Skip to main content
SpringerLink
Log in

Detection of Cell Membrane-Bound CD46 Using Flow Cytometry

  • Protocol
  • First Online:
The Complement System

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1100))

Abstract

CD46 is an important regulator of the complement system by preventing unwanted deposition of the complement activation products and opsonins C3b/C4b onto self-tissue. Recently, intracellular signals mediated by CD46 activation on several distinct human cell types have demonstrated that CD46 also plays decisive roles in immuneregulation. The growing recognition of CD46 as key regulator in several vital biological processes, led to increased demand in sensitive methods for monitoring CD46 expression and changes thereof on cells and in tissues. Here we describe a method, which allows for studying CD46 expression on the surface of cells using specific antibodies in combination with fluorescence-activated cell sorting (FACS) analysis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Purcell D, Deacon N, Andrew S, McKenzie I (1990) Human non-lineage antigen, CD46 (HuLy-m5): purification and partial sequencing demonstrates structural homology with complement-regulating glycoproteins. Immunogenetics 31:21–28

    Article  PubMed  CAS  Google Scholar 

  2. Liszewski M, Post T, Atkinson J (1991) Membrane cofactor protein (MCP or CD46): newest member of the regulators of complement activation gene cluster. Annu Rev Immunol 9:431–455

    Article  PubMed  CAS  Google Scholar 

  3. Liszewski MK, Atkinson JP (1996) Membrane cofactor protein (MCP; CD46). Isoforms differ in protection against the classical pathway of complement. J Immunol 156:4415–4421

    PubMed  CAS  Google Scholar 

  4. Hakulinen J, Junnikkala S, Sorsa T, Meri S (2004) Complement inhibitor membrane cofactor protein (MCP; CD46) is constitutively shed from cancer cell membranes in vesicles and converted by a metalloproteinase to a functionally active soluble form. Eur J Immunol 34:2620–2629

    Article  PubMed  CAS  Google Scholar 

  5. Seya T, Hara T, Iwata K, Kuriyama S, Hasegawa T, Nagase Y et al (1995) Purification and functional properties of soluble forms of membrane cofactor protein (CD46) of complement: identification of forms increased in cancer patients’ sera. Int Immunol 7:727–736

    Article  PubMed  CAS  Google Scholar 

  6. Kawano M (2000) Complement regulatory proteins and autoimmunity. Arch Immunol Ther Exp (Warsz) 48:367–372

    CAS  Google Scholar 

  7. Fett AL, Hermann MM, Muether PS, Kirchhof B, Fauser S (2012) Immunohistochemical localization of complement regulatory proteins in the human retina. Histol Histopathol 27:357–364

    PubMed  Google Scholar 

  8. Saunders R, Abarrategui-Garrido C, Frémeaux-Bacchi V, Goicoechea de Jorge E, Goodship T, López Trascasa M et al (2007) The interactive Factor H-atypical hemolytic uremic syndrome mutation database and website: update and integration of membrane cofactor protein and Factor I mutations with structural models. Hum Mutat 28(3):222–234

    Article  PubMed  CAS  Google Scholar 

  9. Westra D, Volokhina E, van der Heijden E, Vos A, Huigen M, Jansen J et al (2010) Genetic disorders in complement (regulating) genes in patients with atypical haemolytic uraemic syndrome (aHUS). Nephrol Dial Transplant 25:2195–2202

    Article  PubMed  CAS  Google Scholar 

  10. Riley RC, Kemper C, Leung M, Atkinson JP (2002) Characterization of human membrane cofactor protein (MCP; CD46) on spermatozoa. Mol Reprod Dev 62:534–546

    Article  PubMed  CAS  Google Scholar 

  11. Kawamoto A, Ohashi K, Kishikawa H, Zhu LQ, Azuma C, Murata Y (1999) Two-color fluorescence staining of lectin and anti-CD46 antibody to assess acrosomal status. Fertil Steril 71:497–501

    Article  PubMed  CAS  Google Scholar 

  12. Ruseva MM, Hughes TR, Donev RM, Sivasankar B, Pickering MC, Wu X et al (2009) Crry deficiency in complement sufficient mice: C3 consumption occurs without associated renal injury. Mol Immunol 46:803–811

