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Results for “Ku Autoantigen”

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PubMed2000

Ku86 variant expression and function in multiple myeloma cells is associated with increased sensitivity to DNA damage.

Ku is a heterodimer of Ku70 and Ku86 that binds to double-stranded DNA breaks (DSBs), activates the catalytic subunit (DNA-PKcs) when DNA is bound, and is essential in DSB repair and V(D)J recombination. Given that abnormalities in Ig gene rearrangement and DNA damage repair are hallmarks of multiple myeloma (MM) cells, we have characterized Ku expression and function in human MM cells. Tumor cells (CD38(+)CD45RA(-)) from 12 of 14 (86%) patients preferentially express a 69-kDa variant of Ku86 (Ku86v). Immunoblotting of whole cell extracts (WCE) from MM patients shows reactivity with Abs targeting Ku86 N terminus (S10B1) but no reactivity with Abs targeting Ku86 C terminus (111), suggesting that Ku86v has a truncated C terminus. EMSA confirmed a truncated C terminus in Ku86v and further demonstrated that Ku86v in MM cells had decreased Ku-DNA end binding activity. Ku86 forms complexes with DNA-PKcs and activates kinase activity, but Ku86v neither binds DNA-PKcs nor activates kinase activity. Furthermore, MM cells with Ku86v have increased sensitivity to irradiation, mitomycin C, and bleomycin compared with patient MM cells or normal bone marrow donor cells with Ku86. Therefore, this study suggests that Ku86v in MM cells may account for decreased DNA repair and increased sensitivity to radiation and chemotherapeutic agents, whereas Ku86 in MM cells confers resistance to DNA damaging agents. Coupled with a recent report that Ku86 activity correlates with resistance to radiation and chemotherapy, these results have implications for the potential role of Ku86 as a novel therapeutic target.

Antigens, Nuclear↗
PubMed2000

Quantitative radioligand assays using de novo-synthesized recombinant autoantigens in connective tissue diseases: new tools to approach the pathogenic significance of anti-RNP antibodies in rheumatic diseases.

OBJECTIVE: To describe new assays for the detection and quantification of antibodies to RNPs in rheumatic diseases, using soluble nuclear antigens synthesized de novo in reticulocyte lysates. METHODS: Sera from 381 patients with various rheumatic diseases, including 212 patients with systemic lupus erythematosus (SLE), were analyzed in order to evaluate the sensitivity and specificity of serum autoantibody reactivities to several recombinant soluble autoantigens: U1-A RNP, Sm-B, SSA/Ro 52 and SSA/Ro 60, SSB/La, and Ku. Radioligand assays (RLAs) were performed following the in vitro transcription and translation of each autoantigen from the corresponding complementary DNA, labeled with 35S-methionine. The radiolabeled protein was then bound by the specific serum autoantibody, forming immune complexes that were captured by protein A-Sepharose beads and quantified by counting the radioactivity. RESULTS: Among the SLE patients, 44% were positive for anti-U1-A RNP activity, 34% for anti-Sm-B, 44% for anti-SSA (32% for Ro 52 and 46% for Ro 60), 32% for anti-SSB/La, and 11% for anti-Ku reactivities. SSA antibodies had a high frequency in patients with mixed connective tissue disease (MCTD) (80%); 65% of these patient sera reacted with Ro 52, 45% with Ro 60, and 45% with U1-A RNP. Twenty percent of the MCTD patients also exhibited antibodies to Sm-B and Ku. In patients with Sjögren's syndrome, anti-SSA was the main anti-RNP antibody (63%), together with SSB/La antibodies (44%). Among patients with inflammatory myopathy, only antibodies against Ro 52 (36%) and Ro 60 (36%) were present. These new RLA allowed observation of a strong correlation (P < 0.0001) between Sm-B antibody levels and the severity of SLE (as measured by the SLE Disease Activity Index), and establishment of a correlation between anti-U1-A RNP antibodies and the occurrence of SLE nephritis (P < 0.02). All RLAs were highly specific for the antigen tested and displayed, in the disease groups studied, a higher sensitivity than conventional immunodiffusion assays. CONCLUSION: These highly sensitive, specific, and quantitative RLAs represent new tools for the detection of autoantibodies to RNP antigens in rheumatic diseases, and may be useful for (differential) diagnosis in clinical practice.

