Anti-centromere Autoantibodies Research Articles

Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis

The relationship between the kinetochore and the centromeric heterochromatin that surrounds it is unknown. Anti-centromere autoantibodies (ACAs) that recognize antigens found in the heterochromatin beneath the kinetochore disrupt mitotic events when microinjected into human cells. We show here that ACAs interfere with two different stages of centromere assembly during interphase, resulting in abnormal kinetochore structures during mitosis. Antibody injection prior to late G2 results in the subsequent failure to assemble a trilaminar kinetochore. Such chromosomes bind microtubules but are incapable of movement. Antibody disruption of events during G2 produces unstable kinetochores that prevent the normal transition into anaphase. These experiments present a novel way to examine events in the pathway of kinetochore assembly that occur during interphase, at a time when this structure cannot be visualized directly.

Idiotypic analysis of human anti-topoisomerase I autoantibodies.

Anti-topoisomerase I autoantibodies (anti-topo I) are associated with proximal scleroderma and are of prognostic significance in patients with Raynaud's phenomenon. Polyclonal anti-idiotypic sera were raised against affinity-purified anti-topo I from 2 patients with scleroderma (EM, SG) and 1 healthy individual (NM). All 3 anti-topo I preparations expressed immunodominant private Ids in or near the antigen binding site of the autoantibody. Further analysis of Id-EM showed isotypic restriction to IgG and a stable Id-expression over the course of 9 years. Id-SG and Id-NM were expressed on IgG and on IgA. The idiotypic character of anti-topo I closely resembles that of anti-centromere autoantibodies which are associated with the CREST syndrome of scleroderma. The data suggest an antigen-driven process in the origin of autoantibodies in scleroderma.

The igg, igm, and iga isotypes of anti‐topoisomerase i and anticentromere autoantibodies

We studied the expression of IgG, IgM, and IgA autoantibodies in the anti-topoisomerase I and anticentromere immune responses by enzyme-linked immunosorbent assay, immunoblotting, and immunofluorescence. While IgG autoantibodies were most common, IgA autoantibodies were also frequently found, but IgM autoantibodies were rare. This is the first report of IgA autoantibodies in scleroderma.

Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B.

A cDNA clone encoding CENP-B, the 80-kDa human centromere autoantigen, was used to construct a panel of hybrid proteins containing four different regions of CENP-B. These have allowed us to identify three independent epitopes on CENP-B that are targets of autoantibodies. Two of these are recognized concurrently in greater than or equal to 90% of patient sera containing anticentromere autoantibodies (ACA), conclusively demonstrating that this autoimmune response is polyclonal. When present and previous data are combined, ACA are shown to recognize at least five independent epitopes on CENP-B. A radioimmunoassay based on cloned CENP-B has demonstrated that sera from greater than or equal to 96% of patients with ACA recognize the cloned antigen, thus defining a region of the protein that is recognized by virtually all patients with ACA. These findings have significant implications for models that seek to explain the origin of ACA and for the future detection of this group of autoantibodies in the clinical setting.

Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen

We have isolated a series of overlapping cDNA clones for approximately 95% of the mRNA that encodes CENP-B, the 80-kD human centromere autoantigen recognized by patients with anticentromere antibodies. The cloned sequences encode a polypeptide with an apparent molecular mass appropriate for CENP-B. This polypeptide and CENP-B share three non-overlapping epitopes. The first two are defined by monoclonal antibodies elicited by injection of cloned fusion protein. Epitope 1 corresponds to a major antigenic site recognized by the anticentromere autoantibody used to obtain the original clone. Epitope 2 is a novel one not recognized by the autoantibody. These epitopes were shown to be distinct both by competitive binding experiments and by their presence or absence on different subcloned portions of the fusion protein. The third independent epitope, recognized by a subset of anticentromere-positive patient sera, maps to a region substantially closer to the amino terminus of the fusion protein. DNA and RNA blot analyses indicate that CENP-B is unrelated to CENP-C, a 140-kD centromere antigen also recognized by these antisera. CENP-B is the product of a 2.9-kb mRNA that is encoded by a single genetic locus. This mRNA is far too short to encode a polypeptide the size of CENP-C. The carboxy terminus of CENP-B contains two long domains comprised almost entirely of glutamic and aspartic acid residues. These domains may be responsible for anomalous migration of CENP-B on SDS-polyacrylamide gels, since the true molecular mass of CENP-B is approximately 65 kD, 15 kD less than the apparent molecular mass deduced from gel electrophoresis. Quite unexpectedly, immunofluorescence analysis using antibodies specific for CENP-B reveals that the levels of antigen vary widely between chromosomes.

