Hiraizumi S, Spohr U, Spiro R G
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02215.
J Biol Chem. 1993 May 5;268(13):9927-35.
Endo-alpha-D-mannosidase is a Golgi-located processing enzyme that achieves deglucosylation of N-linked carbohydrate units through its unique property of cleaving the oligosaccharide chain internally with the release of glucose-substituted mannose (Glc1-3Man). By chemically modifying the characteristic disaccharide product, Glc alpha 1-->3Man, a number of potent inhibitors of the endomannosidase were obtained, foremost among which were Glc alpha 1-->3(1-deoxy)mannojirimycin (Glc alpha 1-->3DMJ) and Glc alpha 1-->3(1,2-dideoxy)mannose (IC50 = 1.7 and 3.8 microM, respectively), which, while blocking the in vitro action of the enzyme, had negligible effect on other endoplasmic reticulum- and Golgi-processing glycosidases. Although preparation of a large number of Glc alpha 1-->3DMJ derivatives did not yield a more effective endomannosidase inhibitor it provided valuable information relating to the structural requirements for the enzyme-substrate interaction. Glc alpha 1-->3DMJ was found to be active not only on rat liver endomannosidase but also on the enzyme from a number of other sources including mouse lymphoma (BW5147.3), HepG2, baby hamster kidney, and Madin-Darby canine kidney cell lines. When tested in vivo in lymphoma and Madin-Darby canine kidney cells during a castanospermine-imposed glucosidase blockade, Glc alpha 1-->3DMJ interrupted the endomannosidase processing pathway as evident from a concomitant inhibition of complex oligosaccharide formation and Glc3Man release; similarly the capacity of the glucosidase II-deficient mouse lymphoma cell line (PHAR2.7) to synthesize complex oligosaccharides was blocked by Glc alpha 1-->3DMJ. Endomannosidase could not be detected in Chinese hamster ovary cells by in vitro assay and consistent with this these cells produced only glucosylated polymannose N-linked oligosaccharides during glucosidase blockade. It would appear that by acting in conjunction with a glucosidase inhibitor, Glc alpha 1-->3DMJ and related endomannosidase-blocking agents could have the potential of influencing the exit of glycoproteins from the endoplasmic reticulum and interfering with viral replication.
内切α-D-甘露糖苷酶是一种位于高尔基体的加工酶，它通过其独特的特性实现N-连接碳水化合物单元的去糖基化，即在内切寡糖链时释放葡萄糖取代的甘露糖（Glc1-3Man）。通过对特征性二糖产物Glcα1→3Man进行化学修饰，获得了许多强力的内切甘露糖苷酶抑制剂，其中最主要的是Glcα1→3(1-脱氧)甘露基野尻霉素（Glcα1→3DMJ）和Glcα1→3(1,2-二脱氧)甘露糖（IC50分别为1.7和3.8 microM），它们在阻断该酶的体外作用时，对其他内质网和高尔基体加工糖苷酶的影响可忽略不计。尽管制备大量的Glcα1→3DMJ衍生物并未产生更有效的内切甘露糖苷酶抑制剂，但它提供了有关酶-底物相互作用结构要求的有价值信息。发现Glcα1→3DMJ不仅对大鼠肝脏内切甘露糖苷酶有活性，而且对来自许多其他来源的酶也有活性，包括小鼠淋巴瘤（BW5147.3）、HepG2、幼仓鼠肾和Madin-Darby犬肾细胞系。当在栗精胺引起的糖苷酶阻断期间在淋巴瘤和Madin-Darby犬肾细胞中进行体内测试时，Glcα1→3DMJ中断了内切甘露糖苷酶加工途径，这从对复杂寡糖形成和Glc3Man释放的同时抑制中可以明显看出；同样，Glcα1→3DMJ阻断了缺乏葡糖苷酶II的小鼠淋巴瘤细胞系（PHAR2.7）合成复杂寡糖的能力。通过体外测定在中国仓鼠卵巢细胞中未检测到内切甘露糖苷酶，与此一致的是，这些细胞在糖苷酶阻断期间仅产生糖基化的多聚甘露糖N-连接寡糖。看来，通过与糖苷酶抑制剂协同作用，Glcα1→3DMJ和相关的内切甘露糖苷酶阻断剂可能有影响糖蛋白从内质网输出并干扰病毒复制的潜力。
