Enomoto-Okawa Yurie, Maeda Yuka, Harashima Nozomi, Sugawara Yumika, Katagiri Fumihiko, Hozumi Kentaro, Hui Kam Man, Nomizu Motoyoshi, Ito Yuji, Kikkawa Yamato
Graduate School of Science and Engineering, Kagoshima University, Kagoshima, Japan.
Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
PLoS One. 2017 Jan 6;12(1):e0167860. doi: 10.1371/journal.pone.0167860. eCollection 2017.
The Lutheran glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is an Ig superfamily (IgSF) transmembrane receptor for laminin α5. Although Lu is not present in normal hepatocytes, its expression is significantly increased in hepatocellular carcinoma (HCC). In this study, we isolated thirteen phage antibodies to Lu from a phage library of peripheral blood from HCC patients, suggesting that these patients produced autoantibodies against endogenous Lu. To characterize the phage antibodies, we determined the Lu domains they recognize. The extracellular domain of Lu contains five IgSF domains, D1-D2-D3-D4-D5. The epitope of one phage antibody (A7) was localized to the D5 domain. The other phage antibodies recognized the D2 domain, which is also recognized by a function blocking mouse monoclonal antibody. One of the antibodies to D2 (C7) inhibited the binding of Lu to ligand, and it also prevented tumor cell migration on laminin-511 (LM-511). However, the C7 scFv purified from the periplasm fraction of bacteria did not exhibit the inhibitory effects, indicating that the scFv form could not sterically inhibit the binding of Lu to LM-511. We also identified the amino acid residues that form the epitope recognized by the C7 phage antibody. Mutagenesis studies showed that Arg247 is necessary for forming the epitope. The C7 phage antibody and its epitope may be useful for developing drugs to prevent HCC progression and/or metastasis.
路德糖蛋白（Lu），也称为基底细胞粘附分子（B-CAM），是层粘连蛋白α5的免疫球蛋白超家族（IgSF）跨膜受体。虽然Lu在正常肝细胞中不存在，但其在肝细胞癌（HCC）中的表达显著增加。在本研究中，我们从HCC患者外周血噬菌体文库中分离出13种针对Lu的噬菌体抗体，表明这些患者产生了针对内源性Lu的自身抗体。为了表征这些噬菌体抗体，我们确定了它们识别的Lu结构域。Lu的细胞外结构域包含五个IgSF结构域，即D1-D2-D3-D4-D5。一种噬菌体抗体（A7）的表位定位于D5结构域。其他噬菌体抗体识别D2结构域，一种功能阻断小鼠单克隆抗体也能识别该结构域。其中一种针对D2的抗体（C7）抑制了Lu与配体的结合，并且还阻止了肿瘤细胞在层粘连蛋白-511（LM-511）上的迁移。然而，从细菌周质部分纯化的C7单链抗体片段（scFv）未表现出抑制作用，这表明scFv形式不能在空间上抑制Lu与LM-511的结合。我们还确定了构成C7噬菌体抗体识别表位的氨基酸残基。诱变研究表明，Arg247是形成该表位所必需的。C7噬菌体抗体及其表位可能有助于开发预防HCC进展和/或转移的药物。
