Skip to main content
SpringerLink
Log in

Antigen-binding abilities of anti-nephrin antibody are prescribed by signal sequence of expression vector in genetic immunization

  • Original Article
  • Published:
Clinical and Experimental Nephrology Aims and scope Submit manuscript

Abstract

Background

Nephrin is an essential protein for maintaining the normal structure of podocyte foot processes and the glomerular filtration barrier. To analyze the mechanism of proteinuria and nephrotic syndrome, we previously reported on a new method of producing polyclonal anti-nephrin antibody by genetic immunization. In the present paper, we investigate the effect of signal peptide sequence cDNA on the characteristics of polyclonal anti-nephrin antibodies induced by genetic immunization.

Methods

Five fragments of nephrin cDNA with or without signal peptide sequence were inserted into the pTARGET™ vector. Rats were immunized with these vectors by the gene gun method. Sera obtained from rats at 14 weeks following immunization were analyzed by Western blotting, immunoprecipitation, flow cytometry and immunohistochemistry.

Results

Four different antibodies induced by cDNA encoding nephrin protein fragments without signal peptide showed antigen site-specific binding to fragmented glycosylation-disturbed nephrin proteins. These antibodies also reacted to a glycosylation-disturbed full-length nephrin protein (inhibited by tunicamycin), but did not react to either a native nephrin protein or a fully glycosylated conformational nephrin protein. Four different antibodies induced by cDNA encoding nephrin fragments with signal peptide showed an antigen site-specific binding to glycosylation-disturbed nephrin protein fragments. In addition, these antibodies reacted to both a native nephrin protein and a full-length glycosylated conformational nephrin protein.

Conclusions

The absence of signal peptide sequence cDNA in the expression vector produced antibodies specific for glycosylation-disturbed proteins, while its presence produced antibodies that bound to native or fully glycosylated conformational protein.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Tryggvason K, Patrakka J, Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria. N Engl J Med. 2006;354:1387–401.

    Article  CAS  Google Scholar 

  2. Yan K, Khoshnoodi J, Ruotsalainen V, Tryggvason K. N-linked glycosylation is critical for the plasma membrane localization of nephrin. J Am Soc Nephrol. 2002;13:1385–9.

    Article  CAS  Google Scholar 

  3. Khoshnoodi J, Hill S, Tryggvason K, Friedman DB. Identification of N-linked glycosylation sites in human nephrin using mass spectrometry. J Mass Spectrom. 2007;42:370–9.

    Article  CAS  Google Scholar 

  4. Gerace L, Gilmore R, Johnson A, Lazarow P, Neupert W, O’Shea E, et al. The endoplasmic reticulum. In: Alberts B, Johnson A, Lewis J, Raff M, et al., editors. Molecular biology of the cell. 4th ed. New York: Newton Press; 2004. p. 689–709.

    Google Scholar 

  5. Kitamura M. Endoplasmic reticulum stress in the kidney. Clin Exp Nephrol. 2008;12:317–25.

    Article  CAS  Google Scholar 

  6. Aoyama T, Kamata K, Yamanaka N, Takeuchi Y, Higashihara M, Kato S. Characteristics of polyclonal anti-human nephrin antibodies induced by genetic immunization using nephrin cDNA. Nephrol Dial Transplant. 2006;21:1073–81.

    Article  CAS  Google Scholar 

  7. Emr S, Kelly R, Pelham HR, Schekman G, Schiavo JL, Schwarth K. What is the purpose of glycosylation? In: Alberts B, Johnson A, Lewis J, et al., editors. Molecular biology of the cell. 4th ed. New York: Newton Press; 2004. p. 735–6.

    Google Scholar 

  8. Harlow E, Lane D. Epitope mapping. In: Harlow E, Lane D, editors. Using antibody: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 1999. pp. 382–385.

  9. Nakajo A, Khoshnoodi J, Takenaka H, Hagiwara E, Watanabe T, Kawakami H, et al. Mizoribine corrects defective nephrin biogenesis by restoring intracellular energy balance. J Am Soc Nephrol. 2007;18:2554–64.

    Article  CAS  Google Scholar 

  10. Inagi R, Kumagai T, Nishi H, Kawakami T, Miyata T, Fujita T, et al. Preconditioning with endoplasmic reticulum stress ameliorates mesangioproliferative glomerulonephritis. J Am Soc Nephrol. 2008;19:915–22.

    Article  CAS  Google Scholar 

  11. Cybulsky AV, Takano T, Papillon J, Bijian K. Role of the endoplasmic reticulum unfolded protein response in glomerular epithelial cell injury. J Biol Chem. 2005;280:24396–403.

    Article  CAS  Google Scholar 

  12. Cybulsky AV, Takano T, Papillon J, Khadir A, Liu J, Peng H. Complement C5b-9 membrane attack complex increases expression of endoplasmic reticulum stress proteins in glomerular epithelial cells. J Biol Chem. 2002;277:41342–51.

    Article  CAS  Google Scholar 

  13. Inagi R, Nangaku M, Onogi H, Ueyama H, Kitao Y, Nakazato K, et al. Involvement of endoplasmic reticulum (ER) stress in podocyte injury induced by excessive protein accumulation. Kidney Int. 2005;68:2639–50.

    Article  CAS  Google Scholar 

  14. Bijian K, Cybulsky AV. Stress proteins in glomerular epithelial cell injury. Contrib Nephrol. 2005;148:8–20.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

S. Naito and K. Kamata equally contributed to this article. We thank Dr. Michihito Okubo for his critically reading and helpful suggestion to this article, and Mrs. Naoko Ishigaki for technical assistance.

Author information

Authors and Affiliations

  1. Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, Sagamihara, Kanagawa, 252-0374, Japan

    Shokichi Naito, Kouju Kamata, Togo Aoyama, Tomoko Okamoto & Hiromi Tazaki

  2. Department of Nephrology in Internal Medicine, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0374, Japan

    Shokichi Naito, Kouju Kamata, Togo Aoyama, Tomoko Okamoto & Hiromi Tazaki

  3. Department of Surgery, National Defense Medical Collage, Tokorozawa, Saitama, Japan

    Nozomu Yamanaka

Authors
  1. Shokichi Naito
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kouju Kamata
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Togo Aoyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nozomu Yamanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tomoko Okamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hiromi Tazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kouju Kamata.

About this article

Cite this article

Naito, S., Kamata, K., Aoyama, T. et al. Antigen-binding abilities of anti-nephrin antibody are prescribed by signal sequence of expression vector in genetic immunization. Clin Exp Nephrol 15, 339–345 (2011). https://doi.org/10.1007/s10157-010-0399-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10157-010-0399-4

Keywords

Search

Navigation