Novel human autoantibodies to phosphoepitopes on mitotic chromosomal autoantigens (MCAs)

Background: Human autoantibodies to proteins of the mitotic apparatus have demonstrated clinical utility and usefulness as molecular probes for identi fication and characterization of novel autoantigens, as exemplified by auto antibodies to centromere proteins. In contrast, there have been very few re ports of autoantibodies with reactivity to antigens located along mitotic c hromosome arms, but not in interphase nuclei. The purpose of this study tva s to identify and characterize autoantibodies with reactivity to mitotic ch romosomal antigens (MCAs) located exclusively on mitotic chromosome arms, a nd to determine a patients with these autoantibodies have common clinical f eatures. Methods: Routine immunofluorescence screening of serum samples referred for antinuclear antibody investigation over a 10-year period was used to ident ify autoantibodies to MCAs. MCAs were identified by exclusive immunofluores cence staining of mitotic chromosome arm with no staining of interphase nuc lei. MCA-reactive sera mere further characterized for patterns of staining on mitotic chromosome arms and sensitivities to chemical and enzymatic trea tments, and for one of these sera, its ability to abrogate progression thro ugh mitosis when microinjected into cells. Results: Of 60,000 sera screened for antinuclear antibodies by immunofluore scence, we identified three IgG autoantibodies reacting exclusively to MCAs . The anti-MCA autoantibodies did not react with condensed chromatin in spe rmatozoa or in apoptotic HeLa cells. Reactivity of all three sera was abrog ated by treatment,vith protease, but not RNase, indicating that the MCAs ar e protein in nature and do not contain RNA epitopes, The three anti-MCA ant ibodies seem to react to three different antigens because they gave differe nt patterns of staining of chromosome arms, reacted with chromosomes in dif ferent stages of mitosis, and displayed different sensitivities to treatmen t crith DNase I, salt, and phosphatases, Phosphatase treatment suggests tha t MCA1 and MCA2 contain serine/threonine phospheopitope(s) and MCA3 tyrosin e phosphoepitope(s), Loss of MCA2 reactivity to DNase I treatment and its r etention after salt extraction suggests that it is a chromosomal scaffold p rotein. Sensitivity of all three MCAs to acid suggests that they are histon e-like or histone-associated proteins. Conclusions: We report die identification of three novel MCA-reactive sera. Patient diagnoses included discoid lupus erythematosus, chronic lymphocyti c leukemia Sjogren's syndrome, and polymyalgia rheumatica The reactivity of anti-MCA antibodies with phosphoepitopes is likely to explain restriction of immunofluorescence staining to chromosome arms during mitosis, Microinje ction of MCA1-reactive antibodies led to metaphase arrest, without any chan ge in morphology of the mitotic spindle or metaphase chromosomes suggesting that MCA1 may have a role in sister chromatid separation.