Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn's disease
Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn's disease
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DOI：
10.1136/gut.2008.162495
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发表时间：
2009-12-01
期刊：
影响因子：
24.5
通讯作者：
Conrad, K.
中科院分区：
文献类型：
--
作者：
Roggenbuck, D.;Hausdorf, G.;Conrad, K.
背景与目的：克罗恩病是一种炎症性肠病，其发病机制尚不完全清楚。自身免疫机制被认为在克罗恩病的发生发展中起作用，但靶抗原和潜在的途径尚未被充分识别。方法：基于免疫印迹和基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱仪的数据，确定了针对克罗恩病的胰腺自身抗体(PAB)的主要抗原靶。用纯化的大鼠和瞬时转基因哺乳动物HEK 293细胞合成的人重组GP2，用酶联免疫吸附试验(ELISA)和间接免疫荧光(IIF)证实自身抗体反应的特异性。采用实时定量聚合酶链式反应(RT-PCR)和间接免疫荧光(IIF)检测结肠活检组织中Pabs的mRNA和抗原定位。结果：主要酶原颗粒膜糖蛋白2(GP2)是克隆氏病患者Pabs的自身抗原。42例克罗恩病患者PAb阳性血清对大鼠GP2Ig G反应性显著高于PAb阴性血清(n=31)、溃疡性结肠炎患者(n=49)和献血员血清(n=69)(p<；0.0001)。在42份PAB阳性血清中，28份(66%)和18份(43%)在IIF中分别表现出对人重组GP2的Ig G和Ig A反应。PAB阴性的克罗恩病患者(n=31)无反应。克罗恩病患者(n=4)的结肠活检组织中GP2mRNA转录水平显著高于溃疡性结肠炎(n=4)患者(p=0.0286)。结论：抗GP2自身抗体构成了新的克罗恩病特异性标志物，其定量检测可显著提高IBD的血清学诊断水平。人肠上皮细胞中GP2的表达提示抗GP2反应在克罗恩病的发病机制中具有重要作用。
Backround and aims: The aetiopathogenesis of Crohn's disease, an inflammatory bowel disease (IBD), is not yet fully understood. Autoimmune mechanisms are thought to play a role in the development of Crohn's disease, but the target antigens and the underlying pathways have not been sufficiently identified.Methods: Based on data from immunoblotting and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry, the major antigenic target of pancreatic autoantibodies (PABs), which are specific for Crohn's disease, was identified. Specificity of autoantibody reactivity was confirmed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) using purified rat and human recombinant GP2 synthesised in transiently transfected mammalian HEK 293 cells. Real-time polymerase chain reaction (rt-PCR) and IIF were used to detect mRNA and antigen localisation in human colon biopsies.Results: The major zymogen granule membrane glycoprotein 2 (GP2) was identified as the autoantigen of PABs in Crohn's disease. PAB-positive sera from patients with Crohn's disease (n = 42) displayed significantly higher IgG reactivity to rat GP2 in ELISA than either PAB-negative sera (n = 31), or sera from patients with ulcerative colitis (n = 49), or sera from blood donors (n = 69) (p < 0.0001, respectively). Twenty-eight (66%) and 18 (43%) of 42 PAB-positive sera demonstrated IgG and IgA reactivity to human recombinant GP2 in IIF, respectively. Patients with PAB-negative Crohn's disease (n = 31) were not reactive. GP2 mRNA transcription was significantly higher in colon biopsies from patients with Crohn's disease (n = 4) compared to patients with ulcerative colitis (n = 4) (p = 0.0286). Immunochemical staining confirmed GP2 expression in human colon biopsies from patients with Crohn's disease.Conclusion: Anti-GP2 autoantibodies constitute novel Crohn's disease-specific markers, the quantification of which could significantly improve the serological diagnosis of IBD. The expression of GP2 in human enterocytes suggests an important role for anti-GP2 response in the pathogenesis of Crohn's disease.