Direct evidence for the non-random localization of mammalian chromosomes in the interphase nucleus

Indirect immunofluorescence staining with human anti-centromere autoantibodies from a patient (LU 851) suffering from the CREST form of scleroderma was used to analyse chromosome topology in interphase nuclei of rat-kangaroo (PTO) and Indian muntjac (IM) cells. In some cells, centromeres were arranged in pairs suggesting association of homologous chromosomes. Clustering of centromeres at one pole of the nucleus (Rabl configuration) and other patterns suggesting higher order organization were also observed. In one fifth of the IM cells it was possible to identify the intranuclear location of each single chromosome on the basis of the morphology of the immunostained centromeres. In 30% of the IM cells in which centromeres could be identified, homologous chromosomes occupied adjacent territories within the interphase chromatin.

A highly conserved 72,000 dalton centromeric antigen reactive with autoantibodies from patients with progressive systemic sclerosis.

An autoantibody reactive with a 72,000 dalton centromeric antigen was detected by immunoblotting with the use of a nuclear enriched HeLa cell preparation in 42 of 77 patients with progressive systemic sclerosis (PSS). Reactivity with the 72,000 dalton polypeptide was associated with anti-centromere autoantibodies (ACA) detected by immunofluorescence (IF), and the antigen was highly conserved, being present in both human cells and Leishmania tropica. Thirty-five (83%) of the 42 sera reactive with the 72,000 dalton polypeptide also reacted with a 19,500 dalton polypeptide, and antibodies eluted from both the 72,000 dalton and the 19,500 dalton polypeptides reacted with the centromere when retested by IF on intact HEp2 cells, demonstrating that both polypeptides are antigenic components of the centromere. Only one of the 42 sera had precipitating antibodies to the Scl-70 antigen detected by counterimmunoelectrophoresis, indicating that the 72,000 dalton polypeptide was not related to the previously described Scl-70 antigen. The other 35 of the 77 sera tested were negative for ACA, although all had ANA, with the main patterns of IF being fine speckling of the nucleus (18 sera) and homogeneous or speckled staining of the nucleolus (17 sera). Anti-Scl-70 antibodies were detected in 17 of these 35 patients, 15 (88%) of whom reacted with an 89,000 dalton polypeptide, one with a 140,000 dalton polypeptide, and one with a 74,000 dalton polypeptide. Ten of the 15 sera reacting with the 89,000 dalton polypeptide also reacted with a 74,000 dalton polypeptide, and 2-D gel analysis suggested a relationship between the two molecules. Clinically defined types of scleroderma tended to associate with antibodies to particular molecular antigenic specificities. Thirty-seven (88%) of the 42 patients reactive with the 72,000 dalton polypeptide had sclerodactyly and features of the CREST syndrome, whereas patients reactive with the 89,000 dalton polypeptide and with Scl-70 tended to have more extensive cutaneous and visceral involvement.

Human anticentromere antibodies: Distribution, characterization of antigens, and effect on microtubule organization

Properties of human anticentromere autoantibodies were analyzed. In intact cells or isolated cell fractions, these sera stain the centromeres of mitotic chromosomes and discrete speckles (prekinetochores) in nuclei. Staining is also retained in matrix preparations from nuclei or chromosomes. Immunoprecipitation or immunoblotting demonstrates protein antigens of 14, 20, 23, and 34 kd in HeLa nuclei and chromosomes; immunoprecipitates of nuclei also contain a protein of 15.5 kd. Matrix preparations contain only the 20, 23, and 34 kd species. Absorption of the anticentromere serum with any one of the four nuclear antigens immobilized on nitrocellulose is sufficient to eliminate centromere staining. Using a lysed cell model for microtubule nucleation, anticentromere sera are shown to inhibit specifically the organization of microtubules at the kinetochore.