Epitope mapping of recombinant HeLa SmB and B' peptides obtained by the polymerase chain reaction.
SmB and SmB' are the major antigenic proteins contained within small nuclear RNP particles that are recognized by both human SLE and MRL mouse anti-Sm sera. We amplified cDNA obtained from HeLa cells by using the polymerase chain reaction and identified two clones, U2 and L13, that encode SmB and SmB', respectively. The nucleotide sequences of these two clones were identical except for the insertion of a 145-bp sequence in U2 that contained an early in frame termination codon and a potential 3' consensus splice site. The predicted amino acid sequences of HeLa SmB and B' proteins were therefore identical except for the COOH terminal 2 (U2) and 11 (L13) amino acids. U2, L13, and four subclones of U2 (F-B, B-R, F-X, and X-B) were ligated to pATH vectors and expressed as trpE fusion proteins. Epitope mapping with 12 human SLE and 12 MRL/lpr mouse anti-SmB/B' sera revealed that antibodies directed against the X-B peptide accounted for most (65.5 +/- 15.4 and 63.2 +/- 25.3%), B-R intermediate levels (51.5 +/- 30.8 and 18 +/- 19.6%), and F-X none of the anti-SmB activity in human and mouse sera, respectively. Ten human and two mouse sera contained antibodies that cross-reacted with epitopes located within the proline-rich, COOH-terminal, 27-residue peptide encoded by B-R and the NH2-proximal F-B peptide. These observations suggest that a) the polymerase chain reaction is a powerful ancillary method to synthesize autoantigens, b) SmB and B' in HeLa cells are derived from alternative splicing of a common RNA transcript, and c) both SLE and MRL anti-SmB/B' sera recognize multiple epitopes (some shared and some unique) on these proteins.
Hospital for Special Surgery, Cornell Medical Center, New York, NY 10021.
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Elkon
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Hines
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Chu
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Parnassa
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D000939:Epitopes; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012261:Ribonucleoproteins / Q000276:immunology*; D017411:Ribonucleoproteins, Small Nuclear
D001324:Autoantigens; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear