The identification of mammalian centrosomal antigens using human autoimmune anticentrosome antisera

Abstract

Human autoimmune sera were screened for the presence of anticentrosome autoantibodies. Two high titer sera were identified that reacted with HeLa, CHO, and PtK₂ centrosomes by immunofluorescence, although the fluorescent patterns that were obtained using the two antisera were separate and distinct. Serum obtained from patient IJ contained antibodies that reacted with epitopes present only in mitotic centrosomes; staining of interphase centrosomes was never detected uing IJ antiserum. Immunoblot analysis demonstrated that antibodies present in IJ antiserum reacted with a 190 kD spindle pole antigen. Immunofluorescent staining of cultured mammalian cells demonstrated that antibodies present in serum obtained from patient SPJ reacted with both interphase and mitotic centrosomes. Characterization of SPJ antiserum by immunoblotting demonstrated that antibodies present in the SPJ serum recognized proteins of M_rs of 39, 185, and 220 kD, although the possibility that the 185 kD polypeptide was a proteolytic breakdown product of the 220 kD protein has not been eliminated. Neither antiserum was able to inhibit microtubule nucleation from centrosomes in a lysed cell system in which pure 6S tubulin was added to permeabilized cells following pretreatment of the cells with either SPJ or IJ antiserum. These antisera should be useful probes for studying the biochemistry of the mammalian centrosome.