Autoantibodies↗
PubMed2004

Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK(CS). Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK(CS) is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK(CS) enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK(CS) is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK(CS) is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK(CS) is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK(CS) during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.

Caspase 3↗
PubMed2003

Ultraviolet B radiation-induced cell death: critical role of ultraviolet dose in inflammation and lupus autoantigen redistribution.

The nuclear self-Ags targeted in systemic lupus erythematosus translocate to the cell membrane of UV-irradiated apoptotic keratinocytes and may represent an important source of self-immunization. It is hard to understand how the noninflammatory milieu accompanying most apoptosis might provoke an immunogenic response leading to autoantibodies. We have found that the precise amount of keratinocyte UV exposure is crucial in determining the rate of apoptosis, the amount of inflammatory cytokine production, and the degree of autoantigen translocation. Low doses of UVB (</=15 mJ/cm(2)) promptly induced a normal, caspase-dependent apoptosis, while intermediate doses of UV-B (35 mJ/cm(2)) caused apoptosis with altered morphology, slower DNA fragmentation, and poly(ADP-ribose) polymerase degradation accompanied by increased Bcl-2. High doses of UVB (80 mJ/cm(2)) induced instead necrosis. We observed IL-1 production upon intermediate and high UVB doses. Nuclear Ag redistribution was also markedly UV dose dependent: at low doses, Sm, Ku, and DNA translocated to the surfaces of early apoptotic cells. At intermediate doses, these Ags concentrated on the cell membrane when the nucleus was still visible. At high doses, these autoantigens diffused into the cytoplasm and were released into the supernatant. Taken together, the results show that low-dose UVB induces prompt noninflammatory apoptosis. In contrast, intermediate and high doses of UVB induce proinflammatory apoptosis and necrosis, where the production of inflammatory cytokines is accompanied by exposure and release of autoantigens. The key importance of the UV dose on the fate of apoptotic keratinocytes and on their potential immunogenicity should help clarify the role of UVB in inducing systemic lupus erythematosus autoimmunity.

Antigens, Nuclear↗
PubMed1992

Autoantibodies to small nuclear and cytoplasmic ribonucleoproteins in Japanese patients with inflammatory muscle disease.

OBJECTIVE: To elucidate the profile and clinical significance of autoantibodies to small nuclear and small cytoplasmic ribonucleoproteins in Japanese patients with polymyositis and dermatomyositis (PM/DM). METHODS: Radioimmunoprecipitation assays were performed with sera from 91 patients with various inflammatory muscle diseases and from 254 control patients with other rheumatic diseases. Patients with PM/DM were categorized according to autoantibody specificities, and clinical comparisons were made. RESULTS: Antibodies to aminoacyl transfer RNA (tRNA) synthetases and the signal recognition particle (SRP) were found to be specific for PM/DM: PM/DM patients with antibodies to 3 aminoacyl-tRNA synthetases (histidyl, threonyl, and glycyl) appeared to form a distinct clinical subset, associated with chronic interstitial lung disease, polyarthritis, and myositis, while 3 patients with antibodies that bound the SRP had severe myositis that was resistant to corticosteroid therapy. In general, the frequency and clinical correlations of anti-aminoacyl-tRNA synthetase antibodies in Japanese patients were similar to those in North American populations. In contrast, among this Japanese cohort, the occurrence of anti-PM/Scl appeared to be reduced in frequency, whereas anti-Ku and anti-U2 RNP antibodies were accentuated in patients with overlap syndromes. CONCLUSION: We conclude that racial groups differ somewhat in their characteristic autoimmune responses to individual autoantigens. These differences could reflect variation in immunogenetic background or different etiologic mechanisms which might occur at different geographic locations.

