Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry
Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry
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DOI：
10.1016/j.jim.2007.07.014
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发表时间：
2007-09-30
影响因子：
2.2
通讯作者：
Jefferis, Roy
中科院分区：
文献类型：
--
作者：
Mimura, Yusuke;Ashton, Peter R.;Jefferis, Roy
人IgG分子的保守结构特征是Fc区Asn 297处存在寡糖部分。此外，15-20%的正常多克隆IgG分子在轻链（L）和/或重链（H）的可变（V）区中携带N-连接的寡糖。电喷雾电离质谱（ESI-MS）已被应用于两个IgG 1骨髓瘤蛋白（Wid和Cri）的聚糖分析后，温和的还原和酸化。观察到Wid的H和L链的不均匀离子峰，而Cri的L链峰是均匀的。IgG Wid的H和L链的位点特异性去糖基化在天然条件下分别用肽-N-糖苷酶F和内切糖苷酶F2实现。两种蛋白质之间的Fc糖型不同之处在于Cri-Fc具有完全核心岩藻糖基化和部分唾液酸化的双触角复合型聚糖，而Wid-Fc聚糖是非岩藻糖基化、部分半乳糖基化和非唾液酸化的。与Fc聚糖相反，Wid的L链聚糖显示为岩藻糖基化、完全半乳糖基化和唾液酸化，表明产生Wid的骨髓瘤细胞的糖基化机制是完整的。因此，两种内切糖苷酶的组合可以提供通过ESI-MS对Fab和Fc进行聚糖分析的简单方法，这可能有助于开发具有定制聚糖谱的治疗性IgG。(c)2007 Elsevier B. V.保留所有权利。
A conserved structural feature of human IgG molecules is the presence of an oligosaccharide moiety within the Fc region at Asn297. In addition, 15-20% of normal polyclonal IgG molecules bear N-linked oligosaccharides in the variable (V) regions of the light (L) and/or heavy (H) chains. Electrospray ionization mass spectrometry (ESI-MS) has been applied to the glycan analysis of two IgG 1 myeloma proteins (Wid and Cri) after mild reduction and acidification. Heterogeneous ion peaks were observed for both the H and L chains of Wid in contrast to Cri whose L chain peak was homogeneous. Site-specific deglycosylation of the H and L chains of IgG Wid was achieved under native conditions with peptide-N-glycosidase F and endoglycosidase, F2, respectively. The Fc glycoforms differed between the two proteins in that Cri-Fc bears diantennary complex-type glycans that are fully core-fucosylated and partially sialylated while Wid-Fc glycans are non-fucosylated, partially galactosylated and non-sialylated. In contrast to the Fc glycans, the L chain glycans of Wid were shown to be fucosylated, fully galactosylated and sialylated, indicating that the glycosylation machinery of the Wid-producing myeloma cells is intact. Thus, combination of the two endoglycosidases can provide a simple means of glycan analysis of both Fab and Fc by ESI-MS, which may contribute to the development of therapeutic IgG with customized glycan profiles. (c) 2007 Elsevier B.V. All rights reserved.