Weng S, Spiro R G
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts, USA.
Arch Biochem Biophys. 1996 Jan 1;325(1):113-23. doi: 10.1006/abbi.1996.0014.
Studies were undertaken to evaluate the relationship of the recently described (S. Weng and R. G. Spiro, 1993, J. Biol. chem. 268, 25656-25663) rat liver kifunensine (KIF)-resistant mannosidase (ER mannosidase II) to the mannose-trimming enzyme of cytosol. We observed that the ER mannosidase II manifests a large number of catalytic and immunological properties similar to those of the cytosolic alpha-mannosidase, which contrast with the quite different characteristics of the KIF-sensitive enzyme (ER mannosidase I). In addition to a mutual resistance to KIF inhibition, the cytosolic enzyme and ER mannosidase II have comparable susceptibility to blocking by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, and the latter agent was found to function effectively both in vitro and in vivo. The cytosolic and ER II mannosidases were alike in specifically excising the terminal mannose of the alpha 1,6-linked chain of Man9GlcNAc to yield Man8GlcNAc isomer C; in preferentially hydrolyzing polymannose-GlcNAc1 over polymannose-GlcNAc2 substrates; and in cleaving p-nitrophenyl alpha-D-mannoside. An immunological cross-reactivity between cytosolic mannosidase (M(r) 105 kDa) and ER mannosidase II (M(r) 82 kDa), neither of which is N-glycosylated, was established, suggesting that the latter is translocated posttranslationally into the lumen of the ER compartment in which we found it to be present as a soluble protein. Since antibodies directed against a sequence near the C-terminal end of the cytosolic enzyme reacted with ER mannosidase II while those against a sequence close to the N-terminus did not, it is likely that a proteolytic cleavage of the latter segment takes place during or after translocation. The absence in ER mannosidase II of the pronounced cobalt activation of the cytosolic enzyme suggests that the portion of the polypeptide chain removed during the 105- to 82-kDa conversion includes the binding domain for this ion.
开展了多项研究，以评估最近描述的（翁世伟和R.G.斯皮罗，1993年，《生物化学杂志》268卷，25656 - 25663页）大鼠肝脏抗 kifunensine（KIF）甘露糖苷酶（内质网甘露糖苷酶II）与胞质溶胶中甘露糖修剪酶的关系。我们观察到，内质网甘露糖苷酶II表现出大量与胞质α - 甘露糖苷酶相似的催化和免疫特性，这与对KIF敏感的酶（内质网甘露糖苷酶I）的截然不同的特性形成对比。除了对KIF抑制具有相互抗性外，胞质酶和内质网甘露糖苷酶II对苦马豆素和1,4 - 二脱氧 - 1,4 - 亚氨基 - D - 甘露糖醇的阻断具有相当的敏感性，并且发现后者在体外和体内均有效发挥作用。胞质和内质网II型甘露糖苷酶在特异性切除Man9GlcNAc的α1,6 - 连接链的末端甘露糖以产生Man8GlcNAc异构体C方面相似；在优先水解多聚甘露糖 - GlcNAc1而非多聚甘露糖 - GlcNAc2底物方面相似；以及在切割对硝基苯基α - D - 甘露糖苷方面相似。在未进行N - 糖基化的胞质甘露糖苷酶（分子量105 kDa）和内质网甘露糖苷酶II（分子量82 kDa）之间建立了免疫交叉反应性，这表明后者在翻译后转运到内质网腔中，我们发现它以可溶性蛋白的形式存在于内质网腔中。由于针对胞质酶C末端附近序列的抗体与内质网甘露糖苷酶II发生反应，而针对靠近N末端序列的抗体则不发生反应，因此很可能在转运期间或之后发生了后者片段的蛋白水解切割。内质网甘露糖苷酶II中不存在胞质酶明显的钴激活作用，这表明在105 kDa至82 kDa转化过程中去除的多肽链部分包括该离子的结合结构域。
