Boudin H, Grauz-Guyon A, Faure M P, Forgez P, Lhiaubet A M, Dennis M, Beaudet A, Rostene W, Pelaprat D
Institut National de la Santé et de la Recherche Médicale, Unité 339, Hôpital St Antoine, Paris, France.
Biochem J. 1995 Jan 1;305 ( Pt 1)(Pt 1):277-83. doi: 10.1042/bj3050277.
In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied in the visualization of the receptor.
在本研究中，利用针对受体第三细胞内环片段产生的抗肽抗体，通过免疫方法检测了转染的中国仓鼠卵巢（CHO）细胞、感染的Sf9昆虫细胞以及大鼠大脑皮层中表达的大鼠神经降压素受体（NTR）的分子形式。针对融合蛋白的免疫印迹实验表明，该抗肽抗体在变性条件下可识别NTR内相应的氨基酸序列。在对NTR转染的CHO细胞膜进行免疫印迹分析时，在60至72 kDa之间观察到高水平的免疫反应性，而在大鼠NTR cDNA推导的分子量47 kDa处仅观察到微弱的条带。在用肽N - 糖苷酶F对膜蛋白进行去糖基化后，高分子量条带不再出现，表明它们代表受体的糖基化形式。来自表达NTR的杆状病毒感染的Sf9昆虫细胞膜提取物提供了一种截然不同的免疫印迹模式，因为在这种情况下检测到的主要条带位于47 kDa，即非糖基化受体的分子大小。综上所述，这些数据表明，虽然NTR蛋白在CHO细胞中大多被糖基化，但在Sf9昆虫细胞中未被糖基化。此外，在这两种细胞系中观察到的受体蛋白分子大小与在7日龄大鼠大脑皮层中内源性表达的NTR不同，在大鼠大脑皮层中检测到56和54 kDa的条带。对杆状病毒感染的Sf9细胞制备的膜进行的结合实验表明，当受体嵌入细胞膜时，免疫原性序列仍可被抗体识别。对转染的CHO细胞和感染的Sf9细胞进行的免疫组织化学研究证实了这一解释，并进一步表明该抗体可用于受体的可视化。
