Human recombinant anti-La (SS-B) autoantibodies demonstrate the accumulation of phosphoserine-366-containing La isoforms in nucleoplasmic speckles

• Published in: European journal of cell biology 2003-03, Vol.82 (3), p.131-141
• Main Authors: Raats, Jos M.H., Roeffen, Will F., Litjens, Sandy, Bulduk, Ibrahim, Mans, Gijs, van Venrooij, Walther J., Pruijn, Ger J.M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, Antinuclear - genetics; Antibodies, Antinuclear - immunology; apoptosis; Apoptosis - immunology; Autoantibodies - genetics; Autoantibodies - immunology; Autoimmune Diseases - genetics; Autoimmune Diseases - immunology; Cell Nucleus - metabolism; COS Cells; Epitope Mapping; HeLa Cells; Humans; La (SS-B); Molecular Sequence Data; phosphorylation; Phosphoserine - metabolism; Protein Isoforms - genetics; Protein Isoforms - immunology; Recombinant antibody fragments; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Sequence Analysis, DNA; systemic lupus erythematosus (SLE)
• ISSN: 0171-9335; 1618-1298
• DOI: 10.1078/0171-9335-00304

Using the recombinant La (SS-B) protein or a phosphorylated peptide derived thereof 27 La-specific human recombinant autoantibodies were selected from anti-La-positive systemic lupus erythematosus and systemic sclerosis patient-derived combinatorial phage display antibody libraries. Binding of these anti-La antibodies to various isoforms of the La protein present in normal and apoptotic cell extracts was analysed by Western blotting. Twenty-four of the selected antibodies recognize most, if not all isoforms of La, whereas three are exclusively reactive with the protein phosphorylated at serine-366. Sequence analysis of the selected antibodies showed a restricted spectrum of diversity in their VH germline gene usage. Remarkably, the recombinant antibodies recognizing exclusively the phosphoserine-366-containing isoform of La displayed a spleckled nucleoplasmic staining pattern in immunofluorescence analysis of HeLa and HEp-2 cells. This pattern differed markedly from those obtained with anti-La antibodies recognizing all isoforms of the La protein. Colocalization experiments with marker antibodies for spliceosomal UsnRNPs and RNA polymerase III subunits revealed that the anti-phosphorylated La antibodies stain the same nucleoplasmic speckles as anti-UsnRNP antibodies. In contrast to anti-UsnRNP antibodies the anti-phosphorylated La antibodies did not stain the Cajal bodies. In addition, no colocalization of phosphorylated La with RNA polymerase III was observed. Potential functional implications of the accumulation of phosphorylated La in nucleoplasmic speckles are discussed.