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

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Article numbere0130670
JournalPloS one
Volume10
Issue number6
DOIs
Publication statusPublished - 2015 Jun 23

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Epitope Mapping
embryonic stem cells
Embryonic Stem Cells
Stem cells
topology
B-lymphocytes
Epitopes
B-Lymphocytes
Cells
Topology
Membranes
receptors
antibodies
Antibodies
Proteins
proteins
cells
Mutant Proteins
epitope mapping
Endoplasmic Reticulum

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

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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.",
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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. / Kim, Won Tae; Choi, Hong Seo; Hwang, Hyo Jeong; Jung, Han Sung; Ryu, Chun Jeih.

In: PloS one, Vol. 10, No. 6, e0130670, 23.06.2015.

Research output: Contribution to journalArticle

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AU - Choi, Hong Seo

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AU - Jung, Han Sung

AU - Ryu, Chun Jeih

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