    Article  PubMed  CAS  Google Scholar 

  13. Segerman A, Atkinson J, Marttila M, Dennerquist V, Wadell G, Arnberg N (2003) Adenovirus type 11 uses CD46 as a cellular receptor. J Virol 77:9183–9191

    Article  PubMed  CAS  Google Scholar 

  14. Santoro F, Greenstone H, Insinga A, Liszewski M, Atkinson J, Lusso P et al (2003) Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46. J Biol Chem 278:25964–25969

    Article  PubMed  CAS  Google Scholar 

  15. Cattaneo R (2004) Four viruses, two bacteria, and one receptor: membrane cofactor protein (CD46) as pathogens’ magnet. J Virol 78:4385–4388

    Article  PubMed  CAS  Google Scholar 

  16. Astier A, Trescol-Biémont M, Azocar O, Lamouille B, Rabourdin-Combe C (2000) Cutting edge: CD46, a new costimulatory molecule for T cells, that induces p120CBL and LAT phosphorylation. J Immunol 164:6091–6095

    PubMed  CAS  Google Scholar 

  17. Kemper C, Chan A, Green J, Brett K, Murphy K, Atkinson J (2003) Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421:388–392

    Article  PubMed  CAS  Google Scholar 

  18. Cardone J, Le Friec G, Vantourout P, Roberts A, Fuchs A, Jackson I et al (2010) Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells. Nat Immunol 11:862–871

    Article  PubMed  CAS  Google Scholar 

  19. Le Friec G, Sheppard D, Whiteman P, Karsten CM, Al-Tilib Shamoun S et al (2012) The novel Jagged1 and CD46 interaction on T lymphocytes mediates a critical defence mechanism of human adaptive Th1 immunity. Nat Immunol 13:1213–1221

    Article  PubMed  Google Scholar 

  20. Astier AL, Meiffren G, Freeman S, Hafler DA (2006) Alterations in CD46-mediated Tr1 regulatory T cells in patients with multiple sclerosis. J Clin Invest 116:3252–3257

    Article  PubMed  CAS  Google Scholar 

  21. Cardone J, Al-Shouli S, Kemper C (2011) A novel role for CD46 in wound repair. Front Immunol 2:28

    Article  PubMed  Google Scholar 

  22. Hofman P, Hsi B, Manie S, Fenichel P, Thyss A, Rossi B (1994) High expression of the antigen recognized by the monoclonal antibody GB24 on human breast carcinomas: a preventive mechanism of malignant tumor cells against complement attack? Breast Cancer Res Treat 32:213–219

    Article  PubMed  CAS  Google Scholar 

  23. Cho SW, Oglesby TJ, Hsi BL, Adams EM, Atkinson JP (1991) Characterization of three monoclonal antibodies to membrane co-factor protein (MCP) of the complement system and quantification of MCP by radioassay. Clin Exp Immunol 83:257–261

    Article  PubMed  CAS  Google Scholar 

  24. Manchester M, Valsamakis A, Kaufman R, Liszewski MK, Alvarez J, Atkinson JP et al (1995) Measles virus and C3 binding sites are distinct on membrane cofactor protein (CD46). Proc Natl Acad Sci U S A 92:2303–2307

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work is supported by an MRC Research Grant (Grant no. G1002165 awarded to CK), the Medical Research Council Centre for Transplantation, Guy’s Hospital, King’s College and the Department of Health, National Institute for Health Research comprehensive Biomedical Research Centre award to Guy’s & St. Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust.

Author information

Authors and Affiliations

  1. Division of Transplantation Immunology and Mucosal Biology, MRC Centre for Transplantation, King’s College London, Guy’s Hospital, London, UK

    Martin Kolev & Claudia Kemper

Authors
  1. Martin Kolev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claudia Kemper
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Division of Infectious Diseases, Brigham and Women’s Hospital, Harvard University Harvard Medical School, Boston, Massachusetts, USA

    Mihaela Gadjeva

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Kolev, M., Kemper, C. (2014). Detection of Cell Membrane-Bound CD46 Using Flow Cytometry. In: Gadjeva, M. (eds) The Complement System. Methods in Molecular Biology, vol 1100. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-724-2_27

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-724-2_27

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-723-5

  • Online ISBN: 978-1-62703-724-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation