Dairaku K, Spiro R G
Department of Biological Chemistry, Harvard Medical School, Boston, MA USA.
Glycobiology. 1997 Jun;7(4):579-86. doi: 10.1093/glycob/7.4.579.
Endo-alpha-D-mannosidase is a processing enzyme which in contrast to other glycosidases involved in the trimming of N-linked oligosaccharides of glycoproteins acts at an internal position by cleaving the linkage between the glucose-substituted mannose and the internal portion of the polymannose unit and thereby provides an alternate deglucosylating pathway. In order to evaluate at what stage in evolution this unusual enzyme first emerged, we have carried out a phylogenetic survey of its distribution among a broad group of eukaryotes ranging from unicellular organisms to highly developed animals and plants, all of which are known to have the capacity to N-glycosylate proteins and subsequently trim the nascent glucosylated polymannose oligosaccharides. It became evident from enzyme assays and in vivo studies that endomannosidase is limited in its distribution to members of the chordate phylum, including placental and marsupial mammals, birds, reptiles, amphibians, and fish, with the single except of the Mollusca in which it was detected in three distinct classes. The enzyme's absence in all other invertebrates examined as well as in yeast, various protozoa and higher plants, stands in contrast to glucosidase II and alpha 1,2-mannosidase which were found to be present in all eukaryotes studied. The observation that endomannosidase activity was not present in insects was confirmed by radiolabeling experiments with Sf9 cells in culture. These cells, which are widely employed for the expression of mammalian genes, were in distinction to mouse cells unable to circumvent a castanospermine (CST)-induced glucosidase blockade. Moreover we observed that Tetrahymenae, which synthesize glycoproteins with truncated N-linked oligosaccharides, could not process these beyond the Glc3Man5GlcNAc2 stage in the presence of CST. The late appearance of endomannosidase during evolution suggests a need for an alternate deglucosylation route in higher animals which parallels the development of elaborate complex N-linked oligosaccharides. Such carbohydrate units are believed to carry out vital biological functions and deglucosylation is a prerequisite to the further processing steps required for their formation.
内切α-D-甘露糖苷酶是一种加工酶，与参与糖蛋白N-连接寡糖修剪的其他糖苷酶不同，它通过切割葡萄糖取代的甘露糖与多聚甘露糖单元内部之间的连接，在内切位置起作用，从而提供了一条替代的去糖基化途径。为了评估这种不寻常的酶在进化的哪个阶段首次出现，我们对其在从单细胞生物到高度发达的动植物等广泛的真核生物群体中的分布进行了系统发育调查，所有这些生物都已知具有N-糖基化蛋白质并随后修剪新生的糖基化多聚甘露糖寡糖的能力。从酶分析和体内研究中可以明显看出，内切甘露糖苷酶的分布仅限于脊索动物门的成员，包括胎盘哺乳动物和有袋哺乳动物、鸟类、爬行动物、两栖动物和鱼类，唯一的例外是软体动物，在其中三个不同的类别中检测到了该酶。在所有其他检查的无脊椎动物以及酵母、各种原生动物和高等植物中均未发现该酶，这与在所有研究的真核生物中都存在的葡糖苷酶II和α1,2-甘露糖苷酶形成了对比。用培养的Sf9细胞进行的放射性标记实验证实了昆虫中不存在内切甘露糖苷酶活性。这些细胞被广泛用于表达哺乳动物基因，与小鼠细胞不同，它们无法绕过栗精胺（CST）诱导的葡糖苷酶阻断。此外，我们观察到，合成具有截短的N-连接寡糖的糖蛋白的四膜虫，在存在CST的情况下，无法将这些寡糖加工到Glc3Man5GlcNAc2阶段之后。内切甘露糖苷酶在进化过程中的较晚出现表明，高等动物需要一条替代的去糖基化途径，这与复杂的复杂N-连接寡糖的发展并行。据信这种碳水化合物单元具有重要的生物学功能，而去糖基化是其形成所需的进一步加工步骤的先决条件。
