Rizou C, Ioannidis J P, Panou-Pomonis E, Sakarellos-Daitsiotis M, Sakarellos C, Moutsopoulos H M, Vlachoyiannopoulos P G
Department of Organic Chemistry and Biochemistry, School of Natural Sciences, University of Ioannina.
Am J Respir Cell Mol Biol. 2000 Mar;22(3):344-51. doi: 10.1165/ajrcmb.22.3.3850.
We hypothesized that B-cell epitope mapping of DNA Topoisomerase I (type-I topoisomerase, or Topo I) may define epitopes strongly associated with pulmonary interstitial fibrosis (PIF) in systemic sclerosis (SSc). B-cell epitope mapping of Topo I was performed using 63 20-mer peptides overlapping by eight residues and spanning the entire length of the Topo I sequence. These peptides, coupled to polystyrene pins, were tested for antibody binding by enzyme-linked immunosorbent assays (ELISAs) using immunoglobulin G fractions from anti-Topo I, anticentromere, anti-U3RNP-positive, and normal sera. Four major epitopes were recognized by anti-Topo I sera, but not from the control sera: WWEEERYPEGIKWKFLEHKG (205-224, epitope I), RIANFKIEPPGLFRGRGNHP (349-368, epitope II), PGHKWKEVRHDNKVTWLVSW (397-416, epitope III), and ELDGQEYVVEFDFLGKDSIR (517-536, epitope IV). Peptide-epitopes were then synthesized in their soluble forms and ELISA systems were developed. Epitopes II to IV are localized at highly exposed sites of the Topo I tertiary structure, whereas epitope I is localized at a less accessible site. In a cohort of 81 patients with SSc with clinical data on the evolution of their disease, patients with antibodies in their sera recognizing at least three of the four epitopes had 3.1 times (P = 0.02) the hazard of developing PIF compared with patients whose sera recognized no epitopes or only one or two of the four epitopes. The discrimination was much stronger than that achieved by the simple determination of Topo I antibodies by counterimmunoelectrophoresis and immunoblot (hazard ratio 1.7, P = 0.30) in the same patients. B-cell epitope mapping of the anti-Topo I response has identified four major epitopes which cumulatively show a strong association with the development of PIF in SSc.
我们推测，DNA拓扑异构酶I（I型拓扑异构酶，即Topo I）的B细胞表位图谱可能会确定与系统性硬化症（SSc）中的肺间质纤维化（PIF）密切相关的表位。使用63个20肽进行Topo I的B细胞表位图谱分析，这些肽段相互重叠8个残基，覆盖Topo I序列的全长。将这些肽段偶联到聚苯乙烯针上，通过酶联免疫吸附测定（ELISA），使用来自抗Topo I、抗着丝粒、抗U3RNP阳性血清和正常血清的免疫球蛋白G组分检测抗体结合情况。抗Topo I血清识别出四个主要表位，但对照血清未识别：WWEEERYPEGIKWKFLEHKG（205 - 224，表位I）、RIANFKIEPPGLFRGRGNHP（349 - 368，表位II）、PGHKWKEVRHDNKVTWLVSW（397 - 416，表位III）和ELDGQEYVVEFDFLGKDSIR（517 - 536，表位IV）。然后合成这些肽表位的可溶形式，并建立ELISA系统。表位II至IV位于Topo I三级结构的高度暴露位点，而表位I位于较难接近的位点。在一组81例有疾病进展临床数据的SSc患者中，血清中抗体识别四个表位中至少三个的患者发生PIF的风险是血清未识别表位或仅识别四个表位中的一个或两个的患者的3.1倍（P = 0.02）。在同一组患者中，这种区分能力比通过对流免疫电泳和免疫印迹简单测定Topo I抗体所达到的区分能力（风险比1.7，P = 0.30）要强得多。抗Topo I反应的B细胞表位图谱分析确定了四个主要表位，这些表位累积显示与SSc中PIF的发生密切相关。
