Expression of rat β(1,4)-N-acetylglucosaminyltransferase III in Nicotiana tabacum remodels the plant-specific N-glycosylation
Expression of rat β(1,4)-N-acetylglucosaminyltransferase III in Nicotiana tabacum remodels the plant-specific N-glycosylation
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DOI：
10.1111/j.1467-7652.2008.00370.x
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发表时间：
2009-01-01
影响因子：
13.8
通讯作者：
Kallio, Pauli T.
中科院分区：
文献类型：
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作者：
Frey, Alexander D.;Karg, Saskia R.;Kallio, Pauli T.
植物N-连接聚糖与其哺乳动物对应物显著不同，主要是关于核心聚糖的修饰，其通常含有β（1，2）-木糖和α（1，3）-岩藻糖。已知通过β（1，4）-N-乙酰葡糖胺转移酶III（GnTIII）添加平分的N-乙酰葡糖胺残基可控制哺乳动物中N-连接聚糖的加工，例如通过防止核心聚糖的α（1，6）-岩藻糖基化。为了胜过植物特异性的β（1，2）-木糖和α（1，3）-岩藻糖修饰，大鼠GnTIII用其天然定位结构域（GnTIII）或用拟南芥甘露糖苷酶II（ManII）（GnTIII（A.th.））的细胞质尾区、跨膜结构域和茎区（CTS）表达。两种CTS都将增强型黄色荧光蛋白（eYFP）靶向布雷菲德菌素A敏感区室，指示高尔基体定位。GnTIII表达使缺乏木糖和岩藻糖的N-聚糖的分数从野生型植物中的13% +/-7%增加到表达GnTIII（A.th.）的植物中的60% +/-8%。表达大鼠GnTIII的植物的N-聚糖包含复合二等分型、复合二等分型或杂合二等分型三种主要聚糖结构，占总N-聚糖的70%-85%。在GnTIII（A.th.）表达时，N-聚糖具有较高的异质性，且为杂合型。共表达A.相对于表达GnTIII或GnTIII（A.th.）的植物，拟南芥ManII显著增加了复杂二等分结构的量，而人ManII的共表达没有将混合结构的库重定向到复合型结构。所述方法提供的优点是，它可以在任何所需的植物系统中实施，以有效地从N-聚糖中除去β（1，2）-木糖和α（1，3）-岩藻糖。
Plant N-linked glycans differ substantially from their mammalian counterparts, mainly with respect to modifications of the core glycan, which typically contains a beta(1,2)-xylose and an alpha(1,3)-fucose. The addition of a bisecting N-acetylglucosamine residue by beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII) is known to control the processing of N-linked glycans in mammals, for example by preventing alpha(1,6)-fucosylation of the core glycan. In order to outcompete plant-specific beta(1,2)-xylose and alpha(1,3)-fucose modifications, rat GnTIII was expressed either with its native localization domain (GnTIII) or with the cytoplasmic tail, transmembrane domain and stem region (CTS) of Arabidopsis thaliana mannosidase II (ManII) (GnTIII(A.th.)). Both CTSs targeted enhanced yellow fluorescent protein (eYFP) to a brefeldin A-sensitive compartment, indicative of Golgi localization. GnTIII expression increased the fraction of N-glycans devoid of xylose and fucose from 13% +/- 7% in wild-type plants to 60% +/- 8% in plants expressing GnTIII(A.th.). N-Glycans of plants expressing rat GnTIII contained three major glycan structures of complex bisected, complex, or hybrid bisected type, accounting for 70%-85% of the total N-glycans. On expression of GnTIII(A.th.), N-glycans displayed a higher heterogeneity and were of hybrid type. Co-expression of A. thaliana ManII significantly increased the amount of complex bisected structures relative to the plants expressing GnTIII or GnTIII(A.th.), whereas co-expression of human ManII did not redirect the pool of hybrid structures towards complex-type structures. The method described offers the advantage that it can be implemented in any desired plant system for effective removal of beta(1,2)-xylose and alpha(1,3)-fucose from the N-glycan.