Lubas W A, Spiro R G
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02215.
J Biol Chem. 1988 Mar 15;263(8):3990-8.
Golgi membranes from rat liver have been shown to contain an endo-alpha-D-mannosidase which can convert Glc1Man9GlcNAc to Man8GlcNAc with the release of Glc alpha 1----3Man (Lubas, W. A., and Spiro, R. G. (1987) J. Biol. Chem. 262, 3775-3781). We now report that this enzyme has the capacity to cleave the alpha 1----2 linkage between the glucose-substituted mannose residue and the remainder of the polymannose branch in a wide range of oligosaccharides (Glc3Man9GlcNAc to Glc1Man4GlcNAc) as well as glycopeptides and oligosaccharide-lipids. Whereas the tri- and diglucosylated species (Glc3Man9GlcNAc and Glc2Man9GlcNAc), which yielded Glc3Man and Glc2Man, respectively, were processed more slowly than Glc1Man9GlcNAc, the monoglucosylated components with truncated mannose chains (Glc1Man8GlcNAc to Glc1Man4GlcNAc) were trimmed at an increased rate which was inversely related to the number of mannose residues present. The endomannosidase was not inhibited by a number of agents which are known to interfere with N-linked oligosaccharide processing by exoglycosidases, including 1-deoxynojirimycin, castanospermine, bromoconduritol, 1-deoxymannojirimycin, swainsonine, and EDTA. However, Tris and other buffers containing primary hydroxyl groups substantially decreased its activity. After Triton solubilization, the endomannosidase was observed to be bound to immobilized wheat germ agglutinin, indicating the presence of a type of carbohydrate unit consistent with Golgi localization of the enzyme. The Man8GlcNAc isomer produced by endomannosidase action was found to be processed by Golgi enzymes through a different sequence of intermediates than the rough endoplasmic reticulum-generated Man8GlcNAc variant, in which the terminal mannose of the middle branch is absent. Whereas the latter oligosaccharide is converted to Man5GlcNAc via Man7GlcNAc and Man6GlcNAc at an even rate, the processing of the endomannosidase-derived Man8GlcNAc stalls at the Man6GlcNAc stage due to the apparent resistance to Golgi mannosidase I of the alpha 1,2-linked mannose of the middle branch. The results of our study suggest that the Golgi endomannosidase takes part in a processing route for N-linked oligosaccharides which have retained glucose beyond the rough endoplasmic reticulum; the distinctive nature of this pathway may influence the ultimate structure of the resulting carbohydrate units.
已证明大鼠肝脏的高尔基体膜含有一种内切α-D-甘露糖苷酶，该酶可将Glc1Man9GlcNAc转化为Man8GlcNAc，并释放出Glcα1----3Man（卢巴斯，W. A.，和斯皮罗，R. G.（1987年）《生物化学杂志》262，3775 - 3781）。我们现在报告，这种酶能够切割多种寡糖（Glc3Man9GlcNAc至Glc1Man4GlcNAc）以及糖肽和寡糖脂中葡萄糖取代的甘露糖残基与多聚甘露糖分支其余部分之间的α1----2键。三葡糖基化和二葡糖基化的物种（Glc3Man9GlcNAc和Glc2Man9GlcNAc）分别产生Glc3Man和Glc2Man，其加工速度比Glc1Man9GlcNAc慢，而具有截短甘露糖链的单葡糖基化成分（Glc1Man8GlcNAc至Glc1Man4GlcNAc）的修剪速度加快，且与存在的甘露糖残基数量成反比。多种已知会干扰外切糖苷酶对N-连接寡糖加工的试剂，包括1-脱氧野尻霉素、栗精胺、溴代昆布醇、1-脱氧甘露野尻霉素、苦马豆素和乙二胺四乙酸，均未抑制该内切甘露糖苷酶。然而，含有伯羟基的Tris和其他缓冲液会大幅降低其活性。经Triton增溶后，观察到该内切甘露糖苷酶与固定化的麦胚凝集素结合，表明存在一种与该酶的高尔基体定位一致的碳水化合物单元类型。发现由内切甘露糖苷酶作用产生的Man8GlcNAc异构体经高尔基体酶加工的中间产物序列与糙面内质网产生的Man8GlcNAc变体不同，后者中间分支的末端甘露糖缺失。虽然后者寡糖以均匀的速度通过Man7GlcNAc和Man6GlcNAc转化为Man5GlcNAc，但由于中间分支α1,2-连接的甘露糖对高尔基体甘露糖苷酶I具有明显抗性，内切甘露糖苷酶衍生出的Man8GlcNAc的加工在Man6GlcNAc阶段停滞。我们的研究结果表明，高尔基体内切甘露糖苷酶参与了N-连接寡糖的加工途径，这些寡糖在糙面内质网之后仍保留葡萄糖；该途径的独特性质可能会影响最终产生的碳水化合物单元的结构。
