DOI Page / Citation link:
https://doi.org/10.11588/diglit.48120#0047
Abstracts
29
Molecular Cloning and Expression of cDNAs Encoding snRNP
Polypeptide Autoantigens
S.O. Hoch, J. A. Haselby, M. Jannatipour, and L.A. Rokeach
The Agouron Institute, La Jolla, CA 92037, USA
Anti-Sm is an antibody specificity associated with the autoimmune disease
systemic lupus erythematosus. Sm antibodies precipitate the small nuclear
ribonucleoprotein (SnRNP) complexes which contain the Ul, U2, U4, U5 and
U6 snRNAs and a minimum of eleven polypetides. Functionally the Sm snRNP
are involved in the maturation of the nascent mRNA transcript. Each snRNP con-
tains a common core of six polypeptides of which three, the B' (27000 molecular
weight), B (26000 m.w.) and D (13 000m.w.) proteins, are synonymous with the
major Sm autoantigens. The Sm snRNP have proven readily amenable to isola-
tion. Once isolated, the snRNP are resistant to chromatographic fractionation in-
to the individual polypeptides; however, there are compelling reasons to have such
moieties relative to these proteins as snRNP components and as autoantigens.
Thus, we undertook to circumvent this problem by cloning the cDNAs encoding
the three Sm polypeptides of interest, B', B and D. Amino-terminal sequences
were obtained for the three polypeptides. These sequences were used to design
oligonucleotide probes to successfully screen a human B-lymphocyte cDNA
library for clones encoding Sm-D (1) and Sm-B7B (2). Evaluation of the
predicted amino acid sequences suggested the importance of the carboxy-terminal
sequence of both Sm-D and Sm-B7B relative to their immunoreactivity and func-
tion as snRNP components. In order to study these features we have undertaken
the heterologous expression of these Sm autoantigens in different prokaryotic and
eukaryotic hosts. Sm-B7B was expressed in an immunoreactive form as a fusion
protein with TrpE (anthranilate synthase) of E. coli. Multiple translational fu-
sions between this bacterial trpE gene and fragments encompassing the length of
the Sm-B7B coding sequence were constructed. All were screened by im-
munoblotting against a number of autoimmune sera. These analyses showed that
the major Sm determinant was located at the carboxyterminus of the protein, in
the region containing a proline-rich repetitive unit that is shared with other
snRNP and nucleic acid binding proteins. The same approach, using TrpE fusion,
has been utilized for Sm-D epitope mapping, underscoring the importance of the
glycine-arginine repeated motif at its carboxy terminus. Experiments continue to
allow for the expression of these polypeptides in different host systems to study
the possible effect of posttranslational modification on immunoreactivity and to
generate non-fusion entities to facilitate analyses of the assembly of the core
snRNA structure.
29
Molecular Cloning and Expression of cDNAs Encoding snRNP
Polypeptide Autoantigens
S.O. Hoch, J. A. Haselby, M. Jannatipour, and L.A. Rokeach
The Agouron Institute, La Jolla, CA 92037, USA
Anti-Sm is an antibody specificity associated with the autoimmune disease
systemic lupus erythematosus. Sm antibodies precipitate the small nuclear
ribonucleoprotein (SnRNP) complexes which contain the Ul, U2, U4, U5 and
U6 snRNAs and a minimum of eleven polypetides. Functionally the Sm snRNP
are involved in the maturation of the nascent mRNA transcript. Each snRNP con-
tains a common core of six polypeptides of which three, the B' (27000 molecular
weight), B (26000 m.w.) and D (13 000m.w.) proteins, are synonymous with the
major Sm autoantigens. The Sm snRNP have proven readily amenable to isola-
tion. Once isolated, the snRNP are resistant to chromatographic fractionation in-
to the individual polypeptides; however, there are compelling reasons to have such
moieties relative to these proteins as snRNP components and as autoantigens.
Thus, we undertook to circumvent this problem by cloning the cDNAs encoding
the three Sm polypeptides of interest, B', B and D. Amino-terminal sequences
were obtained for the three polypeptides. These sequences were used to design
oligonucleotide probes to successfully screen a human B-lymphocyte cDNA
library for clones encoding Sm-D (1) and Sm-B7B (2). Evaluation of the
predicted amino acid sequences suggested the importance of the carboxy-terminal
sequence of both Sm-D and Sm-B7B relative to their immunoreactivity and func-
tion as snRNP components. In order to study these features we have undertaken
the heterologous expression of these Sm autoantigens in different prokaryotic and
eukaryotic hosts. Sm-B7B was expressed in an immunoreactive form as a fusion
protein with TrpE (anthranilate synthase) of E. coli. Multiple translational fu-
sions between this bacterial trpE gene and fragments encompassing the length of
the Sm-B7B coding sequence were constructed. All were screened by im-
munoblotting against a number of autoimmune sera. These analyses showed that
the major Sm determinant was located at the carboxyterminus of the protein, in
the region containing a proline-rich repetitive unit that is shared with other
snRNP and nucleic acid binding proteins. The same approach, using TrpE fusion,
has been utilized for Sm-D epitope mapping, underscoring the importance of the
glycine-arginine repeated motif at its carboxy terminus. Experiments continue to
allow for the expression of these polypeptides in different host systems to study
the possible effect of posttranslational modification on immunoreactivity and to
generate non-fusion entities to facilitate analyses of the assembly of the core
snRNA structure.