Kawamoto S, Uchino S, Hattori S, Hamajima K, Mishina M, Nakajima-Iijima S, Okuda K
Department of Bacteriology, Yokohama City University School of Medicine, Japan.
Brain Res Mol Brain Res. 1995 May;30(1):137-48. doi: 10.1016/0169-328x(95)00005-d.
Using a baculovirus expression vector system, the zeta 1 subunit of the mouse N-methyl-D-aspartate (NMDA) receptor channel was expressed in Spodoptera frugiperda insect cells. The peptide corresponding to the C-terminus of the zeta 1 subunit was synthesized by using the multiple antigen peptide (MAP) system, and an antibody to the synthetic peptide was produced. Immunoblotting using the newly developed antibody revealed the major 122-kDa and the minor 104-kDa protein bands. The effect of tunicamycin on the immunoblots and [35S]methionine/[35S]cysteine metabolic radiolabeling suggested that the two bands corresponded to glycosylated and non-N-glycosylated forms, respectively. Membranes prepared from insect cells infected with the recombinant virus had the binding activity of antagonist ligand 5,7-[3-3H]dichlorokynurenate (DCKA) of a glycine recognition domain of the receptor. Both immunofluorescence labeling and the [3H]DCKA binding assays also showed a greater level of expression (Bmax = 51 pmol/mg protein) in the insect cells. The ligand binding characteristics of the receptors expressed in insect cells suggested that the single zeta 1 subunit protein has glycine antagonist binding properties comparable to those of the native NMDA receptor channels. The lack of DCKA-binding activity of the non-N-glycosylated NMDA receptor expressed in the presence of tunicamycin suggested that N-linked oligosaccharide is essentially required for expression of a functional receptor in insect cells. This is the first report describing the importance of N-glycosylation for the acquisition of ligand binding to NMDA receptor channel subunit protein.
利用杆状病毒表达载体系统，小鼠N-甲基-D-天冬氨酸（NMDA）受体通道的ζ1亚基在草地贪夜蛾昆虫细胞中得以表达。通过使用多抗原肽（MAP）系统合成了与ζ1亚基C末端相对应的肽，并制备了针对该合成肽的抗体。使用新开发的抗体进行免疫印迹分析，揭示了主要的122 kDa和次要的104 kDa蛋白条带。衣霉素对免疫印迹和[35S]甲硫氨酸/[35S]半胱氨酸代谢性放射性标记的影响表明，这两条带分别对应糖基化和非N-糖基化形式。用重组病毒感染昆虫细胞制备的膜具有受体甘氨酸识别域的拮抗剂配体5,7-[3-3H]二氯犬尿氨酸（DCKA）的结合活性。免疫荧光标记和[3H] DCKA结合试验均显示昆虫细胞中的表达水平更高（Bmax = 51 pmol/mg蛋白）。在昆虫细胞中表达的受体的配体结合特性表明，单个ζ1亚基蛋白具有与天然NMDA受体通道相当的甘氨酸拮抗剂结合特性。在衣霉素存在下表达的非N-糖基化NMDA受体缺乏DCKA结合活性，这表明N-连接寡糖对于昆虫细胞中功能性受体的表达至关重要。这是第一份描述N-糖基化对于NMDA受体通道亚基蛋白获得配体结合重要性的报告。
