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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Stable expression of human #beta#l,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns
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Stable expression of human #beta#l,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns

机译：人＃beta＃l，4-半乳糖基转移酶在植物细胞中的稳定表达修饰了N-联糖基化模式

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摘要

#BETA#1,4-Galactosyltransferase (UDP galactose: #beta#-N-acetylglucosaminide: #beta#l,4-galactosyltransferase; EC 2.4.1.22) catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine in the penultimate stages of the terminal glycosylation of N-linked complex oligosaccharides in mam- malian cells. Tobacco BY2 cells lack this Golgi enzyme. To determine to what extent the production of a mammalian glycosyltransferase can alter the glycosylation pathway of plant cells, tobacco BY2 suspension-cultured cells were stably transformed with the full-length human galactosyltransferase gene placed under the control of the cauliflower mosaic virus 35S promoter. The expression was confirmed by assaying enzymatic activity as well as by Southern and Western blot- ting. The transformant with the highest level of enzymatic activity has glycans with galactose residues at the terminal nonreducing ends, indicating the successful modification of the plant cell N-glycosylation pathway. Analysis of the oligo- saccharide structures shows that the galactosylated N-glycans account for 47.3 of the total sugar chains. In addition, the absence of the dominant xylosidated- and fucosylated-type sugar chains confirms that the trausformed cells can be used to produce glycoproteins without the highly immunogenic glycans typically found in plants. These results demonstrate the synthesis in plants of N-linked glycans with modified and defined sugar chain structures similar to mammalian glyco- proteins.
机译：＃BETA＃1,4-半乳糖基转移酶（UDP半乳糖：＃beta＃-N-乙酰氨基葡萄糖：＃beta＃1,4-半乳糖基转移酶； EC 2.4.1.22）催化将半乳糖从UDP-Gal转移到倒数第二个中的N-乙酰氨基葡萄糖N-连接的复杂寡糖在哺乳动物细胞中末端糖基化的各个阶段。烟草BY2细胞缺乏这种高尔基酶。为了确定哺乳动物糖基转移酶的产生可以在多大程度上改变植物细胞的糖基化途径，用在花椰菜花叶病毒35S启动子控制下的全长人半乳糖基转移酶基因稳定转化了烟草BY2悬浮培养的细胞。通过测定酶活性以及Southern和Western印迹证实了表达。具有最高酶活性水平的转化体具有在末端非还原端带有半乳糖残基的聚糖，表明植物细胞N-糖基化途径的成功修饰。对寡糖结构的分析表明，半乳糖基化的N-聚糖占总糖链的47.3。另外，不存在显性的木糖基化和岩藻糖基化型糖链，这证实了该转化的细胞可用于产生糖蛋白，而没有通常在植物中发现的高度免疫原性的聚糖。这些结果证明了在植物中合成具有类似于哺乳动物糖蛋白的修饰的和限定的糖链结构的N-连接的聚糖。

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