Hanson Robert, Evilia Caryn, Gilmer John, Woods Linda, Black Brad, Flores Raja, Pfau Jean C
Department of Biological Sciences, Idaho State University, Pocatello, Idaho.
Department of Chemistry, Idaho State University, Pocatello, Idaho.
Physiol Rep. 2016 Aug;4(15). doi: 10.14814/phy2.12881.
Lamellar pleural thickening (LPT) is a fibrotic disease induced by exposure to Libby amphibole (LA) asbestos that causes widespread scarring around the lung, resulting in deterioration of pulmonary function. Investigating the effects of autoantibodies to mesothelial cells (MCAA) present in the study populations has been a major part of the effort to understand the mechanism of pathogenesis. It has been shown in vitro that human mesothelial cells (Met5a) exposed to MCAA increase collagen deposition into the extracellular matrix (ECM). In this study, we sought to further elucidate how MCAA drive increased collagen deposition by identifying the protein targets bound by MCAA on the cellular surface using biotinylation to label and isolate surface proteins. Isolated surface protein fractions were identified as containing MCAA targets using ELISA The fractions that demonstrated binding by MCAA were then analyzed by tandem mass spectrometry (MS/MS) and MASCOT analysis. The most promising result from the MASCOT analysis, plasminogen (PLG), was tested for MCAA binding using purified human PLG in an ELISA We report that serum containing MCAA bound at an optical density (OD) 3 times greater than that of controls, and LA-exposed subjects had a high frequency of positive tests for anti-PLG autoantibodies. This work implicates the involvement of the plasminogen/plasmin system in the mechanism of excess collagen deposition in Met5a cells exposed to MCAA Elucidating this mechanism could contribute to the understanding of LPT.
板层状胸膜增厚（LPT）是一种因接触利比角闪石（LA）石棉而引发的纤维化疾病，会导致肺部周围广泛瘢痕形成，进而致使肺功能恶化。研究研究人群中存在的抗间皮细胞自身抗体（MCAA）的作用，一直是理解发病机制努力的重要组成部分。体外研究表明，暴露于MCAA的人源间皮细胞（Met5a）会增加胶原蛋白向细胞外基质（ECM）的沉积。在本研究中，我们试图通过使用生物素化标记和分离表面蛋白来鉴定MCAA在细胞表面结合的蛋白靶点，从而进一步阐明MCAA如何驱动胶原蛋白沉积增加。使用酶联免疫吸附测定（ELISA）将分离的表面蛋白组分鉴定为含有MCAA靶点。然后通过串联质谱（MS/MS）和MASCOT分析对显示与MCAA结合的组分进行分析。MASCOT分析中最有前景的结果——纤溶酶原（PLG），在ELISA中使用纯化的人PLG测试其与MCAA的结合情况。我们报告称，含有MCAA的血清结合时的光密度（OD）比对照组高3倍，且接触LA的受试者抗PLG自身抗体检测呈阳性的频率较高。这项工作表明纤溶酶原/纤溶酶系统参与了暴露于MCAA的Met5a细胞中胶原蛋白过度沉积的机制。阐明这一机制有助于对LPT的理解。
