Davis S J, Davies E A, Barclay A N, Daenke S, Bodian D L, Jones E Y, Stuart D I, Butters T D, Dwek R A, van der Merwe P A
MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
J Biol Chem. 1995 Jan 6;270(1):369-75. doi: 10.1074/jbc.270.1.369.
The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.
免疫球蛋白超家族（IgSF）在进化上的成功被认为反映了IgSF蛋白结构域形成稳定结构单元的能力。然而，糖基化在稳定这些结构域中的作用存在争议。在本研究中，我们对糖基化对细胞间识别分子CD2（由两个IgSF结构域组成）的配体结合特性的影响进行了系统分析。通过在中国仓鼠卵巢细胞表达过程中用N-丁基脱氧野尻霉素抑制葡糖苷酶I，并使用内切糖苷酶H消化表达的人可溶性CD2（hsCD2），产生了一种在每个糖基化位点带有单个N-乙酰葡糖胺残基的hsCD2形式。通过表面等离子体共振分析确定，这种形式的hsCD2的配体和抗体结合特性与完全糖基化的hsCD2没有区别。该蛋白还形成了具有衍射质量的晶体，对2.5埃分辨率晶体结构的分析表明，结构域1上存在的单个N-乙酰葡糖胺残基不太可能稳定hsCD2的配体结合面。hsCD2的第二种完全去糖基化形式也能结合配体和抗体，尽管这种形式的蛋白容易聚集。与先前的研究结果相反，目前的数据表明人CD2的结构完整性和配体结合功能不依赖于糖基化。
