Bischoff J, Liscum L, Kornfeld R
J Biol Chem. 1986 Apr 5;261(10):4766-74.
The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.
甘露糖类似物1-脱氧甘露基野尻霉素可抑制高尔基体α-甘露糖苷酶I，但不抑制内质网（ER）α-甘露糖苷酶，已被用于确定内质网α-甘露糖苷酶在完整细胞中糖蛋白上天冬酰胺连接寡糖加工过程中的作用。在没有抑制剂的情况下，UT-1细胞中内质网糖蛋白3-羟基-3-甲基戊二酰辅酶A还原酶上发现的主要寡糖结构是Man6GlcNAc和Man8GlcNAc的单一异构体。在存在150μM 1-脱氧甘露基野尻霉素的情况下，Man8GlcNAc2异构体积累，表明对1-脱氧甘露基野尻霉素有抗性的内质网α-甘露糖苷酶负责还原酶上Man9GlcNAc2向Man8GlcNAc2的转化。然而，Man8GlcNAc2向Man6GlcNAc2的加工过程一定归因于对1-脱氧甘露基野尻霉素敏感的α-甘露糖苷酶。当细胞在存在1-脱氧甘露基野尻霉素的情况下用[2-(3)H]甘露糖进行15小时的放射性标记，然后在无抑制剂的非放射性培养基中再孵育3小时时，在标记期间积累的Man8GlcNAc2寡糖部分被修剪为Man6GlcNAc。这一发现表明，第二种对1-脱氧甘露基野尻霉素敏感的α-甘露糖苷酶存在于晶体内质网中，负责将还原酶寡糖链从Man8GlcNAc2修剪为Man6GlcNAc2。为了确定内质网α-甘露糖苷酶是否负责将所有糖蛋白的寡糖从Man9GlcNAc修剪为Man8GlcNAc，检测了在存在或不存在1.0 mM 1-脱氧甘露基野尻霉素的情况下用[2-(3)H]甘露糖标记的大鼠肝细胞的总天冬酰胺连接寡糖。该抑制剂阻止了复合寡糖的形成，并使分泌糖蛋白上的Man9GlcNAc2量增加了30倍，Man8GlcNAc2量增加了13倍。这一结果表明，只有三分之一的分泌糖蛋白最初由内质网α-甘露糖苷酶加工，三分之二由高尔基体α-甘露糖苷酶I或另一种对1-脱氧甘露基野尻霉素敏感的α-甘露糖苷酶加工。该抑制剂使细胞糖蛋白上的Man9GlcNAc2量仅增加了2.6倍，这表明这些糖蛋白中较高比例最初由内质网α-甘露糖苷酶加工。我们得出结论，一些但不是所有的肝细胞糖蛋白是内质网α-甘露糖苷酶的底物，该酶催化从Man9GlcNAc2上去除一个特定的甘露糖残基以形成Man8GlcNAc2的单一异构体。
