A calreticulin-like protein co-purifies with a '60 kD' component of Ro/SSA, but is not recognized by antibodies in Sjögren's syndrome sera.
In this study, we used human tonsils for the isolation of the 60 kD component of the Ro/SSA autoantigen, following the method described by Wu et al. (J Immunol Methods 1989; 121:219-24). Western blot analyses were carried out using Ro/SSA-reactive human Sjögren's syndrome sera, to follow the autoantigen through the purification procedure. A 60 kD Ro/SSA component was eluted as a broad peak from a Mono Q column. Within this peak, a much more abundant protein, co-migrating with the Ro/SSA component on SDS-PAGE, was also eluted. The more abundant protein was further purified on a Superose 12 column and its N-terminal sequence was shown to be identical to that of human calreticulin. The 60 kD Ro/SSA autoantigen was also further purified on the Superose 12 column and was eluted as an asymmetric peak, with the majority being eluted at a position corresponding to 60 kD, whereas the calreticulin-like protein was eluted from the same column as an apparent dimer of approximately 120 kD. A panel of five Ro/SSA-reactive human sera reacted with the purified Ro/SSA antigen, but not with the calreticulin-like protein. Therefore, it is clear that the calreticulin-like protein is not a Ro autoantigen and is distinct from the 60 kD Ro/SSA antigen. As the calreticulin-like protein is a much more abundant protein than the 60 kD Ro/SSA component, its co-purification with the autoantigen on ion-exchange and its close migration with the autoantigen on SDS-PAGE may explain why peptide sequences for human calreticulin were derived from apparent 60 kD Ro/SSA antigen preparations.
Lu
J
J
Department of Biochemistry, University of Oxford, UK.
Willis
A C
AC
Sim
R B
RB
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology / Q000302:isolation & purification*; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000302:isolation & purification*; D037282:Calreticulin; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014066:Palatine Tonsil / Q000737:chemistry; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins / Q000276:immunology / Q000302:isolation & purification*; D012859:Sjogren's Syndrome / Q000276:immunology*; D000099207:SS-A Antigen
D001323:Autoantibodies; D001324:Autoantigens; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C116006:RO60 protein, human