Purification and characterization of the Ro and La antigens. Modulation of their binding affinities to poly(U) by phosphorylation and the presence of ATP.
Both the La and the Ro antigen (the latter for the first time) were purified to apparent homogeneity. Ro was found to be a 94 (90)-kDa and La a 50-kDa polypeptide. Both antigens bind to RNA with a high preference for poly(U). The binding hierarchy is U much greater than G greater than A greater than C for La and U much greater than C greater than G greater than A for Ro. Only 15% of the total amount of La or 21% of that of Ro, present in the L5178y cell extract, is able to bind to poly(U), indicating the existence of RNA binding and nonbinding subclasses of La and Ro. The purified antigens were used for the isolation of monospecific antibodies. These antibodies were specific for their respective antigen and did not cross-react. Both the Ro and the La antigen are phosphorylated in vitro by the cytoplasmic protein kinase CII, whereas the nuclear protein kinases NI and NII are unable to phosphorylate the antigens. After phosphorylation or in the presence of ATP the binding affinity of both antigens to poly(U) strongly decreases. The phosphorylation reaction together with the immunoprecipitation by the monospecific antibodies represents a highly sensitive and specific assay which was used during purification and characterization of the Ro and La antigen.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000906:Antibodies / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001324:Autoantigens / Q000032:analysis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D011072:Poly U / Q000378:metabolism*; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen
D000255:Adenosine Triphosphate; D000906:Antibodies; D001324:Autoantigens; D011072:Poly U; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen; C116006:RO60 protein, human