Efficient isolation of cDNA clones encoding rheumatoid arthritis autoantigens by lambda phage surface display

Bacteriophage lambda surface display was used to isolate cDNA clones encoding autoantigens recognized by synovial fluid (SF) or sera from patients with rheumatoid arthritis (RA). We constructed cDNA libraries from human synovial sarcoma cells and synovial tissue, using the surface display vector lambdafoo. The cDNA libraries were screened by affinity selection using 40 SF and 44 sera as probes separately immobilized in microtiter wells. Phage clones isolated encode 13 different autoantigens; an unknown protein, two proteins previously unanalyzed as autoimmune antigens, three proteins previously unknown to be recognized by RA sera, and seven known RA antigens. When analyzed their sensitivity and specificity for RA by phage enzyme-linked immunosorbent assay, frequencies of sera that recognize the newly-isolated autoantigens ranged from 20.5 to 6.8% of a panel of RA sera, and 13.6-0% of other autoimmune disease sera. These results indicate that the lambda phage surface display may be powerful for the isolation of cDNA clones encoding autoantigens recognized by SF or sera from patients with not only RA but also other autoimmune diseases.     

Proteomic analysis of human brain identifies alpha-enolase as a novel autoantigen in Hashimoto's encephalopathy

Hashimoto's encephalopathy (HE) is a rare autoimmune disease associated with Hashimoto's thyroiditis (HT). To identify the HE-related autoantigens, we developed a human brain proteome map using two-dimensional electrophoresis and applied it to the immuno-screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI-TOF-MASS analysis, immuno-positive spots of 48 kDa (pI 7.3-7.8) detected from HE patient sera were identified as a novel autoimmuno-antigen, alpha-enolase, harboring several modifications. Specific high reactivities against human alpha-enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut-off level. Although a few HT patients showed faint reactions to alpha-enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti-alpha-enolase antibody is useful for defining HE-related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.     

Identification of AHNAK as a novel autoantigen in systemic lupus erythematosus

To identify candidate autoantigens associated with arthritis, a rat chondrocyte cDNA library was immunoscreened with serum from a patient with rheumatoid arthritis. One isolated cDNA encoded part of AHNAK, a 700-kDa phosphoprotein with DNA binding properties, that appears to be involved in several signal transduction pathways. Immunoreactivity against an in vitro translated human AHNAK fragment was detected in 4.6% (5/109) of patients with rheumatoid arthritis, 29.5% (18/61) of patients with systemic lupus erythematosus (SLE), and 1.2% (2/172) of blood donors. Anti-AHNAK antibodies reacted with a recombinant human AHNAK fragment and with native AHNAK from C32 cell lysates. In vitro translated AHNAK fragment could be cleaved by granzyme B and caspase-3. Anti-AHNAK positive SLE patients had a higher frequency of homogeneous antinuclear antibody staining patterns and a lower frequency of recent mucosal ulcerations. This is the first report that AHNAK can be targeted by the immune system in autoimmune disease.     

日本血吸虫童虫裂解物对东方田鼠肝脏T7噬菌体展示cDNA文库的筛选及阳性克隆分析

东方田鼠对血吸虫具有天然抗性。为筛选和分析东方田鼠抗血吸虫抗性相关基因, 以日本血吸虫童虫可溶性裂解物为探针, 筛选东方田鼠肝脏噬菌体展示cDNA文库。经三轮筛选, 特异性噬菌体得到有效富集(375倍)。随机挑取92个克隆进行序列测定, 获得了19条有效EST序列。其中13个条EST序列与已知基因或表达序列标签同源, 6个EST序列与已知基因或表达序列标签均无同源性, 为新的表达序列标签。将19个EST序列的阳性噬菌体克隆和血吸虫童虫共培养, 其中4号(GenBank Accession No.: EW968294)、13号(GenBank Accession No.: EW968303)、14号(GenBank Accession No.: EW968304)、15号(GenBank Accession No.: EW968305)、18号(GenBank Accession No.: EW968308)克隆均诱导了显著的杀虫效果。综合生物信息学分析结果及体外杀伤试验结果, 编码CASP8和FADD类似性细胞程序性死亡调节蛋白、a-2-HS-糖蛋白、M4蛋白、具有R3H结构域的一种mRNA结合蛋白以及三种未知蛋白的编码基因(14、15、18号克隆)可能是东方田鼠抗血吸虫病抗性相关基因。为进一步研究东方田鼠抗血吸虫机理奠定了基础。     

