Comparison of the N-linked glycosylation of human β1,3-N-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae
Comparison of the N-linked glycosylation of human β1,3-N-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae
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DOI：
10.1016/j.jbiotec.2009.06.013
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发表时间：
2009-08-10
影响因子：
4.1
通讯作者：
Park, Enoch Y.
中科院分区：
文献类型：
--
作者：
Dojima, Takashi;Nishina, Takuya;Park, Enoch Y.
人β 1，3 N-乙酰葡糖胺转移酶2（β 3GnT 2）的N-糖基化对其生物学功能是必不可少的。用重组BmNPV杆粒在稳定转化的昆虫细胞和家蚕幼虫中通过非病毒表达系统产生与GFP（uv）融合的β 3GnT 2（GFP（uv）-β 3GnT 2），并纯化用于N-糖基化分析。通过糖酰胺酶A消化、2-氨基吡啶（PA）标记和HPLC图谱鉴定β 3GnT 2的N-聚糖结构。稳定转化的粉纹夜蛾细胞中以少甘露糖苷N-聚糖结构为主（73.2%）。相反，在家蚕幼虫中表达的β 3 GnT 2上检测到具有连接至Man α（1，3）分支的Gal（21.3%）和GlcNAc（16.2%）末端残基的N-聚糖。末端Gal和二等分GlcNAc残基如Gal β 1，4GlcNAc β 1，2 Man α 1，3（GlcNAc β 1，4）（Man α 1，6）Man β 1，4GlcNAc的存在不是鳞翅目昆虫N-糖基化的典型结构。尽管在Tni细胞中发现了变应原性α 1，3-岩藻糖残基，但在家蚕幼虫中仅α 1，6-岩藻糖残基附着于β 3GnT 2聚糖。因此，家蚕幼虫可能是一种生产人糖蛋白的有用宿主。(C)2009爱思唯尔有限公司版权所有。
N-Glycosylation of human beta 1,3N-acetylglucosaminyltransferase 2 (beta 3GnT2) is essential for its biological function. beta 3GnT2 fused to GFP(uv) (GFP(uv)-beta 3GnT2) was produced by non-virus expression systems in stably transformed insect cells and silkworm larvae using a recombinant BmNPV bacmid, and purified for analysis of N-glycosylation. The N-glycan structure of beta 3GnT2 was identified by glycoamidase A digestion, labeling with 2-aminopyridine (PA), and HPLC mapping. The paucimannosidic N-glycan structure (73.2%) was predominant in stably transformed Trichoplusia ni cells. In contrast, N-glycan with Gal (21.3%) and GlcNAc (16.2%) terminal residues linked to Man alpha(1,3) branch were detected on beta 3GnT2 expressed in silkworm larvae. The presence of terminal Gal and bisecting GlcNAc residues such as Gal beta 1, 4GlcNAc beta 1, 2Man alpha 1,3(GlcNAc beta 1,4)(Man alpha 1,6)Man beta 1, 4GlcNAc is not typical structure for lepidopteran insect N-glycosylation. Although allergenic alpha 1,3-fucose residues have been found in T ni cells, only alpha 1,6-fucose residues were attached to the beta 3GnT2 glycan in silkworm larvae. Therefore, silkworm larvae might be a useful host for producing human glycoproteins. (C) 2009 Elsevier B.V. All rights reserved.