Mapping of epitopes recognized by PM/Scl autoantibodies with gene-fragment phage display libraries.
Sera from patients suffering from the polymyositis/scleroderma overlap syndrome (PM/Scl) recognize two antigenically non-related proteins with apparent molecular masses of 100 kDa and 75 kDa respectively. The two proteins are part of a particle termed PM/Scl localized in the granular component of the nucleolus. The predominant immunoreactivity of the PM/Scl sera was shown to be directed against the 100 kDa protein. The cDNA of the 100 kDa protein has been cloned recently and its immunogenic regions have been partially mapped using recombinant proteins. Thus far the localization of antigenic determinants on polypeptides has been done by expressing defined cDNA fragments in bacteria or by synthesizing overlapping short peptides and probing their immunoreactivity with antibodies. Here we present an alternative approach to localize autoimmune epitopes using sera containing polyclonal antibodies and gene-fragment phage display libraries. For epitope fine mapping of the PM/Scl-100 protein random fragments of the corresponding cDNA were cloned into the PIII protein of fUSE-5. These gene-fragment phage display libraries were incubated with affinity purified anti-PM/Scl-100 antibodies to enrich for epitope-displaying phages. All PM/Scl sera tested recognized 23 consecutive amino acids (229-251) encoded by four overlapping fUSE-5 clones, suggesting that a major epitope is contained within the 23 amino acids. In addition a minor epitope was localized in a region of 21 amino acids (775-795) encoded by two overlapping fUSE-5 clones since only three out of the seventeen sera reacted with this amino acid sequence. Additional fine mapping of the major epitope was done using synthetic oligopeptides. Thus, a stretch of 16 amino acids at position 229-244 could be identified as a major epitope on the deduced PM/Scl-100 amino acid sequence.
Blüthner
M
M
Institute of Molecular Genetics, University of Heidelberg, Germany.
Bautz
E K
EK
Bautz
F A
FA
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions / Q000235:genetics; D001323:Autoantibodies / Q000737:chemistry*; D017104:Bacteriophage M13 / Q000737:chemistry / Q000235:genetics*; D018604:Epitope Mapping / Q000379:methods*; D015723:Gene Library; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D008969:Molecular Sequence Data; D017285:Polymyositis / Q000276:immunology*; D012595:Scleroderma, Systemic / Q000276:immunology*; D017384:Sequence Deletion / Q000276:immunology
D001323:Autoantibodies; D007106:Immune Sera