日本血吸虫童虫裂解物对东方田鼠肝脏T7噬菌体展示cDNA文库的筛选及阳性克隆分析

东方田鼠对血吸虫具有天然抗性。为筛选和分析东方田鼠抗血吸虫抗性相关基因, 以日本血吸虫童虫可溶性裂解物为探针, 筛选东方田鼠肝脏噬菌体展示cDNA文库。经三轮筛选, 特异性噬菌体得到有效富集(375倍)。随机挑取92个克隆进行序列测定, 获得了19条有效EST序列。其中13个条EST序列与已知基因或表达序列标签同源, 6个EST序列与已知基因或表达序列标签均无同源性, 为新的表达序列标签。将19个EST序列的阳性噬菌体克隆和血吸虫童虫共培养, 其中4号(GenBank Accession No.: EW968294)、13号(GenBank Accession No.: EW968303)、14号(GenBank Accession No.: EW968304)、15号(GenBank Accession No.: EW968305)、18号(GenBank Accession No.: EW968308)克隆均诱导了显著的杀虫效果。综合生物信息学分析结果及体外杀伤试验结果, 编码CASP8和FADD类似性细胞程序性死亡调节蛋白、a-2-HS-糖蛋白、M4蛋白、具有R3H结构域的一种mRNA结合蛋白以及三种未知蛋白的编码基因(14、15、18号克隆)可能是东方田鼠抗血吸虫病抗性相关基因。为进一步研究东方田鼠抗血吸虫机理奠定了基础。     

A major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein autoantigen

Apoptotically modified forms of autoantigens have been hypothesized to participate in lupus immunopathogenesis. This study identifies a major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein lupus autoantigen (70k). Human autoimmune sera with strong recognition of apoptotic 70k and minimal recognition of intact 70k were identified and tested for reactivity to truncated forms of 70k by immunoblot and ELISA. Patient sera that preferentially recognized apoptotic 70k were specific for an epitope dependent on residues 180-205 of the protein. This epitope was also recognized by 19 of 28 (68%) intact anti-70k-positive autoimmune human sera with Abs also recognizing apoptotic but not the intact form 70k, but only 1 of 9 (11%) intact 70k-positive sera without such Abs (Fisher's exact, p = 0.0055). Immunization of HLA-DR4-transgenic C57BL/6 mice with a peptide containing this epitope induced anti-70k immunity in 13 of 15 mice, including Abs recognizing apoptotic but not intact forms of autoantigens in 12 of 15 mice. Anti-70k responder mice also developed spreading of immunity to epitopes on the endogenous form of 70k, and proliferative lung lesions consistent with those described in patients with anti-70k autoimmunity. Thus, a major epitope in the B cell response to U1-70 kDa localizes to the RNA binding domain of the molecule, overlaps with the most common T cell epitope in the anti-70k response, and is not present on the intact form of the 70k molecule. Immunization of mice against this epitope induces an immune response with features seen in human anti-70k autoimmune disease.     

Evolutionarily conserved antigens in autoimmune disease: implications for an infective aetiology

The immune system has evolved to eliminate or inactivate infectious organisms. An inappropriate response against self-components (autoantigens) can result in autoimmune disease. Here we examine the hypothesis that some evolutionarily conserved proteins, present in pathogenic and commensal organisms and their hosts, provide the stimulus that initiates autoimmune disease in susceptible individuals. We focus on seven autoantigens, of which at least four, glutamate decarboxylase, pyruvate dehydrogenase, histidyl-tRNA synthetase and alpha enolase, have orthologs in bacteria. Citrullinated alpha-enolase, a target for autoantibodies in 40% of patients with rheumatoid arthritis, is our main example. The major epitope is highly conserved, with over 90% identity to human in some bacteria. We propose that this reactivity of autoantibodies to shared sequences provides a model of autoimmunity in rheumatoid arthritis, which may well extend to other autoimmune disease in humans.     

