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.
Highlights
Progesterone receptor membrane component 1 (PGRMC1) is a multifunctional protein with a C-terminal cytochrome b5 domain[1]
The present study found that two monoclonal antibodies (MAbs), 108-B6 and 4A68, recognized csPGRMC1 (Fig. 1), and their epitopes were located between residues [183–195, 171–182], respectively, of Progesterone receptor membrane component1 (PGRMC1) (Fig. 3d–f)
The present results suggest that the C-terminal domain of csPGRMC1 is exposed on the cell surface. csPGRMC1 was recognized by C2C3, a commercially available polyclonal anti-PGRMC1 antibody raised against the C-terminal domain of PGRMC1 (Fig. 4 and Supplementary Fig. 3)
Summary
Progesterone receptor membrane component 1 (PGRMC1) is a multifunctional protein with a C-terminal cytochrome b5 domain[1]. Flow cytometric analysis found that an anti-PGRMC1 antibody recognizing the N-terminal domain (residues 1–46) of PGRMC1 was not able to bind to csPGRMC1 on cancer cells, it was able to recognize intracellular PGRMC1 in saponin-treated cells. Flow cytometric analysis showed that 108-B6 and 4A68 binding to csPGRMC1 was inhibited by trypsin treatment, suggesting that the epitopes of 108-B6 and 4A68 is outside the N-terminal domain and have trypsin-sensitive sites within them. This observation led us to investigate the epitope of two MAbs on PGRMC1. Epitope analysis of PGRMC1 antibodies reveals that csPGRMC1 has a different membrane topology from that of intracellular PGRMC1
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