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Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens.

Publication ,  Journal Article
Wang, J; Chou, CH; Blankson, J; Satoh, M; Knuth, MW; Eisenberg, RA; Pisetsky, DS; Reeves, WH
Published in: Mol Biol Rep
June 1993

The Ku autoantigen is a DNA binding factor consisting of 70 and approximately 80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factors in vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients' sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of approximately 1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610-705, 8-221, and 1-374, respectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506-541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Duke Scholars

Published In

Mol Biol Rep

DOI

ISSN

0301-4851

Publication Date

June 1993

Volume

18

Issue

1

Start / End Page

15 / 28

Location

Netherlands

Related Subject Headings

  • Tumor Cells, Cultured
  • Recombinant Proteins
  • Precipitin Tests
  • Nuclear Proteins
  • Molecular Sequence Data
  • Mice, Inbred BALB C
  • Mice
  • Male
  • Ku Autoantigen
  • Immunoblotting
 

Citation

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Wang, J., Chou, C. H., Blankson, J., Satoh, M., Knuth, M. W., Eisenberg, R. A., … Reeves, W. H. (1993). Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens. Mol Biol Rep, 18(1), 15–28. https://doi.org/10.1007/BF01006891
Wang, J., C. H. Chou, J. Blankson, M. Satoh, M. W. Knuth, R. A. Eisenberg, D. S. Pisetsky, and W. H. Reeves. “Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens.Mol Biol Rep 18, no. 1 (June 1993): 15–28. https://doi.org/10.1007/BF01006891.
Wang J, Chou CH, Blankson J, Satoh M, Knuth MW, Eisenberg RA, et al. Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens. Mol Biol Rep. 1993 Jun;18(1):15–28.
Wang, J., et al. “Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens.Mol Biol Rep, vol. 18, no. 1, June 1993, pp. 15–28. Pubmed, doi:10.1007/BF01006891.
Wang J, Chou CH, Blankson J, Satoh M, Knuth MW, Eisenberg RA, Pisetsky DS, Reeves WH. Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens. Mol Biol Rep. 1993 Jun;18(1):15–28.
Journal cover image

Published In

Mol Biol Rep

DOI

ISSN

0301-4851

Publication Date

June 1993

Volume

18

Issue

1

Start / End Page

15 / 28

Location

Netherlands

Related Subject Headings

  • Tumor Cells, Cultured
  • Recombinant Proteins
  • Precipitin Tests
  • Nuclear Proteins
  • Molecular Sequence Data
  • Mice, Inbred BALB C
  • Mice
  • Male
  • Ku Autoantigen
  • Immunoblotting