Asian People↗
PubMed1997

Revisiting autoantibody profiles in systemic lupus erythematosus.

OBJECTIVE: To obtain more definitive assays of the spectrum of soluble autoantigens targeted by individual patients with systemic lupus erythematosus (SLE) and to determine whether the autoimmune response is restricted in specificity. Although there are many reports of a broad spectrum of autoantibody specificities in SLE, none has considered the diversity of autoantibody sets, which may more accurately describe the autoimmune response. METHODS: Sera of 68 patients with SLE were assayed for autoantibodies by ELISA and/or immunoprecipitation. Specificities were grouped into sets, including double stranded (ds) DNA and/or histone, U1 RNP and/or Sm, Ro and/or La, ribosomes, Ku, Ki, and others. An analysis was also performed of reported SLE autoantibody profiles. RESULTS: The prevalences of autoantibody sets included: dsDNA and/or histone, 59%; U1 RNP and/or Sm, 40%; Ro and/or La, 41%; ribosomes, 4.4%; Ku, 4.4%; Ki, 2.9%. On average, autoantibody positive patients had 2-3 autoantibodies (median = 2) and about 2-3 autoantibody sets (median = 2), consistent with a retrospective analysis of past studies. CONCLUSION: Immune dysregulation in SLE generally involves a multiplicity of autoantibody specificities. These data further support a model in which global immune dysregulation in SLE leads to organ-nonspecific autoimmunity against particular ubiquitous autoantigens.

Adult↗
PubMed2003

Allolymphocyte apoptosis induced by soluble liver specific antigen.

OBJECTIVE: To investigate the alloimmunogenicity of liver specific antigen and its effects on allolymphocytes. METHODS: Liver specific antigen isolated from inbred F344 rats was used as immunogen to immunize inbred Lew rats through different immunization pathways such as low-dose long-term hind footpad, high-dose portal vein and thymus immunization. Western blotting, DNA fragments gel electrophoresis, mixed lymphocyte culture (MLC) and mixed lymphocyte hepatocyte culture (MLHC) were employed to analyze the immune state after immunization. RESULTS: At the time point of sampling, different degree of specific low immunoresponses appeared in all immunized groups as well as cyclophosphamide (CY) treated group. Compared with group I, other groups expressed caspase-3 significantly as detected by using Western blotting. DNA fragment gel electrophoresis of splenocytes showed lymphocyte apoptosis. Compared with the group I, MLC of the experimental groups showed no significant changes except that of the group V, whereas MLHC decreased markedly (P<0.05). CONCLUSIONS: Liver specific antigen not only has alloimmunogenicity to induce alloimmunoreaction but induce antigen specific low immunoresponses and antigen specific lymphocyte apoptosis by high-dose or low-dose long-term immunization. It may be an important transplantation antigen that may lead to a novel way to liver transplantation immunotolerance.

Animals↗
PubMed1994

Characterization of the Su antigen, a macromolecular complex of 100/102 and 200-kDa proteins recognized by autoantibodies in systemic rheumatic diseases.

The Su autoantigen was characterized biochemically using human and murine autoimmune sera and the clinical significance of anti-Su antibodies was studied in 236 Japanese and 160 American patients with systemic rheumatic diseases. Anti-Su in immunodiffusion (ID) was strongly associated with immunoprecipitation of one or more 100- to 102-kDa proteins by MRL/lpr mouse sera (27/32 of ID positive vs 4/20 of ID negative, P = 0.000016), and all four human anti-Su reference sera immunoprecipitated the 100/102-kDa protein(s). In addition, all sera immunoprecipitated a less efficiently labeled approximately 200-kDa protein that comigrated on sucrose density gradients with the 100/102-kDa proteins. Based on these data, a complex of the 100/102-kDa and 200-kDa proteins is likely to be the main target of anti-Su antibodies. Three of four anti-Su monospecific sera were negative for immunofluorescent antinuclear antibodies (ANA), suggesting anti-Su antibodies may be associated with a negative ANA in some cases. Autoantibodies to Su were detected frequently by immunoprecipitation in systemic lupus erythematosus (17-21%), scleroderma (13-20%), and overlap syndrome (22-40%) and were associated with autoantibodies to Ku.

