Kato Toshihiko, Kitamura Kumiko, Maeda Megumi, Kimura Yoshinobu, Katayama Takane, Ashida Hisashi, Yamamoto Kenji
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Kyoto, Japan.
J Biol Chem. 2007 Jul 27;282(30):22080-8. doi: 10.1074/jbc.M700805200. Epub 2007 May 30.
Free oligosaccharides (FOSs) in the cytosol of eukaryotic cells are mainly generated during endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded glycoproteins. We analyzed FOS of the nematode Caenorhabditis elegans to elucidate its detailed degradation pathway. The major FOSs were high mannose-type ones bearing 3-9 Man residues. About 94% of the total FOSs had one GlcNAc at their reducing end (FOS-GN1), and the remaining 6% had two GlcNAc (FOS-GN2). A cytosolic endo-beta-N-acetylglucosaminidase mutant (tm1208) accumulated FOS-GN2, indicating involvement of the enzyme in conversion of FOS-GN2 into FOS-GN1. The most abundant FOS in the wild type was Man(5)GlcNAc(1), the M5A' isomer (Manalpha1-3(Manalpha1-6)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAc), which is different from the corresponding M5B' (Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAc) in mammals. Analyses of FOS in worms treated with Golgi alpha-mannosidase I inhibitors revealed decreases in Man(5)GlcNAc(1) and increases in Man(7)GlcNAc(1). These results suggested that Golgi alpha-mannosidase I-like enzyme is involved in the production of Man(5-6)-GlcNAc(1), which is unlike in mammals, in which cytosolic alpha-mannosidase is involved. Thus, we assumed that major FOSs in C. elegans were generated through Golgi trafficking. Analysis of FOSs from a Golgi alpha-mannosidase II mutant (tm1078) supported this idea, because GlcNAc(1)Man(5)GlcNAc(1), which is formed by the Golgi-resident GlcNAc-transferase I, was found as a FOS in the mutant. We concluded that significant amounts of misfolded glycoproteins in C. elegans are trafficked to the Golgi and are directly or indirectly retro-translocated into the cytosol to be degraded.
真核细胞胞质溶胶中的游离寡糖（FOSs）主要在错误折叠糖蛋白的内质网（ER）相关降解（ERAD）过程中产生。我们分析了线虫秀丽隐杆线虫的FOS，以阐明其详细的降解途径。主要的FOS是带有3 - 9个甘露糖残基的高甘露糖型。总FOS中约94%在其还原端有一个N - 乙酰葡糖胺（FOS - GN1），其余6%有两个N - 乙酰葡糖胺（FOS - GN2）。一种胞质内切β - N - 乙酰葡糖胺酶突变体（tm1208）积累了FOS - GN2，表明该酶参与了FOS - GN2向FOS - GN1的转化。野生型中最丰富的FOS是Man(5)GlcNAc(1)，即M5A'异构体（Manα1 - 3(Manα1 - 6)Manα1 - 6(Manα1 - 3)Manβ1 - 4GlcNAc），这与哺乳动物中相应的M5B'（Manα1 - 2Manα1 - 2Manα1 - 3(Manα1 - 6)Manβ1 - 4GlcNAc）不同。用高尔基体α - 甘露糖苷酶I抑制剂处理线虫后对FOS的分析显示，Man(5)GlcNAc(1)减少，Man(7)GlcNAc(1)增加。这些结果表明，高尔基体α - 甘露糖苷酶I样酶参与了Man(5 - 6)-GlcNAc(1)的产生，这与哺乳动物不同，在哺乳动物中胞质α - 甘露糖苷酶参与其中。因此，我们推测秀丽隐杆线虫中的主要FOS是通过高尔基体运输产生的。对高尔基体α - 甘露糖苷酶II突变体（tm1078）的FOS分析支持了这一观点，因为由高尔基体驻留的N - 乙酰葡糖胺转移酶I形成的GlcNAc(1)Man(5)GlcNAc(1)在该突变体中被发现为一种FOS。我们得出结论，秀丽隐杆线虫中大量错误折叠的糖蛋白被运输到高尔基体，并直接或间接逆向转运到胞质溶胶中进行降解。
