Epitope Mapping of Anti-PGRMC1 Antibodies Reveals the Non-conventional Membrane Topology of PGRMC1 on the Cell Surface

Overview

Journal Sci Rep

Specialty Science

Date 2019 Jan 26

PMID 30679694

Citations 6

Authors

Ji Yea Kim

So Young Kim

Hong Seo Choi

Sungkwan An

Chun Jeih Ryu

Affiliations

Soon will be listed here.

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.

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References

Chen M, Zhang J . Topogenesis of cystic fibrosis transmembrane conductance regulator (CFTR): regulation by the amino terminal transmembrane sequences. Biochemistry. 1999; 38(17):5471-7. DOI: 10.1021/bi982153t. View

Wang Z, Raifu M, Howard M, Smith L, Hansen D, Goldsby R . Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3' to 5' exonuclease activity. J Immunol Methods. 2000; 233(1-2):167-77. DOI: 10.1016/s0022-1759(99)00184-2. View

Lu Y, Turnbull I, Bragin A, Carveth K, Verkman A, Skach W . Reorientation of aquaporin-1 topology during maturation in the endoplasmic reticulum. Mol Biol Cell. 2000; 11(9):2973-85. PMC: 14969. DOI: 10.1091/mbc.11.9.2973. View

Krebs C, Jarvis E, Chan J, Lydon J, Ogawa S, Pfaff D . A membrane-associated progesterone-binding protein, 25-Dx, is regulated by progesterone in brain regions involved in female reproductive behaviors. Proc Natl Acad Sci U S A. 2000; 97(23):12816-21. PMC: 18847. DOI: 10.1073/pnas.97.23.12816. View

von Heijne G . Recent advances in the understanding of membrane protein assembly and structure. Q Rev Biophys. 2001; 32(4):285-307. DOI: 10.1017/s0033583500003541. View

Shin B, Wang H, Yim A, Le Naour F, Brichory F, Jang J . Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function. J Biol Chem. 2002; 278(9):7607-16. DOI: 10.1074/jbc.M210455200. View

Min L, Takemori H, Nonaka Y, Katoh Y, Doi J, Horike N . Characterization of the adrenal-specific antigen IZA (inner zone antigen) and its role in the steroidogenesis. Mol Cell Endocrinol. 2004; 215(1-2):143-8. DOI: 10.1016/j.mce.2003.11.025. View

Losel R, Dorn-Beineke A, Falkenstein E, Wehling M, Feuring M . Porcine spermatozoa contain more than one membrane progesterone receptor. Int J Biochem Cell Biol. 2004; 36(8):1532-41. DOI: 10.1016/j.biocel.2004.01.003. View

Beausoleil S, Jedrychowski M, Schwartz D, Elias J, Villen J, Li J . Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A. 2004; 101(33):12130-5. PMC: 514446. DOI: 10.1073/pnas.0404720101. View

10.

Sadlish H, Skach W . Biogenesis of CFTR and other polytopic membrane proteins: new roles for the ribosome-translocon complex. J Membr Biol. 2005; 202(3):115-26. DOI: 10.1007/s00232-004-0715-6. View

11.

Peluso J, Pappalardo A, Losel R, Wehling M . Expression and function of PAIRBP1 within gonadotropin-primed immature rat ovaries: PAIRBP1 regulation of granulosa and luteal cell viability. Biol Reprod. 2005; 73(2):261-70. DOI: 10.1095/biolreprod.105.041061. View

12.

von Heijne G . Membrane-protein topology. Nat Rev Mol Cell Biol. 2006; 7(12):909-18. DOI: 10.1038/nrm2063. View

13.

Cahill M . Progesterone receptor membrane component 1: an integrative review. J Steroid Biochem Mol Biol. 2007; 105(1-5):16-36. DOI: 10.1016/j.jsbmb.2007.02.002. View

14.

Zeng C, Vangveravong S, Xu J, Chang K, Hotchkiss R, Wheeler K . Subcellular localization of sigma-2 receptors in breast cancer cells using two-photon and confocal microscopy. Cancer Res. 2007; 67(14):6708-16. DOI: 10.1158/0008-5472.CAN-06-3803. View

15.

Peluso J, Romak J, Liu X . Progesterone receptor membrane component-1 (PGRMC1) is the mediator of progesterone's antiapoptotic action in spontaneously immortalized granulosa cells as revealed by PGRMC1 small interfering ribonucleic acid treatment and functional analysis of.... Endocrinology. 2007; 149(2):534-43. PMC: 2219306. DOI: 10.1210/en.2007-1050. View

16.

Fernandes M, Brosens J, Gellersen B . Honey, we need to talk about the membrane progestin receptors. Steroids. 2008; 73(9-10):942-52. DOI: 10.1016/j.steroids.2007.12.004. View

17.

Choi H, Kim H, Won A, Kim J, Son C, Kim K . Development of a decoy immunization strategy to identify cell-surface molecules expressed on undifferentiated human embryonic stem cells. Cell Tissue Res. 2008; 333(2):197-206. DOI: 10.1007/s00441-008-0632-6. View

18.

Rohe H, Ahmed I, Twist K, Craven R . PGRMC1 (progesterone receptor membrane component 1): a targetable protein with multiple functions in steroid signaling, P450 activation and drug binding. Pharmacol Ther. 2008; 121(1):14-9. PMC: 2659782. DOI: 10.1016/j.pharmthera.2008.09.006. View

19.

Peluso J, Liu X, Gawkowska A, Lodde V, Wu C . Progesterone inhibits apoptosis in part by PGRMC1-regulated gene expression. Mol Cell Endocrinol. 2010; 320(1-2):153-61. PMC: 2844455. DOI: 10.1016/j.mce.2010.02.005. View

20.

Ahmed I, Rohe H, Twist K, Craven R . Pgrmc1 (progesterone receptor membrane component 1) associates with epidermal growth factor receptor and regulates erlotinib sensitivity. J Biol Chem. 2010; 285(32):24775-82. PMC: 2915713. DOI: 10.1074/jbc.M110.134585. View