Accessibility of epitopes on the 52-kD Ro/SSA protein (Ro52) and on the RoRNP associated Ro52 protein as determined by anti-peptide antibodies.
The Ro ribonucleoprotein particle (RoRNP) is the target of a variety of specific anti-protein autoantibodies produced by patients with systemic autoimmune diseases. RoRNPs, the function of which remains elusive, appear to be rather heterogeneous in terms of their molecular composition. Among several Ro proteins, a protein of 52 kD (Ro52) has been identified, but its association with RoRNPs remains questionable. In this study, we first mapped the Ro52 regions that are accessible at the surface of the isolated protein, by means of antibodies raised in rabbits, against 39 overlapping synthetic peptides covering the whole Ro52 molecule and using several different methods, including various ELISA formats and Western blotting (based on the use of recombinant Ro52) and immunoprecipitation of in vitro translated Ro52. Then, the whole set of anti-peptide antibodies was used to attempt to immunoprecipitate RoRNPs from a cell extract of K562 cells. RoRNPs, especially Ro(hY3)RNPs, were effectively immunoprecipitated by certain anti-peptide antibodies, indicating that Ro52 protein is associated with at least a subset of RoRNP particles. As expected, a number of epitopes available on the isolated Ro52 molecule are no longer accessible when Ro52 is associated with the RoRNP. Conversely, neotopes, among them at least one corresponding to a previously characterized epitope recognized by patients' antibodies, are only present at the surface of the particle and not detectable on the isolated Ro52 protein.
Ricchiuti
V
V
Institut de Biologie Moléculaire et Cellulaire, UPR 9021, CNRS, Strasbourg, France.
Pruijn
G J
GJ
Thijssen
J P
JP
van Venrooij
W J
WJ
Muller
S
S
D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis / Q000737:chemistry; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D011233:Precipitin Tests; D020733:RNA, Small Cytoplasmic; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012261:Ribonucleoproteins / Q000737:chemistry / Q000276:immunology*; D014407:Tumor Cells, Cultured; D000099207:SS-A Antigen
D000974:Antibodies, Antinuclear; D001324:Autoantigens; D000939:Epitopes; D007106:Immune Sera; D010455:Peptides; D020733:RNA, Small Cytoplasmic; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human; C035356:SS-A antibodies