Characterization of recombinant human autoantibody fragments directed toward the autoantigenic U1-70K protein

The U1-70K protein is specifically bound to stemloop I of the U1 small nucl ear RNA contained in the U1 small nuclear ribonucleoprotein complex (U1 snR NP), which is involved in the splicing of pre-mRNA. All components of the U 1 snRNP complex, including the U1-70K protein, are important autoantigens i n patients with systemic lupus erythematosus (SLE) and mixed connective tis sue disease (MCTD). Here we describe for the first time the selection and c haracterization of recombinant human anti-U1-70K single chain autoantibody fragments (anti-hU1-70K scFv) from autoimmune patient-derived phage display antibody libraries. All scFv specifically recognize parts of the hU1-70K p rotein and its apoptotic 40-kDa cleavage product. In Western blotting assay s a number of scFv preferentially recognize the 40-kDa apoptotic cleavage f ragment of the U1-70K protein, suggesting a possible involvement of this ap optotic cleavage product in the autoimmune response of patients. The germli ne gene usage of these recombinant autoantibodies was also determined. Usin g several U1-70K deletion and point mutants of both human (h) and Drosophil a melanogaster (Dm) origin, it was established that the U1-70K epitope that is recognized by the anti-hU1-70K scFv is located within the RNA binding d omain.