DOI Page / Citation link:
https://doi.org/10.11588/diglit.48120#0071
Abstracts
53
Molecular Cloning of the Human Autoantigen KU (p70/p80),
a DNA-Terminal-Binding Protein Complex
T. Mimori1, N. Hama1, A. Suwa1, Y. Ohsone1, M. Akizuki1, M. Homma1,
A. J. Griffith2, and J. A. Hardin2
’Keio University School of Medicine, Tokyo, Japan
2Yale University School of Medicine, New Haven, Connecticut, USA
Anti-Ku autoantibodies in patients with PSS-PM overlap syndrome recognize
a 70 kD/80 kD protein heterodimer which binds selectively to terminal region of
dsDNA. In the present study, we isolated and characterized cDNA clones that en-
code the Ku autoantigen, and intended to use them for clinical application.
In the first step, a human hepatoma cell cDNA library inserted into the EcoRI
site of kgtll phages were screened with an anti-Ku serum to identify plaques ex-
pressing Ku epitopes. Three positive clones (K 14, K68 and K71) were isolated and
demonstrated to express fusion proteins recognized by anti-Ku antibodies. In the
second step, Okayama-Berg cDNA library was screened by using an isolated
cDNA encoding the partial 80kD-Ku sequence (K71) as a probe for colony
hybridization. The clone Ku80-6 that contained the longest cDNA insert (3.4 kb,
identical with the larger mRNA from two mRNAs hybridized by K71) was
isolated, and its nucleotide sequence was determined by Sanger's dideoxy meth-
od. The Ku 80-6 cDNA contained a single long open reading frame encoding 732
amino acids (Mr = 82713) followed by 1081 bases of 3'-non-coding region and a
poly(A) tail. The putative polypeptide contained a Leu-Ser repeat in every 7
amino acid residue which may contribute the formation of “Leucine zipper”. The
sequence of the Ku 80-6 was compared with previously described sequences using
the computer search, but no significant homology was found either in DNA (Gen-
Bank) or protein (NBRF) data bank. In Southern blot, the Ku 80-6 hybridized
with 4-5 DNA bands from human leukocyte DNA digested with various restric-
tion enzymes. It was noted that RFLP was seen in the 2.8 kb-Hindlll fragment
and likely to associate with SLE patients who had anti-U 1-RNP antibodies. Us-
ing purified fusionproteins expressing from K14 (70 kD) and K71 (80 kD) clones,
we developed ELISA to detect anti-Ku antibodies. When sera from various col-
lagen diseases were screened by this assay, anti-Ku antibodies were mostly
detected in patients with overlap syndrome, consistent with the result of a conven-
tional immunodiffusion assay.
Our cDNA encoding the Ku antigenic proteins will be a powerful tool to study
not only the structure and function of the Ku autoantigen but also pathogenetic
mechanisms of autoimmune diseases.
53
Molecular Cloning of the Human Autoantigen KU (p70/p80),
a DNA-Terminal-Binding Protein Complex
T. Mimori1, N. Hama1, A. Suwa1, Y. Ohsone1, M. Akizuki1, M. Homma1,
A. J. Griffith2, and J. A. Hardin2
’Keio University School of Medicine, Tokyo, Japan
2Yale University School of Medicine, New Haven, Connecticut, USA
Anti-Ku autoantibodies in patients with PSS-PM overlap syndrome recognize
a 70 kD/80 kD protein heterodimer which binds selectively to terminal region of
dsDNA. In the present study, we isolated and characterized cDNA clones that en-
code the Ku autoantigen, and intended to use them for clinical application.
In the first step, a human hepatoma cell cDNA library inserted into the EcoRI
site of kgtll phages were screened with an anti-Ku serum to identify plaques ex-
pressing Ku epitopes. Three positive clones (K 14, K68 and K71) were isolated and
demonstrated to express fusion proteins recognized by anti-Ku antibodies. In the
second step, Okayama-Berg cDNA library was screened by using an isolated
cDNA encoding the partial 80kD-Ku sequence (K71) as a probe for colony
hybridization. The clone Ku80-6 that contained the longest cDNA insert (3.4 kb,
identical with the larger mRNA from two mRNAs hybridized by K71) was
isolated, and its nucleotide sequence was determined by Sanger's dideoxy meth-
od. The Ku 80-6 cDNA contained a single long open reading frame encoding 732
amino acids (Mr = 82713) followed by 1081 bases of 3'-non-coding region and a
poly(A) tail. The putative polypeptide contained a Leu-Ser repeat in every 7
amino acid residue which may contribute the formation of “Leucine zipper”. The
sequence of the Ku 80-6 was compared with previously described sequences using
the computer search, but no significant homology was found either in DNA (Gen-
Bank) or protein (NBRF) data bank. In Southern blot, the Ku 80-6 hybridized
with 4-5 DNA bands from human leukocyte DNA digested with various restric-
tion enzymes. It was noted that RFLP was seen in the 2.8 kb-Hindlll fragment
and likely to associate with SLE patients who had anti-U 1-RNP antibodies. Us-
ing purified fusionproteins expressing from K14 (70 kD) and K71 (80 kD) clones,
we developed ELISA to detect anti-Ku antibodies. When sera from various col-
lagen diseases were screened by this assay, anti-Ku antibodies were mostly
detected in patients with overlap syndrome, consistent with the result of a conven-
tional immunodiffusion assay.
Our cDNA encoding the Ku antigenic proteins will be a powerful tool to study
not only the structure and function of the Ku autoantigen but also pathogenetic
mechanisms of autoimmune diseases.