Anti-La monoclonal antibodies recognizing epitopes within the RNA-binding domain of the La protein show differential capacities to immunoprecipitate RNA-associated La protein.
The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both the nucleus and cytoplasm of eukaryotic cells, where it is found associated with RNA polymerase III transcripts. We have investigated the capacity of anti-La monoclonal antibodies SW1, SW3, and SW5 to immunoprecipitate human La ribonucleoprotein particles. Distinct differences were observed for SW3 in comparison with SW1 and SW5. While SW1 and SW5 precipitated ribonucleoproteins containing pre-tRNA, pre-5S rRNA, hY RNAs, pre-U6 snRNA or the viral EBER1 and VA RNAs, SW3 precipitated only ribonucleoproteins containing VA RNAs or (the precursor of) 7-2 RNA. Mapping of the epitopes recognized by SW1, SW3, and SW5 revealed that all three monoclonal antibodies recognize an epitope within the domain of the protein formed by the ribonucleoprotein motif. Cross-competition studies suggested that the epitope recognized by SW1 and SW5 are identical but distinct from the epitope recognized by SW3. Further analyses of the recognition of La from other species by these monoclonal antibodies revealed that they all reacted with bovine La and were not reactive with La from rodents and Xenopus laevis. Replacement of a single amino acid in the human protein by its murine counterpart abolished recognition by SW1 and SW5, but had no effect on recognition by SW3. Taken together, our results indicate that SW1 and SW5 recognize the same epitope and that SW3 recognizes a distinct epitope, both of which are located in the RNA-binding domain of La, and that the accessibility of these epitopes is differentially influenced by the association of La with various RNA polymerase III transcripts.
Pruijn
G J
GJ
Department of Biochemistry, University of Nijmegen, The Netherlands.
Thijssen
J P
JP
Smith
P R
PR
Williams
D G
DG
Van Venrooij
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D003429:Cross Reactions; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D012313:RNA / Q000378:metabolism; D051381:Rats; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D000097585:SS-B Antigen
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D001324:Autoantigens; D000939:Epitopes; D012313:RNA; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; C035357:SS-B antibodies