title: An Anti-Human Lutheran Glycoprotein Phage Antibody Inhibits Cell Migration on Laminin-511: Epitope Mapping of the Antibody.
abstract: 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.
authors.affiliation: Graduate School of Science and Engineering, Kagoshima University, Kagoshima, Japan.
authors.forename: Yurie
authors.initials: Y
authors.lastname: Enomoto-Okawa
authors.affiliation: Graduate School of Science and Engineering, Kagoshima University, Kagoshima, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Maeda
authors.affiliation: Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Harashima
authors.affiliation: Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
authors.forename: Yumika
authors.initials: Y
authors.lastname: Sugawara
authors.affiliation: Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Katagiri
authors.affiliation: Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Hozumi
authors.affiliation: Division of Cellular and Molecular Research, National Cancer Centre, Singapore, Singapore.
authors.forename: Kam Man
authors.initials: KM
authors.lastname: Hui
authors.affiliation: Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
authors.forename: Motoyoshi
authors.initials: M
authors.lastname: Nomizu
authors.affiliation: Graduate School of Science and Engineering, Kagoshima University, Kagoshima, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.
authors.forename: Yamato
authors.initials: Y
authors.lastname: Kikkawa
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D061505:Cell Surface Display Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D008188:Lutheran Blood-Group System / Q000276:immunology*; D019151:Peptide Library; D011485:Protein Binding / Q000276:immunology; D054730:Protein Interaction Domains and Motifs / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins; D008188:Lutheran Blood-Group System; D019151:Peptide Library; D057127:Single-Chain Antibodies