动物细胞STT3B依赖型N-寡糖转移相关基因的筛选细胞株的构建

发布时间：2018-12-13 13:29

【摘要】：蛋白质天冬酰胺连接的糖基化(N-糖基化)是一种重要的蛋白质翻译后修饰途径。在真核生物中,N-糖基化修饰起始于内质网,首先在磷酸多萜醇上合成寡糖链,然后由寡糖基转移酶(Oligosaccharyltransferase,OST)将其转移至多肽中的糖基化位点NXS/T(X指脯氨酸以外的其他氨基酸,N为天冬酰胺,S为丝氨酸,T为苏氨酸)上,与天冬酰胺侧链氨基上的氮原子共价连接。OST是由4到8个亚基组成的寡聚膜蛋白复合体,其中高度保守的STT3亚基具有糖基转移酶活性。低等真核生物,如酵母的OST复合体含有唯一的STT3亚基。高等真核生物,如动物细胞表达两种OST复合体,按其转移酶活性亚基的不同分为STT3A复合体和STT3B复合体。STT3A与蛋白质转移通道相作用,在多肽延伸的同时将其糖基化,这与酵母中STT3功能相似。而靠近蛋白质两端或者含有半胱氨酸的糖基化位点容易被STT3A跳过。STT3B可以在STT3A跳过的糖基化位点处加上糖链,进一步提高细胞糖基化效率。诸多研究表明,STT3B有其独特的糖基化机制。STT3B功能相关的基因突变会导致诸多严重的基因疾病。我们对细胞内STT3B的功能和作用机制还缺乏全面的了解。为了构建STT3B依赖型N-寡糖转移相关基因的筛选细胞株,本研究通过定点突变在单体增强绿色荧光蛋白(mEGFP)不同位置引入糖基化位点构建可糖基化mEGFP(N-glycosylatable mEGFP,Ng-m EGFP)。并在其N端融合钙网织蛋白的信号肽序列,同时在其C端加上内质网滞留信号序列KDEL,从而将其定位到内质网。在人胚肾293(HEK293)细胞STT3A或者STT3B敲除的细胞株中表达Ng-m EGFP,通过流式细胞仪分析和蛋白质免疫印迹分析,筛选得到三个STT3B依赖型的糖基化突变体mEGFPQ185N,mEGFPK215T,mEGFPQ185N/N186C。这些突变体在STT3B敲除细胞株中无法被糖基化,且细胞荧光明显弱于野生型和STT3A敲除细胞株。我们将荧光差异最明显的mEGFPQ185N/N186C整合到HEK293细胞基因组上,筛选得到一株荧光对N-糖基化敏感的单细胞株。在该细胞株中敲除STT3B,基因敲除的混合细胞株出现两个EGFP信号峰。结果表明该细胞株在STT3B依赖型N-寡糖转移相关基因高通量筛选研究方面的应用潜力。
[Abstract]:Protein asparagine-linked glycosylation (N-glycosylation) is an important post-translational protein modification pathway. In eukaryotes, N-glycosylation begins with the endoplasmic reticulum. First, oligosaccharide chains are synthesized on polyterpene phosphate alcohols, and then oligosaccharide chains are synthesized by oligosaccharide transferase (Oligosaccharyltransferase,). OST) was transferred to the glycosylation site NXS/T (X finger proline, N is asparagine, S is serine, T is threonine). OST is an oligomeric protein complex composed of 4 to 8 subunits, among which the highly conserved STT3 subunit has glycosyltransferase activity. The OST complex of lower eukaryotes, such as yeast, contains a unique STT3 subunit. Higher eukaryotes, such as animal cells, express two kinds of OST complexes, which are divided into STT3A complex and STT3B complex according to the different subunits of their transferase activity. STT3A acts with protein transfer channels and glycosylation of them while extending polypeptides. This is similar to the function of STT3 in yeast. The glycosylation sites near the two ends of the protein or containing cysteine are easily skipped by STT3A. STT3B can add sugar chains to the glycosylation sites skipped by STT3A and further improve the glycosylation efficiency of cells. Many studies have shown that STT3B has its unique glycosylation mechanism. Gene mutations associated with STT3B function can lead to many serious genetic diseases. We do not have a comprehensive understanding of the function and mechanism of intracellular STT3B. In order to construct a screening cell line of STT3B dependent N-oligosaccharide transfer related genes, a glycosylated mEGFP (N-glycosylatable mEGFP,) was constructed by introducing glycosylation sites into monomeric enhanced green fluorescent protein (mEGFP) by site-directed mutagenesis. Ng-m EGFP). The signal peptide sequence of calmodulin was fused at its N-terminal, and the endoplasmic reticulum retention signal sequence (KDEL,) was added to its C terminal to locate it to the endoplasmic reticulum (ER). Expression of Ng-m EGFP, in STT3A or STT3B knockout cell lines of human embryonic kidney 293 (HEK293) cells was screened by flow cytometry and Western blot analysis. Three STT3B dependent glycosylation mutants mEGFPQ185N,mEGFPK215T,mEGFPQ185N/N186C. were obtained. These mutants could not be glycosylated in STT3B knockout cell lines, and the fluorescence of these mutants was significantly weaker than that of wild type and STT3A knockout cell lines. We integrated mEGFPQ185N/N186C, which has the most obvious fluorescence difference, into the genome of HEK293 cells, and selected a single cell strain which was sensitive to N-glycosylation. There were two EGFP signal peaks in the mixed cell line with STT3B, gene knockout. The results showed that the cell line had potential in high-throughput screening of STT3B dependent N-oligosaccharide transfer related genes.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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