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

Won-Tae Kim; Hong Seo Choi; Hyo Jeong Hwang; Han-Sung Jung; Chun Jeih Ryu

doi:10.1371/journal.pone.0130670

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

PLoS One. 2015 Jun 23;10(6):e0130670. doi: 10.1371/journal.pone.0130670. eCollection 2015.

Authors

Won-Tae Kim¹, Hong Seo Choi¹, Hyo Jeong Hwang¹, Han-Sung Jung², Chun Jeih Ryu¹

Affiliations

¹ Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea.
² 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.

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.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Antibodies, Monoclonal / immunology
Antibody Specificity
Antigen-Antibody Reactions
Antigens, Surface / chemistry
Antigens, Surface / immunology
Binding, Competitive
Cell Line
Cell Membrane / immunology*
Embryonic Stem Cells / immunology*
Epitope Mapping
Humans
Membrane Proteins / chemistry*
Membrane Proteins / genetics
Membrane Proteins / immunology
Mice
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Conformation
Protein Translocation Systems
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / immunology
Sequence Deletion
Sequence Homology, Amino Acid
Species Specificity

Substances

Antibodies, Monoclonal
Antigens, Surface
BCAP31 protein, human
Bcap31 protein, mouse
Membrane Proteins
Protein Translocation Systems
Recombinant Fusion Proteins

Grants and funding

This study was supported in part by the National Research Foundation of Korea (2006-2004097 and 2009-0070942) and the Converging Research Center Program funded by the Ministry of Education, Science and Technology (Project No. 2011K000862). This work was also supported by the Korea Foundation for the Advancement of Science & Creativity (KOFAC) and funded by the Korean Government (MOE). This research was also supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C3266).