DOI Seite / Zitierlink:
https://doi.org/10.11588/diglit.48120#0125
Abstracts
107
Epitope Progression in Autoantibody Production Suggested by Epitope
Mappings of SS-B/La and U2snRNP B" Protein
K. Yamamoto1, H. Kohsaka1, H. Miura1, H. Fujii1, Y. Misaki1, N. Miyasaka2,
K. Nishioka2, and T. Miyamoto1
'Department of Medicine and Physical Therapy, Faculty of Medicine, University of
Tokyo, Japan
2 Institute of Rheumatology, Tokyo Women’s Medical College, Tokyo, Japan
In order to understand the mechanisms of autoantibody productions in
autoimmune patients, we cloned cDNAs encoding the SS-B/La and the U2
snRNP B" proteins. The initial cloning was performed with Agtl 1 cDNA libraries
from human fibroblasts and autoimmune patients’ sera. The isolated clones were
characterized with the purified antibodies against the fusion proteins produced
by the cDNAs in E. coli. The clones identified to encode the SS-B/La or
U2snRNP B" protein were then used as probes for obtaining full length cDNAs
from Okayama-Berg cDNA libraries. A clone termed FS4 was found to encode
the entire coding region of the U 2snRNP B" protein. A clone called FSB was also
determined to contain the whole coding region of the SS-B/La protein. To obtain
large amount of the antigen molecules in E. coli, we have subcloned these FS4
and FSB into another expression vector pEX. Positive clones producing fusion
proteins with cro-/Lgalactosidase were selected with patients’ sera.
Enzyme linked immunosorbent assay (ELISA) was established with these fu-
sion proteins. FS4EX and FSBEX. In the case of the SS-B/La protein, it was
found that there is a good correlation between the reactivities of patients sera to
FSBEX and the conventionally determined specificities of the sera. However, for
the U2snRNP B" protein, about 50% of anti-RNP positive sera were found to
react with FS4EX, which is rather high frequency compared to the previous
reports.
For epitope mapping, the pEX plasmids containing the cDNAs were en-
zymatically manipulated to produce deletion mutants. The resultant several dele-
tion mutants were examined for the reactivities with many patients’ sera. It was
found that the U2snRNP B" protein contains two main epitopes closely located
in the C terminus. The SS-B/La protein was also shown to possess two major
epitopes located in the N terminal and C terminal region respectively. Every pa-
tients’ sera possessing the reactivities to these proteins recognized the major
epitope(s). However, there were some patients’ sera which additionally recognized
other epitopes on the molecules. So far, these patients seemed to be rather ad-
vanced in the clinical courses. In addition, in the case of the U2snRNP B" protein,
the main epitopes were found to be cross-reactive to the U1 snRNP A protein.
Thus, it is possible that the initial reactivity to the B" protein is in the main
107
Epitope Progression in Autoantibody Production Suggested by Epitope
Mappings of SS-B/La and U2snRNP B" Protein
K. Yamamoto1, H. Kohsaka1, H. Miura1, H. Fujii1, Y. Misaki1, N. Miyasaka2,
K. Nishioka2, and T. Miyamoto1
'Department of Medicine and Physical Therapy, Faculty of Medicine, University of
Tokyo, Japan
2 Institute of Rheumatology, Tokyo Women’s Medical College, Tokyo, Japan
In order to understand the mechanisms of autoantibody productions in
autoimmune patients, we cloned cDNAs encoding the SS-B/La and the U2
snRNP B" proteins. The initial cloning was performed with Agtl 1 cDNA libraries
from human fibroblasts and autoimmune patients’ sera. The isolated clones were
characterized with the purified antibodies against the fusion proteins produced
by the cDNAs in E. coli. The clones identified to encode the SS-B/La or
U2snRNP B" protein were then used as probes for obtaining full length cDNAs
from Okayama-Berg cDNA libraries. A clone termed FS4 was found to encode
the entire coding region of the U 2snRNP B" protein. A clone called FSB was also
determined to contain the whole coding region of the SS-B/La protein. To obtain
large amount of the antigen molecules in E. coli, we have subcloned these FS4
and FSB into another expression vector pEX. Positive clones producing fusion
proteins with cro-/Lgalactosidase were selected with patients’ sera.
Enzyme linked immunosorbent assay (ELISA) was established with these fu-
sion proteins. FS4EX and FSBEX. In the case of the SS-B/La protein, it was
found that there is a good correlation between the reactivities of patients sera to
FSBEX and the conventionally determined specificities of the sera. However, for
the U2snRNP B" protein, about 50% of anti-RNP positive sera were found to
react with FS4EX, which is rather high frequency compared to the previous
reports.
For epitope mapping, the pEX plasmids containing the cDNAs were en-
zymatically manipulated to produce deletion mutants. The resultant several dele-
tion mutants were examined for the reactivities with many patients’ sera. It was
found that the U2snRNP B" protein contains two main epitopes closely located
in the C terminus. The SS-B/La protein was also shown to possess two major
epitopes located in the N terminal and C terminal region respectively. Every pa-
tients’ sera possessing the reactivities to these proteins recognized the major
epitope(s). However, there were some patients’ sera which additionally recognized
other epitopes on the molecules. So far, these patients seemed to be rather ad-
vanced in the clinical courses. In addition, in the case of the U2snRNP B" protein,
the main epitopes were found to be cross-reactive to the U1 snRNP A protein.
Thus, it is possible that the initial reactivity to the B" protein is in the main