Partial immunochemical characterization of the Ro and La proteins using antibodies from patients with the sicca syndrome and lupus erythematosus.
Ro and La are intracellular ribonucleoproteins that are frequent targets for autoantibodies in the sicca syndrome and in lupus erythematosus. We analyzed the m.w. of the protein (antigen) moieties of Ro and La in saline cell extracts of human spleen by SDS-PAGE and immunoblotting, gel filtration, and sucrose density ultracentrifugation with radioimmunoassay. pI values for Ro and La proteins were established by isoelectric focusing on thin layer agarose gels and immunoblotting on nitrocellulose transfers. The La protein had an m.w. of approximately 43 kd and was heterogeneous in charge, with pI values from 4.2 to 4.8. Composite two-dimensional maps developed by immunoblotting revealed a characteristic set of seven dots of m.w. 43 kd. Ro determinants were identified on polypeptides of 50 and/or 57 kd. Antigenic activity was also detected in the void volume of spleen extract fractionated by Sephadex G-200 and in 8 to 9S and greater than 19S regions of sucrose gradients, suggesting either aggregation of the Ro protein or participation in protein-protein complexes. pI values of 4.3 to 5.5 were obtained for the Ro antigen, and two-dimensional maps revealed that the 57 kd polypeptide had a similar charge heterogeneity to the La protein, whereas the 50 kd polypeptide had a different fingerprint. Immunoblotting of extracts from bovine, rabbit, and dog extracts showed that antibodies to Ro and La reacted with a limited number of polypeptides (m.w. 50 and/or 57 kd for anti-Ro and 43 or 50 kd for anti-La). These studies support the physical independence of the isolated Ro and La polypeptides, although a precursor product or functional relationship in vivo is possible. These studies also suggest that, in addition to Western blotting, techniques involving immunodeletion, isoelectric focusing with capillary immunoblotting, and 2D immunoblotting provide useful approaches to characterize saline-soluble cellular antigens.
D000941:Antigens / Q000276:immunology / Q000302:isolation & purification*; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D012859:Sjogren's Syndrome / Q000276:immunology*; D000097585:SS-B Antigen; D000099207:SS-A Antigen
D000941:Antigens; D001323:Autoantibodies; D001324:Autoantigens; D003101:Collodion; D010455:Peptides; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen; C116006:RO60 protein, human