Sparrow Lindsay G, Lawrence Michael C, Gorman Jeffrey J, Strike Phillip M, Robinson Christine P, McKern Neil M, Ward Colin W
Division of Molecular and Health Technologies, Commonwealth Scientific and Industrial Research Organisation, Parkville, Victoria 3052, Australia.
Proteins. 2008 Apr;71(1):426-39. doi: 10.1002/prot.21768.
The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N-linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N-linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N-linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO-K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR-B numbering); multiple species of high-mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high-mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high-mannose glycan species. Of the remaining five predicted N-linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high-mannose glycans lie at positions of relatively low steric accessibility.
人胰岛素受体（IR）同二聚体高度糖基化，每个单体总共含有19个预测的N-连接糖基化位点。最近IR胞外域的晶体结构显示，电子密度与构建体中大多数位点的N-连接糖基化一致。在此，我们描述了IR胞外域的精细结构，该结构纳入了所有N-连接聚糖，并揭示了附着的聚糖在多大程度上掩盖了IR二聚体的表面，使其无法与抗体或其他潜在的治疗性结合蛋白相互作用。还讨论了Fab复合在高度糖基化蛋白结晶中的作用。通过蛋白酶消化、糖肽纯化、氨基酸序列分析和质谱法测定了在CHO-K1细胞和糖基化缺陷的Lec8细胞系中表达的IR上聚糖的组成。这些数据共同揭示：在残基25、255、295、418、606、624、742、755和893（IR-B编号）处有多种复杂聚糖；在残基111和514处有多种高甘露糖聚糖；在残基671处有单一的复杂聚糖；在残基215处有单一的高甘露糖聚糖。残基16表现出复杂、杂交和高甘露糖聚糖种类的混合物。在其余五个预测的N-连接位点中，通过氨基酸测序证实残基397和906处的位点被糖基化，而残基78处的位点和残基282处的非典型（NKC）位点未被糖基化。含有残基337处最后一个位点的肽未被回收，但在IR胞外域的电子密度图中可见其被糖基化。完全糖基化的IR模型显示，携带高甘露糖聚糖的位点位于空间可及性相对较低的位置。
