Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples

IntroductionA substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC).ResultsThe expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium.ConclusionTaken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.引言 人类基因组中有相当一部分源自转座因子（transposable elements）——古代逆转录病毒感染的遗留产物。人类基因组中约8%的序列由约40万个长末端重复序列（Long Terminal Repeat, LTR）元件组成，其中包含人类内源性逆转录病毒（human endogenous retrovirus, HERV）序列。在多数生理状态下，表观遗传与转录后调控机制的协同作用被认为可沉默HERV的表达。值得注意的是，部分HERV-H位点的异常激活似乎仅特异性出现于结直肠癌（colorectal carcinoma, CRC）中。 结果 本研究采用新型小鼠抗Gag-H单克隆抗体，对HERV-H衣壳蛋白（Gag-H）的表达水平进行检测。通过流式细胞术筛选，在一组原代结直肠癌细胞系中测试了4株抗体克隆，并通过蛋白质印迹（Western blot）验证了表现最优的抗体克隆。最终，通过免疫组织化学分析细胞离心涂片（cytospin）样本及临床组织样本中的Gag-H蛋白表达。结果显示，染色模式呈现异质性：内皮细胞、基质细胞及浸润免疫细胞无背景染色，而阳性肿瘤细胞与结肠上皮的正常隐窝细胞的细胞质呈现弥漫性阳性染色。 结论 综上，该Gag-H抗体克隆可作为检测结肠来源细胞的有效工具，为后续更深入的研究奠定了基础。本研究在结肠上皮隐窝细胞中观察到的Gag-H蛋白染色结果，提示需要对Gag-H在人类肠道正常生理功能中的潜在作用开展深入分析。