Identification of autoantibodies associated with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinuclear antibodies. We performed serological analysis of cDNA expression library (SEREX) to identify autoantibodies associated with SLE. The screening of three different cDNA expression libraries with pooled sera of patients with SLE yielded 11 independent clones that reacted with pooled sera of patients with SLE. In this screening, autoantibodies to poly(ADP-ribose) polymerase (PARP), U1snRNP, and galectin-3 were prevalent in the sera of patients with SLE (26/68, 25/68, 12/63, respectively). The frequency of autoantibody to PARP was significantly higher in SLE than that of healthy donors (0/76) (38.2% vs 0%, p<0.00001). The autoantibody to PARP was infrequently detected in the serum of patients with RA (1/50). However, autoantibody to PARP was not found in the sera of patients with other rheumatic diseases including Sjogren's syndrome (0/19), systemic sclerosis (0/18), and polymyositis/myositis (0/37). The frequency of autoantibody to human galectin-3 (12/63) was significantly higher in SLE than that of healthy donors (0/56) (19% vs 0%, p=0.0006). Autoantibody to galectin-3 was not found in the sera of patients with rheumatoid arthritis (0/50), Sjogren's syndrome (0/18), and systemic sclerosis (0/19). Interestingly, autoantibody to galectin-3 was also prevalent in the sera of patients with polymyositis/dermatomyositis (16/37, 43.2%). Further functional characterization of these autoantibodies would be necessary to determine their value as diagnostic markers or to define clinical subsets of patients with SLE. Statistical analysis revealed that the presence of autoantibody to PARP was inversely related with pleurisy, and the presence of autoantibody to galectin-3 related with renal disease.     

Proteomic surveillance of autoantigens in relapsing polychondritis

Relapsing polychondritis (RP) is a systemic inflammatory disease, in which autoimmunity to cartilage-related components is thought to be involved in its pathogenesis. However, the autoimmune profile in RP has not been studied fully. We therefore investigated autoantibodies/autoantigens in RP comprehensively, by 2-dimensional electrophoresis (2DE), subsequent western blotting (WB) and mass spectrometry, using cell-extracted proteins as the antigen source. As a result, we detected 15 autoantigens on 2DE-WB, and further identified five of them. On average, one RP serum recognized approximately 8 out of the 15 autoantigens. Frequencies of the autoantibodies to the 5 identified antigens of tubulin alpha ubiquitous/6, vimentin, alpha enolase, calreticulin, and colligin-1/-2 were 91%, 46%, 36%, 82%, and 36%, respectively. ELISA using recombinant proteins for them revealed that frequencies of the autoantibodies to tubulin alpha ubiquitous, vimentin, alpha enolase, calreticulin, and colligin-1 were 36%, 64%, 46%, 27%, and 18%, respectively. Our data demonstrated that the autoimmune reaction was not restricted to cartilagerelated components, rather a variety of autoimmune responses occurred in patients with RP, which may be involved in the pathophysiology of RP. In addition, the proteomic approach using cell-extracted proteins would be a powerful way to investigate autoantigens.     

抗肿瘤蛋白AAL与靶蛋白MRG15的共定位研究

一种从食用真菌Agrocybe aegerita中提取的凝集素AAL具有显著的抗肿瘤效果。该蛋白的基因序列已经通过RT-PCR得到。T7噬菌体展示库筛选发现该蛋白可以在体外和细胞核内的细胞周期调控蛋白mortality factor related gene on chro-mosome 15(MRG15)发生相互作用。本实验目的是证实AAL和MRG15在细胞核内的共定位,为AAL与MRG15的体内的互作提供证据。构建质粒,将aal与mrg15分别连接到pEGFP-C1和pDsRed-C1上,共转染Hela细胞。将pEGFP-C1和pD-sRed-C1空载体共转染Hela细胞作为对照。利用激光共聚焦显微镜研究AAL和MRG15在细胞内的共定位。AAL和MRG15均定位在细胞核内,荧光图片的重叠表明AAL和MRG15在核内共定位。而对照组中的EGFP和DS-RED在细胞内呈弥散分布。这些为AAL和MRG15在细胞核内的相互作用提供了证据。     

Antinuclear autoantibodies: probes for defining proteolytic events associated with apoptosis

Antinuclear autoantibodies (ANAs) derived from patients with systemic autoimmune diseases have proven to be powerful tools in cell and molecular biology. The availability of these autoantibodies has been instrumental in the identification and characterization of a wide range of intracellular proteins involved in essential cellular activities. Recently, these autoantibodies have been used in molecular studies of apoptosis, particularly in the identification of substrates cleaved by proteases of the ICE/CED-3 family during this cell death pathway. The identification of these substrates may help to understand the role of proteolysis in apoptosis. Examples of nuclear autoantigens whose cleavage during apoptosis have been defined using ANAs include the 70 kD protein of the U1 small nuclear ribonucleoprotein particle (U1-70 kD), the nuclear mitotic apparatus protein (NuMA), DNA topoisomerase I, the RNA polymerase I upstream binding factor (UBF), and the catalytic subunit of DNA-dependent protein kinase (DNA-PK_cs). The use of ANAs as probes for defining proteolytic events associated with apoptosis promises to yield important insights into the mechanisms driving this cell death pathway.Abbreviations ANAs Antinuclear autoantibodies - DNA-PK_cs DNA-dependent protein kinase - ICE interleukin-1 (IL-1)-converting enzyme - MARs or SARs matrix or scaffold attachment regions - NuMA nuclear mitotic apparatus protein - PARP poly (ADP) ribose polymerase - snRNPs small nuclear ribonucleoproteins - SLE systemic lupus erythematosus - UBF upstream binding factor     

