Kuroiwa Kenji, Torikai Yusuke, Osawa Mafumi, Nakashima Takaaki, Nakashima Mitsuhiro, Endo Hitoshi, Arai Takao
Department of Biochemistry, Jichi Medical University, Tochigi, Japan.
Hybridoma (Larchmt). 2012 Aug;31(4):225-32. doi: 10.1089/hyb.2012.0002.
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.
糖基磷脂酰肌醇锚定蛋白Thy-1在啮齿动物的神经元和T淋巴细胞的细胞表面大量表达。尽管已知Thy-1作为配体与整合素结合，并介导神经突生长和免疫反应，但其确切功能尚未完全了解。此前我们制备了几种抗大鼠Thy-1单克隆抗体，并鉴定出一种2E11，它能诱导PC12细胞神经突生长。在此，我们筛选了抑制2E11诱导的神经突生长以及刺激或抑制大鼠胸腺细胞聚集的抗体。由于Thy-1缺乏细胞内区域，它需要其他膜结合分子进行信号转导。因此，推测这些抗体在Thy-1与信号分子之间的相互作用中起关键作用。为阐明抗体诱导的Thy-1功能机制，我们进行了抗体表征和表位确定。Thy-1的切割和突变表明，这些抗体不仅识别氨基酸序列，还识别由免疫球蛋白样结构域组成的三维结构。有两种抗体被认为在空间上靠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交联及随后的Thy-1信号传导。因此，本文报道的抗体可能作为交联剂、激动剂或拮抗剂来调节Thy-1信号传导。
