Man_8GlcNAc_2糖基化的酿酒酵母菌株的构建

Construction of the Man_8GlcNAc_2 glycosylation Saccharomyces cerevisiae mutant strain

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man8GlcNAc2。

In Saccharomyces cerevisiae,protein glycosylation passed two different N-linked modification pathways after the export of predominantly Man8GlcNAc2-containing glycoproteins from ER to the Golgi. The core oligosaccharide undergoes maturation in the Golgi resulting in a Man 8-13 GlcNAc2 structure. Alternatively,core structures may be hypermannosylated with up to 200 mannose residues composing of a backbone of α1,6-mannosyl residues with branched α1,2-and α1,3-mannosyl side chains. Mnn1p and Och1p play an important role in this process. The null disruption of MNN1,OCH1 was replaced by the S. cerevisiae URA3,HIS3,respectively. To characterize the N-glycosylation in the mnn1 och1 mutant,mannoproteins were obtained by hot citrate buffer extraction after the mnn1 och1 cells were crumbled. The extracted mannoprotein was precipitated by ethanol,and further purified by concanavalin A-sepharose 4B. The N-oligomannose saccharides were released from mannoprotein by PNGase F digestion,and then peptides and detergents were removed by passage through ion exchange columns. For desalting,glycans were applied to porous graphitic-carbon cartridge. 2-aminopyridine pyridylaminated sugars were profiled and purified by size fractionation HPLC with Shim-pack clc-NH_2 column,and result showed dominantly a single peak. MALDI TOF/MS analysis ofthis peak revealed that its molecular weight was 1796.5Da,which corresponds to the calculated mass of Man_8GlcNAc_2-PA. These results indicated that disruptions of MNN1 and OCH1 eliminated the hypermannosylation of the N-linked glycans,and glycoproteins were glycosylated with a single core type glycan,Man8GlcNAc2,in the mnn1 och1 mutant.