title: Epitope Mapping of Antibodies Suggests the Novel Membrane Topology of B-Cell Receptor Associated Protein 31 on the Cell Surface of Embryonic Stem Cells: The Novel Membrane Topology of BAP31.
abstract: When located in the endoplasmic reticulum (ER) membrane, B-cell receptor associated protein 31 (BAP31) is involved in the export of secreted proteins from the ER to the plasma membrane. In a previous study, we generated two monoclonal antibodies (mAbs), 297-D4 and 144-A8, that bound to surface molecules on human embryonic stem cells (hESCs), but not to surface molecules on mouse embryonic stem cells (mESCs). Subsequent studies revealed that the mAbs recognized BAP31 on the surface of hESCs. To investigate the membrane topology of BAP31 on the cell surface, we first examined the epitope specificity of 297-D4 and 144-A8, as well as a polyclonal anti-BAP31 antibody (α-BAP31). We generated a series of GST-fused BAP31 mutant proteins in which BAP31 was serially deleted at the C- terminus. GST-fused BAP31 mutant proteins were then screened to identify the epitopes targeted by the antibodies. Both 297-D4 and 144-A8 recognized C-terminal residues 208-217, while α-BAP31 recognized C-terminal residues 165-246, of BAP31 on hESCs, suggesting that the C-terminal domain of BAP31 is exposed on the cell surface. The polyclonal antibody α-BAP31 bound to mESCs, which confirmed that the C-terminal domain of BAP31 is also exposed on the surface of these cells. Our results show for the first time the novel membrane topology of cell surface-expressed BAP31 as the extracellular exposure of the BAP31 C-terminal domain was not predicted from previous studies.
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea.
authors.forename: Won-Tae
authors.initials: WT
authors.lastname: Kim
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea.
authors.forename: Hong Seo
authors.initials: HS
authors.lastname: Choi
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea.
authors.forename: Hyo Jeong
authors.initials: HJ
authors.lastname: Hwang
authors.affiliation: Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Korea; Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, Hong Kong SAR.
authors.forename: Han-Sung
authors.initials: HS
authors.lastname: Jung
authors.affiliation: Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea.
authors.forename: Chun Jeih
authors.initials: CJ
authors.lastname: Ryu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000954:Antigens, Surface / Q000737:chemistry / Q000276:immunology; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology*; D053595:Embryonic Stem Cells / Q000276:immunology*; D018604:Epitope Mapping; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D000069284:Protein Translocation Systems; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C091247:BCAP31 protein, human; C489642:Bcap31 protein, mouse; D008565:Membrane Proteins; D000069284:Protein Translocation Systems; D011993:Recombinant Fusion Proteins