DOI Seite / Zitierlink:
https://doi.org/10.11588/diglit.48120#0044
26 Molecular and Cell Biology of Autoantibodies and Autoimmunity
Mapping of Autoimmune Reactive Sites on snRNP Proteins
W.J. Habets
Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen,
The Netherlands
Antibodies from patients with SLE react with snRNP particles and inhibit
pre-mRNA splicing in vitro. Therefore it is thought that these autoantibodies
recognize functional sites on snRNA-associated proteins. For this reason, but also
because knowledge of the primary structure of autoimmune epitopes might pro-
vide insight in the etiology of autoimmune diseases, we undertook epitope map-
ping experiments with the U1 snRNP-specific A protein and the U2 snRNP-
specific B" protein.
Random DNAse I fragments of cDNA clones encoding these two closely
related proteins (ref. 1 and 2) were expressed in Agtll and recombinant plaques
were screened with antibodies from an SLE patient. Two antigenic sites were map-
ped on the A protein which we termed epitope 1 and epitope 2 (ref. 3). Epitope
1 (PPPGMIPPPGLAPGQIPPGAM) is situated in a proline-rich region of the A
protein that has no counterpart on the B" protein. When this proline-rich region
was compared with the primary structure of other snRNP proteins, however,
epitope 1-like regions were found in snRNA-associated proteins B', B, C and N.
Screening of a larg number of patients sera revealed that the epitope 1-like regions
on all these snRNP proteins were targeted by specific subsets of autoantibodies.
Epitope 1-reactive antibodies could be affinity purified from anti-Sm and anti-
U(U1, U2)RNP sera and were shown to cross-react with either snRNP proteins
B7B, N and an as yet unidentified 50K non-snRNP protein, or with snRNP pro-
teins B7B and C. To substantiate these findings, we synthesized peptides based
on the epitope 1 region of protein A and the epitope 1-like region of protein N.
These peptides reacted in ELISA with antibodies from SLE sera. Epitope 2
(PGFKEVRLVPGRHDIAFVEFDNEVQ) is situated in a region of the A protein
that is highly similar to a carboxy terminal region in protein B". It was therefore
not surprising that epitope 2 bound A and B" cross-reactive antibodies from anti-
(Ul, U2)RNP patients sera. Epitope 2-reactive antibodies were not found in anti-
Sm or anti-(Ul)RNP sera.
Using a deletion mutagenesis approach, at least three other autoepitopecon-
taining regions on the A protein could be identified. One of these regions contain-
ed an immunodominant epitope reactive with a least 70% of anti-snRNP sera
from patients with different connective tissue diseases.
For the mapping of epitopes on the U2 snRNP-specific B" protein we used
monoclonal anti-U2 RNP antibodies obtained from Balb/c mice immunized with
recombinant /Lgalactosidase-B" fusion protein. Two monoclonal antibodies
Mapping of Autoimmune Reactive Sites on snRNP Proteins
W.J. Habets
Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen,
The Netherlands
Antibodies from patients with SLE react with snRNP particles and inhibit
pre-mRNA splicing in vitro. Therefore it is thought that these autoantibodies
recognize functional sites on snRNA-associated proteins. For this reason, but also
because knowledge of the primary structure of autoimmune epitopes might pro-
vide insight in the etiology of autoimmune diseases, we undertook epitope map-
ping experiments with the U1 snRNP-specific A protein and the U2 snRNP-
specific B" protein.
Random DNAse I fragments of cDNA clones encoding these two closely
related proteins (ref. 1 and 2) were expressed in Agtll and recombinant plaques
were screened with antibodies from an SLE patient. Two antigenic sites were map-
ped on the A protein which we termed epitope 1 and epitope 2 (ref. 3). Epitope
1 (PPPGMIPPPGLAPGQIPPGAM) is situated in a proline-rich region of the A
protein that has no counterpart on the B" protein. When this proline-rich region
was compared with the primary structure of other snRNP proteins, however,
epitope 1-like regions were found in snRNA-associated proteins B', B, C and N.
Screening of a larg number of patients sera revealed that the epitope 1-like regions
on all these snRNP proteins were targeted by specific subsets of autoantibodies.
Epitope 1-reactive antibodies could be affinity purified from anti-Sm and anti-
U(U1, U2)RNP sera and were shown to cross-react with either snRNP proteins
B7B, N and an as yet unidentified 50K non-snRNP protein, or with snRNP pro-
teins B7B and C. To substantiate these findings, we synthesized peptides based
on the epitope 1 region of protein A and the epitope 1-like region of protein N.
These peptides reacted in ELISA with antibodies from SLE sera. Epitope 2
(PGFKEVRLVPGRHDIAFVEFDNEVQ) is situated in a region of the A protein
that is highly similar to a carboxy terminal region in protein B". It was therefore
not surprising that epitope 2 bound A and B" cross-reactive antibodies from anti-
(Ul, U2)RNP patients sera. Epitope 2-reactive antibodies were not found in anti-
Sm or anti-(Ul)RNP sera.
Using a deletion mutagenesis approach, at least three other autoepitopecon-
taining regions on the A protein could be identified. One of these regions contain-
ed an immunodominant epitope reactive with a least 70% of anti-snRNP sera
from patients with different connective tissue diseases.
For the mapping of epitopes on the U2 snRNP-specific B" protein we used
monoclonal anti-U2 RNP antibodies obtained from Balb/c mice immunized with
recombinant /Lgalactosidase-B" fusion protein. Two monoclonal antibodies