腺苷脱氨酶及同工酶对自身免疫病诊断和病情监测的作用

摘要 目的：检测腺苷脱氨酶(ADA)及其同工酶在自身免疫病患者血清中的变化,探讨其诊断和病情监测作用。方法：收集70例系统性红斑狼疮（SLE）、114例类风湿性关节炎(RA)、55例强直性脊柱炎(AS)、及其年龄性别对应的健康血清标本。测定血清总ADA（tADA）、ADA1及ADA2活性。ROC曲线分析其诊断价值。结果：与健康对照相比,ADA活性在SLE患者血清中显著升高[tADA：15（12,20）vs 8（7,10）U/L;ADA1：3.5（2,5）vs 3（2,3）U/L;ADA2：11（8,15）vs 6（5,7）U/L;P<0.01）]。与健康对照相比,RA患者血清中tADA和ADA1活性无显著变化(P>0.05),ADA2活性水平升高[8（5.25,10）vs 7（5,9）U/L,P<0.05）];与健康对照相比,AS患者血清中tADA和ADA1活性无显著变化(P>0.05),ADA2活性升高[7（5,9）vs 6（5,7）U/L,P<0.05）]。ROC分析显示tADA及ADA2对SLE具有较好诊断价值（tADA：88.6%特异性、77.1%敏感性;ADA2：92.9%特异性、68.6%敏感性）。ADA活性对RA及AS患者无诊断价值。spearman相关性分析显示,tADA活性与SLE患者疾病活动度有一定正相关性（r=0.303,P=0.011)。结论：血清tADA活性检测可作为SLE辅助诊断和病情监测指标。     

IgG autoantibodies against deposited C3 inhibit macrophage-mediated apoptotic cell engulfment in systemic autoimmunity

Defective clearance of apoptotic cells has been shown in systemic lupus erythematosus (SLE) and is postulated to enhance autoimmune responses by increasing access to intracellular autoantigens. Until now, research has emphasized inherited rather than acquired impairment of apoptotic cell engulfment in the pathogenesis of SLE. In this study, we confirm previous results that efficient removal of apoptotic cells (efferocytosis) is bolstered in the presence of wild-type mouse serum, through the C3 deposition on the apoptotic cell surface. In contrast, sera from three mouse models of SLE, Mer(KD), MRL(lpr), and New Zealand Black/WF1 did not support and in fact actively inhibited apoptotic cell uptake. IgG autoantibodies were responsible for the inhibition, through the blockade of C3 recognition by macrophages. Consistent with this, IgG removal reversed the inhibitory activity within autoimmune serum, and purified autoimmune IgG blocked both the detection of C3 on apoptotic cells and C3-dependent efferocytosis. Sera from SLE patients demonstrated elevated anti-C3b IgG that blocked detection of C3 on apoptotic cells, activity that was not found in healthy controls or patients with rheumatoid arthritis, nor in mice prior to the onset of autoimmunity. We propose that the suppression of apoptotic cell disposal by Abs against deposited C3 may contribute to increasing severity and/or exacerbations in SLE.     

Molecular cloning and expression of a novel human cDNA related to the diazepam binding inhibitor.

In order to isolate the unidentified autoantigens in autoimmune diabetes, a human pancreatic islet cDNA library was constructed and screened with the sera from the diabetic patients. From the library screening, one clone (DRS-1) that strongly reacted with the sera was isolated. Subsequent sequence analysis revealed that the clone was a novel cDNA related to the diazepam binding inhibitor. DRS-1 was expressed in most tissues including liver, lung, tonsil, and thymus, in addition to pancreatic islets. DRS-1 was in vitro translated and the recombinant DRS-1 protein was expressed in Escherichia coli and purified. The size of the in vitro translated or bacterially expressed DRS-1 protein was in agreement with the conceptually translated polypeptide of DRS-1 cDNA. Further studies are required to test whether or not DRS-1 is a new autoantigen in autoimmune diabetes.