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

Human autoantibodies to proteins of the mitotic apparatus have demonstrated clinical utility and usefulness as molecular probes for identification and characterization of novel autoantigens, as exemplified by autoantibodies to centromere proteins. In contrast, there have been very few reports of autoantibodies with reactivity to antigens located along mitotic chromosome arms, but not in interphase nuclei. The purpose of this study was to identify and characterize autoantibodies with reactivity to mitotic chromosomal antigens (MCAs) located exclusively on mitotic chromosome arms, and to determine if patients with these autoantibodies have common clinical features.

Routine immunofluorescence screening of serum samples referred for antinuclear antibody investigation over a 10-year period was used to identify autoantibodies to MCAs. MCAs were identified by exclusive immunofluorescence staining of mitotic chromosome arms with no staining of interphase nuclei. MCA-reactive sera were further characterized for patterns of staining on mitotic chromosome arms and sensitivities to chemical and enzymatic treatments, and for one of these sera, its ability to abrogate progression through mitosis when microinjected into cells.

Of 60,000 sera screened for antinuclear antibodies by immunofluorescence, we identified three IgG autoantibodies reacting exclusively to MCAs. The anti-MCA autoantibodies did not react with condensed chromatin in spermatozoa or in apoptotic HeLa cells. Reactivity of all three sera was abrogated by treatment with protease, but not RNase, indicating that the MCAs are protein in nature and do not contain RNA epitopes. The three anti-MCA antibodies seem to react to three different antigens because they gave different patterns of staining of chromosome arms, reacted with chromosomes in different stages of mitosis, and displayed different sensitivities to treatment with DNase 1, salt, and phosphatases. Phosphatase treatment suggests that MCA1 and MCA2 contain serine/threonine phosphoepitope(s) and MCA3 tyrosine phosphoepitope(s). Loss of MCA2 reactivity to DNase 1 treatment and its retention after salt extraction suggests that it is a chromosomal scaffold protein. Sensitivity of all three MCAs to acid suggests that they are histone-like or histone-associated proteins.

We report the identification of three novel MCA-reactive sera. Patient diagnoses included discoid lupus erythematosus, chronic lymphocytic leukemia, Sjögren'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. Microinjection of MCA1-reactive antibodies led to metaphase arrest, without any change in morphology of the mitotic spindle or metaphase chromosomes suggesting that MCA1 may have a role in sister chromatid separation.