Prigent S A, Stanley K K, Siddle K
Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, United Kingdom.
J Biol Chem. 1990 Jun 15;265(17):9970-7.
The sequential epitopes on the human insulin receptor recognized by polyclonal and monoclonal antibodies were investigated using a recombinant DNA technique. Short random fragments of receptor cDNA were cloned and expressed in Escherichia coli as beta-galactosidase fusion proteins by using the expression vector pUEX1. Immunoreactive peptides were detected by colony blotting and identified by sequencing the corresponding cDNA inserts. Eleven antigenic determinants were located with rabbit antisera, nine of these being on the alpha-subunit and two on the beta-subunit of which one was intracellular. Two human autoantibodies reacted with the alpha-subunit on blots, but no sequential epitopes could be located. In the rabbit sera, antibody reacting with these linear epitopes represented a substantial fraction (approximately 50%) of antibody reacting with reduced denatured receptor on blots, but a generally smaller fraction (5-40%) of antibody reacting with solubilized native receptor. Epitope-specific subfractions of antibodies were purified by binding to an elution from bacterial fusion proteins. All of these subfractions reacted with denatured receptor on nitrocellulose blots, but only three precipitated native receptor (epitopes between amino acids 190 and 231, 654 and 669, 954 and 982) and none inhibited insulin binding. (The numbering system used in this manuscript is that of Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf. L., Clauser, E., Ou, J., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A., and Rutter, W. J. (1985) Cell 40, 747-758). The binding sites of two monoclonal antibodies were also determined. One of these antibodies (83-14) is insulin-mimetic, but inhibits insulin binding and its epitope on the alpha-subunit (between amino acids 469 and 592) may contribute to the insulin binding site in the folded protein. The other antibody (18-44) binds close to the N terminus of the beta-subunit (amino acids 765-770) and does not inhibit insulin binding, but does mimic insulin action. The identification of epitopes therefore provides information on receptor conformation and allows structural domains to be identified which are involved in the functional effects of different antibodies.
利用重组DNA技术研究了多克隆抗体和单克隆抗体识别的人胰岛素受体上的连续表位。受体cDNA的短随机片段被克隆，并通过使用表达载体pUEX1在大肠杆菌中作为β-半乳糖苷酶融合蛋白表达。通过菌落印迹法检测免疫反应性肽，并通过对相应的cDNA插入片段进行测序来鉴定。用兔抗血清定位了11个抗原决定簇，其中9个位于α亚基上，2个位于β亚基上，其中1个位于细胞内。两种人自身抗体在印迹上与α亚基反应，但无法定位连续表位。在兔血清中，与这些线性表位反应的抗体占与印迹上还原变性受体反应的抗体的很大一部分（约50%），但通常占与可溶性天然受体反应的抗体的较小一部分（5-40%）。通过与细菌融合蛋白的洗脱物结合来纯化抗体的表位特异性亚组分。所有这些亚组分在硝酸纤维素印迹上与变性受体反应，但只有三个沉淀天然受体（氨基酸190至231、654至669、954至982之间的表位），且均不抑制胰岛素结合。（本手稿中使用的编号系统是Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf. L., Clauser, E., Ou, J., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A., and Rutter, W. J. (1985) Cell 40, 747 - 758中的编号系统）。还确定了两种单克隆抗体的结合位点。其中一种抗体（83-14）具有胰岛素模拟作用，但抑制胰岛素结合，其在α亚基上的表位（氨基酸469至592之间）可能有助于折叠蛋白中的胰岛素结合位点。另一种抗体（18-44）结合在β亚基的N端附近（氨基酸765-770），不抑制胰岛素结合，但确实模拟胰岛素作用。因此，表位的鉴定提供了有关受体构象的信息，并允许识别参与不同抗体功能效应的结构域。
