M. Bluthner et al., MAPPING OF EPITOPES RECOGNIZED BY PM SCL AUTOANTIBODIES WITH GENE-FRAGMENT PHAGE DISPLAY LIBRARIES/, Journal of immunological methods, 198(2), 1996, pp. 187-198
Sera from patients suffering from the polymyositis/scleroderma overlap
syndrome (PM/Scl) recognize two antigenically non-related proteins wi
th apparent molecular masses of 100 kDa and 75 kDa respectively. The t
wo proteins are part of a particle termed PM/Scl localized in the gran
ular component of the nucleolus. The predominant immunoreactivity of t
he PM/Sd sera was shown to be directed against the 100 kDa protein. Th
e cDNA of the 100 kDa protein has been cloned recently and its immunog
enic regions have been partially mapped using recombinant proteins. Th
us far the localization of antigenic determinants on polypeptides has
been done by expressing defined cDNA fragments in bacteria or by synth
esizing overlapping short peptides and probing their immunoreactivity
with antibodies, Here we present an alternative approach to localize a
utoimmune epitopes using sera containing polyclonal antibodies and gen
e-fragment phase display libraries, For epitope fine mapping of the PM
/Scl-100 protein random fragments of the corresponding cDNA were clone
d into the PIII protein of fUSE-5, These gene-fragment phage display l
ibraries were incubated with affinity purified anti-PM/Scl-100 antibod
ies to enrich for epitope-displaying phages, All PM/Sd sera tested rec
ognized 23 consecutive amino acids (229-251) encoded by four overlappi
ng fUSE-5 clones, suggesting that a major epitope is contained within
the 23 amino acids, In addition a minor epitope was localized in a reg
ion of 21 amino acids (775-795) encoded by two overlapping fUSE-5 clon
es since only three out of the seventeen sera reacted with this amino
acid sequence, Additional fine mapping of the major epitope was done u
sing synthetic oligopeptides. Thus, a stretch of 16 amino acids at pos
ition 229-244 could be identified as a major epitope on the deduced PM
/Scl-100 amino acid sequence.