Epitope determination of anti Thy-1 monoclonal antibodies that regulate neurite outgrowth
Epitope determination of anti Thy-1 monoclonal antibodies that regulate neurite outgrowth
复制标题
调节神经突生长的抗 Thy-1 单克隆抗体的表位测定
作者：
Kuroiwa;K.;Torkai,Y.;Osawa;M.;Nakashima;T.;Nakashima;M.;Endo;H.;and Arai;T
糖基磷脂酰肌醇锚定蛋白Thy-1在啮齿类动物神经元和T淋巴细胞表面大量表达。虽然已知Thy-1作为配体结合整合素并介导神经突生长和免疫反应，但其确切功能尚不完全清楚。在此之前，我们制备了几种抗大鼠Thy-1单克隆抗体，并鉴定出一种诱导PC12细胞神经突生长的2E11。在这里，我们筛选了抑制2e11诱导的神经突生长和刺激或抑制大鼠胸腺细胞聚集的抗体。由于Thy-1缺乏胞内区，它需要其他膜结合分子来进行信号转导。因此，假设这些抗体在Thy-1和信号分子之间的相互作用中起关键作用。为了阐明抗体诱导Thy-1功能的机制，进行了抗体鉴定和表位测定。y-1的切割和突变表明，抗体不仅识别氨基酸序列，而且识别由免疫球蛋白样结构域组成的三维结构。两种抗体被认为在空间上接近整合素结合位点并交联Thy-1分子，而第三种抗体被认为抑制Thy-1交联和随后的Thy-1信号传导。因此，本文报道的抗体可以作为交联剂、激动剂或拮抗剂来修饰Thy-1信号。
Glycosylphosphatidylinositol-anchored protein Thy-1 is abundantly expressed on the cell surface of neurons and T lymphocytes in rodents. Although Thy-1 is known to bind integrins as a ligand and to mediate neurite outgrowth and immune responses, its precise function is not fully understood. Previously we produced several anti rat Thy-1 monoclonal antibodies and identified one, 2E11, which induces PC12 cell neurite outgrowth. Here we screened antibodies that inhibit 2E11-induced neurite outgrowth and stimulate or inhibit rat thymocyte aggregation. Since Thy-1 lacks an intracellular region, it requires other membrane-bound molecules for the signal transduction. Hence these antibodies are hypothesized to play key roles in the interaction between Thy-1 and signaling molecules. To elucidate the mechanisms of antibody-induced Thy-1 functions, antibody characterization and epitope determination were carried out. Thy-1 cleavage and mutation revealed that the antibodies recognize not only amino acid sequences, but also the three-dimensional structures consisting of immunoglobulin-like domains. Two antibodies were suggested to bind spatially close to the integrin binding site and crosslink Thy-1 molecules, while a third antibody is believed to inhibit Thy-1 crosslinking and subsequent Thy-1 signaling. The antibodies reported here may therefore function as crosslinkers, agonists, or antagonists that modify Thy-1 signaling.