆球蛋白A（ConA）-琼脂糖结合并用0.5Mα-甲基甘露糖苷洗脱的α-甘露糖苷贮积症和对照成纤维细胞的组分，在pH 4.0下与Manα1----6[Manα1----3]Manβ----4-GlcNAc一起孵育。正如预期的那样，使用来自甘露糖苷贮积症成纤维细胞的酶时，Manα1----3Manβ1----4GlcNAc是唯一产物，而来自对照成纤维细胞的主要产物是Manα1----6Manβ1----4GlcNAc。这证实了存在一种不受疾病影响但被苦马豆素抑制的α(1----6)-甘露糖苷酶活性。α(1----6)-甘露糖苷酶和主要溶酶体α-甘露糖苷酶不同的底物特异性表明，α(1----6)-甘露糖苷酶在α-甘露糖苷贮积症患者积累的寡糖生成中起重要作用。