Animals↗
PubMed2005

Internalization and trafficking mechanisms of coxsackievirus B3 in HeLa cells.

Coxsackievirus B3 (CVB3) is nonenveloped and has a single-stranded positive-sense RNA genome. CVB3 induces myocarditis and ultimately dilated cardiomyopathy. Although there are mounting evidences of an interaction between CVB3 particles and the cellular receptors, coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF), very little is known about the mechanisms of internalization and trafficking. In the present study, we used the CVB3 H3 strain, which is CAR-dependent but DAF-independent Woodruff variant and found that during entry, CVB3 particles were colocalized in clathrin, after interacting primarily with CAR, which was not recycled to the plasma membrane. We also found that CVB3 internalization was dependent on the function of dynamin, a large GTPase that has an essential role in endocytosis. Heat-shock cognate protein, Hsc70, which acts as a chaperone in the release of coat proteins from clathrin-coated vesicles (CCV), played a role in CVB3 trafficking processes. Moreover, endosomal acidification was crucial for CVB3 endocytosis. Finally, CVB3 was colocalized in early endosome autoantigen 1 (EEA1) molecules, which are involved in endosome-endosome tethering and fusion. In conclusion, these data together indicate that CVB3 uses clathrin-mediated endocytosis and is transcytosed to early endosomes.

Base Sequence↗
PubMed1981

Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap.

Autoantibodies in the serum from a patient with connective tissue disease have been used to define a high molecule weight acidic nuclear protein antigen. The antigen tentatively termed Ku, after the first two letters of patient's name, has distinct physicochemical properties and immunological specificities that distinguish it from previously reported antigens. The Ku antigen has an apparent 300,000 mol wt as determined by gel filtration and sucrose density gradient ultracentrifugation techniques. The antigen is destroyed by trypsin, mild heating, and pH variations greater than 10 and less than 5. Treatment with ribonuclease or deoxyribonuclease did not affect the antigenic reactivity. The Ku antigen was demonstrated in the soluble extracts of human, calf, and rabbit, but not of rat tissues. Purified antibody localized the Ku antigen within the nuclei of human liver where a "reticular" pattern of immunofluorescence was seen. Of 330 patients with various connective tissue diseases, 9 had precipitating antibodies to the Ku antigen. Preliminary results of clinical analysis indicated that antibody to the Ku antigen might become a useful marker for a group of patients with clinical characteristics of both polymyositis and scleroderma with a good prognosis.

Antigens↗
PubMed1983

Autoantibodies to nonhistone nuclear antigens and their clinical significance.

Identification of the immunologic specificity of antinuclear antibodies (ANAs) in the various systemic rheumatic diseases has become increasingly important. The standard immunofluorescence technique may enable detection of antibodies to nuclear antigens present in abundance in the nucleus, such as DNA, histones, Sm, nuclear ribonucleoprotein (nRNP), and SS-B/La. The nuclear antigens present in low concentrations, such as SS-A, proliferating cell nuclear antigen (PCNA), rheumatoid arthritis nuclear antigen (RANA), and Ku antigens, are unique to cell types, and their detection requires special substrates or reagent systems. Anti-Sm, anti-Scl-70, anticentromere, and anti-PM-1 are characteristic serologic markers for systemic lupus erythematosus, scleroderma, the CREST syndrome of scleroderma, and polymyositis, respectively. Distinct profiles of ANA characterize different rheumatic diseases. A number of ANAs are found in SLE, whereas other diseases are characterized by the presence or absence of a certain ANA or by differences in mean ANA titers. Specific ANAs have been used to isolate and characterize nuclear antigens at molecular and functional levels.

Antibodies, Antinuclear↗