Epitope mapping of anti-PGRMC1 antibodies reveals the non-conventional membrane topology of PGRMC1 on the cell surface.

Abstract

Progesterone receptor membrane component1 (PGRMC1) is a heme-binding protein involved in cancers and Alzheimer's disease. PGRMC1 consists of a short N-terminal extracellular or luminal domain, a single membrane-spanning domain, and a long cytoplasmic domain. Previously, we generated two monoclonal antibodies (MAbs) 108-B6 and 4A68 that recognize cell surface-expressed PGRMC1 (csPGRMC1) on human pluripotent stem cells and some cancer cells. In this study, flow cytometric analysis found that an anti-PGRMC1 antibody recognizing the N-terminus of PGRMC1 could not bind to csPGRMC1 on cancer cells, and 108-B6 and 4A68 binding to csPGRMC1 was inhibited by trypsin treatment, suggesting that the epitopes of 108-B6 and 4A68 are trypsin-sensitive. To examine the epitope specificity of 108-B6 and 4A68, glutathione-S-transferase (GST)-fused PGRMC1 mutants were screened to identify the epitopes targeted by the antibodies. The result showed that 108-B6 and 4A68 recognized C-terminal residues 183-195 and 171-182, respectively, of PGRMC1, where trypsin-sensitive sites are located. A polyclonal anti-PGRMC1 antibody raised against the C-terminus of PGRMC1 could also recognized csPGRMC1 in a trypsin-sensitive manner, suggesting that the C-terminus of csPGRMC1 is exposed on the cell surface. This finding reveals that csPGRMC1 has a non-conventional plasma membrane topology, which is different from that of intracellular PGRMC1.

• Authors: Ji Yea Kim, So Young Kim, Hong Seo Choi, Sungkwan An, Chun Jeih Ryu
• Journal: Scientific reports (Sci Rep) Vol. 9 Issue 1 Pg. 653 (01 24 2019) ISSN: 2045-2322 [Electronic] England
• PMID: 30679694 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Monoclonal
• Membrane Proteins
• PGRMC1 protein, human
• Receptors, Progesterone

Topics

• Antibodies, Monoclonal (immunology)
• Cell Line
• Cell Membrane (metabolism)
• Epitope Mapping
• Humans
• Intracellular Space (metabolism)
• Membrane Proteins (chemistry, immunology, metabolism)
• Receptors, Progesterone (chemistry, immunology, metabolism)