Identification of a Novel Mycobacterial Histone H1 Homologue (HupB) as an Antigenic Target of pANCA Monoclonal Antibody and Serum Immunoglobulin A from Patients with Crohn's Disease

pANCA is a marker antibody associated with inflammatory bowel disease (IBD), including most patients with ulcerative colitis and a subset with Crohn's disease. This study addressed the hypothesis that pANCA reacts with an antigen(s) of microbial agents potentially relevant to IBD pathogenesis. Using a pANCA monoclonal antibody, we have previously identified the C-terminal basic random-coil domain of histone H1 as a pANCA autoantigen. BLAST analysis of the peptide databases revealed H1 epitope homologues in open reading frames of the Mycobacterium tuberculosis genome. Western analysis of extracts from six mycobacterial species directly demonstrated reactivity to a single, conserved ∼32-kDa protein. Direct protein sequencing, followed by gene cloning, revealed a novel 214-amino-acid protein, an iron-regulated protein recently termed HupB. Sequence analysis demonstrated its homology with the mammalian histone H1 gene family, and recombinant protein expression confirmed its reactivity with the 5-3 pANCA monoclonal antibody. Binding activity of patient serum immunoglobulin G (IgG) to HupB did not correlate with reactivity to histone H1 or pANCA, indicating the complex character of the pANCA antigen. However, anti-HupB IgA was strongly associated with Crohn's disease (P < 0.001). These findings indicate that the 5-3 pANCA monoclonal antibody detects a structural domain recurrent among mycobacteria and cross-reactive with a DNA-binding domain of histone H1. The association of HupB-binding serum IgA with IBD provides new evidence for the association of a mycobacterial species with Crohn's disease.

核周抗中性粒细胞胞浆抗体（perinuclear antineutrophil cytoplasmic antibody, pANCA）是与炎症性肠病（inflammatory bowel disease, IBD）相关的标志性抗体，涵盖绝大多数溃疡性结肠炎患者以及一小部分克罗恩病（Crohn's disease）患者。本研究旨在验证下述假说：pANCA可与潜在参与IBD发病机制的微生物源性抗原发生免疫反应。本研究团队此前利用pANCA单克隆抗体，将组蛋白H1（histone H1）的C端碱性无规卷曲结构域鉴定为pANCA自身抗原。对肽数据库进行BLAST（Basic Local Alignment Search Tool）分析后，在结核分枝杆菌（Mycobacterium tuberculosis）基因组的开放阅读框中发现了H1表位同源序列。对6种分枝杆菌提取物开展的免疫印迹分析（Western analysis），直接证实其可与一种保守的约32 kDa单一条带蛋白发生免疫反应。通过直接蛋白质测序结合基因克隆技术，研究人员鉴定出一种全新的214个氨基酸组成的铁调节蛋白，该蛋白新近被命名为HupB。序列分析显示，该蛋白与哺乳动物组蛋白H1基因家族具有同源性；重组蛋白表达实验进一步证实，其可与5-3 pANCA单克隆抗体发生结合反应。患者血清免疫球蛋白G（immunoglobulin G, IgG）与HupB的结合活性，与针对组蛋白H1或pANCA的免疫反应并无关联，这表明pANCA抗原的识别具有复杂性。然而，抗HupB免疫球蛋白A（immunoglobulin A, IgA）水平与克罗恩病存在显著相关性（P < 0.001）。本研究结果表明，5-3 pANCA单克隆抗体可识别分枝杆菌中普遍存在的一种结构域，该结构域与组蛋白H1的DNA结合结构域存在交叉反应性。血清中结合HupB的IgA与炎症性肠病的相关性，为分枝杆菌菌种与克罗恩病的关联提供了新的实验证据。