Orlando R A, Exner M, Czekay R P, Yamazaki H, Saito A, Ullrich R, Kerjaschki D, Farquhar M G
Department of Pathology, University of California, San Diego, La Jolla 92093-0651, USA.
Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2368-73. doi: 10.1073/pnas.94.6.2368.
Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.
巨膜蛋白是一种大型细胞表面受体，它介导脂蛋白和蛋白酶：蛋白酶抑制剂家族中许多结构和功能不同的配体的结合与内化。为了开始研究巨膜蛋白如何能够结合具有独特结构特性的配体，我们在该受体的一级序列中绘制了载脂蛋白E（apoE）-β极低密度脂蛋白（β-VLDL）、脂蛋白脂肪酶、抑肽酶、乳铁蛋白和受体相关蛋白（RAP）的结合位点。已知RAP可抑制所有配体与巨膜蛋白的结合。我们通过制备针对纯化的巨膜蛋白的单克隆抗体（mAb）来鉴定巨膜蛋白上的一个配体结合位点，选择了一种其与巨膜蛋白的结合被RAP抑制的mAb，并绘制了该mAb的表位。mAb AC10以浓度依赖性方式抑制apoE-β-VLDL、脂蛋白脂肪酶、抑肽酶和乳铁蛋白与巨膜蛋白的结合。当在杆状病毒系统中表达编码巨膜蛋白中四个富含半胱氨酸的配体结合重复序列的cDNA片段并用AC10进行免疫印迹时，它仅识别第二组配体结合重复序列。mAb AC10在该结构域内识别的表位位置被精确到氨基酸1111 - 1210。从这些研究中我们得出结论，apoE-β-VLDL、乳铁蛋白、抑肽酶、脂蛋白脂肪酶和RAP与巨膜蛋白的结合被mAb AC10竞争性或空间位阻性抑制，这表明这些配体结合到第二组配体结合重复序列内的相同或紧密重叠位点。
