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

• Published in: European journal of immunology Vol. 30; no. 10; pp. 3029 - 3038
• Main Authors: Degen, Winfried G. J., Pieffers, Martijn, Welin‐Henriksson, Elisabet, van den Hoogen, Frank H. J., van Venrooij, Walther J., Raats, Jos M. H.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY‐VCH Verlag GmbH 01-10-2000
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal - immunology; Apoptosis; Autoantibodies - chemistry; Autoantibodies - genetics; Autoantibodies - immunology; Autoantigens - immunology; Autoimmune Diseases - immunology; Complementarity Determining Regions; DNA, Complementary - genetics; Genes, Immunoglobulin; Humans; Immunoglobulin Fragments - immunology; Immunoglobulin Variable Region - genetics; Jurkat Cells; Lupus Erythematosus, Systemic - immunology; Molecular Sequence Data; Peptide Fragments - pharmacology; Phage display; Recombinant autoantibody; Recombinant Fusion Proteins - immunology; Ribonucleoprotein, U1 Small Nuclear - immunology; Sequence Alignment; Sequence Homology, Amino Acid; Systemic lupus erythematosus/mixed connective tissue disease; U1-70K protein; U1‐70K
• ISSN: 0014-2980; 1521-4141; 1521-4141
• DOI: 10.1002/1521-4141(200010)30:10<3029::AID-IMMU3029>3.0.CO;2-J

The U1‐70K protein is specifically bound to stemloop I of the U1 small nuclear RNA contained in the U1 small nuclear ribonucleoprotein complex (U1 snRNP), which is involved in the splicing of pre‐mRNA. All components of the U1 snRNP complex, including the U1‐70K protein, are important autoantigens in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Here we describe for the first time the selection and characterization 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 protein and its apoptotic 40‐kDa cleavage product. In Western blotting assays a number of scFv preferentially recognize the 40‐kDa apoptotic cleavage fragment of the U1‐70K protein, suggesting a possible involvement of this apoptotic cleavage product in the autoimmune response of patients. The germ‐line gene usage of these recombinant autoantibodies was also determined. Using several U1‐70K deletion and point mutants of both human (h) and Drosophila 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 domain.