Palamarczyk G, Mitchell M, Smith P W, Fleet G W, Elbein A D
Arch Biochem Biophys. 1985 Nov 15;243(1):35-45. doi: 10.1016/0003-9861(85)90771-4.
1,4-Dideoxy-1,4-imino-D-mannitol (DIM) was synthesized chemically from benzyl-alpha-D-mannopyranoside [Fleet et al (1984) J. Chem. Soc. Chem. Commun., 1240-1241], and was tested in vitro as an inhibitor of various alpha-mannosidases and in cell culture as an inhibitor of glycoprotein processing. DIM proved to be an effective inhibitor of jack bean alpha-mannosidase, with 50% inhibition requiring 25 to 50 ng/ml inhibitor. It also inhibited lysosomal alpha-mannosidase, but in this case 50% inhibition required about 1 to 2 micrograms/ml. In both cases, the inhibition was of the competitive type when p-nitrophenyl-alpha-D-mannopyranoside was used as the substrate. The inhibition was better at higher pH values, suggesting that DIM was more effective when the nitrogen in the ring was in the unprotonated form. In addition, rat liver processing mannosidase I was also inhibited by DIM as measured by the release of [3H]mannose from [3H]mannose-labeled Man9GlcNAc. Glycoprotein processing was examined in influenza virus-infected MDCK cells. Infected cells were incubated in various concentrations of DIM and labeled with [2-3H]mannose. Viral and cell pellets were digested with Pronase and glycopeptides were isolated by gel filtration on columns of Bio-Gel P-4. The glycopeptides were then treated with endoglucosaminidase H (Endo H) and rechromatographed on the Bio-Gel column in order to distinguish complex from high-mannose structures. As the DIM concentration in the medium was raised, more and more of the [3H]mannose was incorporated into high-mannose oligosaccharides, and less and less radioactivity was in the complex chains. Most of the Endo H-released oligosaccharides induced by DIM were of the Man9GlcNAc structure, as determined by gel filtration, HPLC, and digestion by alpha-mannosidase. Thus, DIM also appears to inhibit mannosidase I in cell culture. However, about 15% of the Endo H-released oligosaccharides appear to be hybrid types of oligosaccharides, suggesting that DIM may also inhibit mannosidase II.
1,4-二脱氧-1,4-亚氨基-D-甘露糖醇（DIM）由苄基-α-D-甘露吡喃糖苷通过化学方法合成[Fleet等人（1984年），《化学学会化学通讯》，1240 - 1241页]，并在体外作为各种α-甘露糖苷酶的抑制剂进行测试，在细胞培养中作为糖蛋白加工的抑制剂进行测试。DIM被证明是刀豆α-甘露糖苷酶的有效抑制剂，50%抑制率所需的抑制剂浓度为25至50纳克/毫升。它也抑制溶酶体α-甘露糖苷酶，但在这种情况下，50%抑制率所需的抑制剂浓度约为1至2微克/毫升。在这两种情况下，当对硝基苯基-α-D-甘露吡喃糖苷用作底物时，抑制作用均为竞争性类型。在较高pH值下抑制作用更好，这表明当环中的氮处于未质子化形式时，DIM更有效。此外，通过从[³H]甘露糖标记的Man9GlcNAc释放[³H]甘露糖来测定，大鼠肝脏加工甘露糖苷酶I也受到DIM的抑制。在感染流感病毒的MDCK细胞中研究了糖蛋白加工过程。将感染的细胞在不同浓度的DIM中孵育并用[² - ³H]甘露糖进行标记。用链霉蛋白酶消化病毒和细胞沉淀，并通过Bio - Gel P - 4柱上的凝胶过滤分离糖肽。然后用内切葡糖胺聚糖酶H（Endo H）处理糖肽，并在Bio - Gel柱上重新进行色谱分离，以区分复杂型和高甘露糖型结构。随着培养基中DIM浓度的升高，越来越多的[³H]甘露糖被掺入高甘露糖型寡糖中，而复杂链中的放射性越来越少。通过凝胶过滤、高效液相色谱和α-甘露糖苷酶消化测定，DIM诱导的大多数Endo H释放的寡糖为Man9GlcNAc结构。因此，DIM在细胞培养中似乎也抑制甘露糖苷酶I。然而，约15%的Endo H释放的寡糖似乎是寡糖的杂合类型，这表明DIM也可能抑制甘露糖苷酶II。
