Hsu T A, Takahashi N, Tsukamoto Y, Kato K, Shimada I, Masuda K, Whiteley E M, Fan J Q, Lee Y C, Betenbaugh M J
Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218-2694, USA.
J Biol Chem. 1997 Apr 4;272(14):9062-70. doi: 10.1074/jbc.272.14.9062.
Structures of the N-linked oligosaccharide attached to the heavy chain of a heterologous murine IgG2a produced from Trichoplusia ni (TN-5B1-4, High Five) insect cells were characterized. Coexpression of the chaperone immunoglobulin heavy chain-binding protein (BiP) in the baculovirus-infected insect cells increased the soluble intracellular and secreted IgG level. This facilitated the detailed analysis of N-glycans from both intracellular and secreted IgG. Following purification of the immunoglobulins using Protein A-Sepharose, glycopeptides, prepared by trypsin-chymotrypsin digestion, were further digested with glycoamidase from sweet almond emulsin to obtain the oligosaccharide moieties. The resulting oligosaccharides were then reductively aminated with 2-aminopyridine and the structures identified by two-dimensional high performance liquid chromatography mapping (Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. (1988) Anal. Biochem. 171, 73-90). The N-glycans obtained from the secreted IgG contain 35% complex type, some with terminal galactose residues at either alpha1, 3-Man or alpha1,6-Man branches of the Man3GlcNAc2 core. The remaining oligosaccharides detected in the secreted IgG were principally hybrid (30%) and paucimannosidic (35%) type N-glycans. Most (84%) of these secreted glycoforms contained fucose alpha1, 6-linked to the innermost GlcNAc residue and the presence of a potentially allergenic fucose alpha1,3-linked to the innermost GlcNAc residue was also detected. In contrast, the intracellular immunoglobulins included 50% high mannose-type N-glycans with lower levels of complex, hybrid, and paucimannosidic-type structures. Reverse phase one-dimensional high performance liquid chromatography analysis of the IgG N-glycans in the absence of heterologous BiP exhibited a similar distribution of intracellular and secreted glycoforms. These studies indicate that Trichoplusia ni TN-5B1-4 cells are capable of terminal galactosylation. However, the processing pathways in these cell lines appear to diverge from mammalian cells in the formation of paucimannosidic structures, in the presence of alpha1,3-fucose linkages, and in the absence of sialylation.
对从粉纹夜蛾（TN - 5B1 - 4，High Five）昆虫细胞产生的异源鼠源IgG2a重链上连接的N - 连接寡糖结构进行了表征。伴侣免疫球蛋白重链结合蛋白（BiP）在杆状病毒感染的昆虫细胞中共表达，增加了可溶性细胞内和分泌型IgG水平。这便于对细胞内和分泌型IgG的N - 聚糖进行详细分析。使用蛋白A - 琼脂糖纯化免疫球蛋白后，通过胰蛋白酶 - 糜蛋白酶消化制备的糖肽，再用甜杏仁乳化素的糖酰胺酶进一步消化以获得寡糖部分。然后将所得寡糖用2 - 氨基吡啶进行还原性胺化，并通过二维高效液相色谱图谱鉴定结构（Tomiya，N.，Awaya，J.，Kurono，M.，Endo，S.，Arata，Y.，和Takahashi，N.（1988）Anal. Biochem. 171，73 - 90）。从分泌型IgG获得的N - 聚糖包含35%的复合型，其中一些在Man3GlcNAc2核心的α1,3 - Man或α1,6 - Man分支处带有末端半乳糖残基。在分泌型IgG中检测到的其余寡糖主要是杂合型（30%）和寡甘露糖型（35%）N - 聚糖。这些分泌型糖型中的大多数（约84%）含有与最内层GlcNAc残基α1,6 - 连接的岩藻糖，并且还检测到存在与最内层GlcNAc残基α1,3 - 连接的潜在致敏性岩藻糖。相比之下，细胞内免疫球蛋白包含50%的高甘露糖型N - 聚糖，复合型、杂合型和寡甘露糖型结构的水平较低。在没有异源BiP的情况下，对IgG N - 聚糖进行反相一维高效液相色谱分析，细胞内和分泌型糖型的分布相似。这些研究表明粉纹夜蛾TN - 5B1 - 4细胞能够进行末端半乳糖基化。然而，这些细胞系中的加工途径在寡甘露糖型结构的形成、α1,3 - 岩藻糖连接的存在以及唾液酸化的缺失方面似乎与哺乳动物细胞不同。
