人類表皮生長因子受體的硫酸化研究

Abstract

蛋白質酪胺酸硫酸化是由酪胺酸亞硫酸基轉移酶(tyrosylprotein sulfotransferase,TPST)所催化的一種常見蛋白質轉譯後修飾，調控蛋白質的交互作用，進而影響生理反應和致病機制。目前雖然已確知蛋白質酪胺酸硫酸化之重要性，但許多重要的硫酸化的蛋白質受質仍亟待研究。經由預測軟體(Sulfinator)預測，發現人類表皮生長因子受體(epidermal growth factor receptor, EGFR)可能為有四個蛋白質酪胺酸硫酸化可能的催化位。表皮生長因子受體影響許多生理現象，當它被訊號分子活化，將會觸發細胞增生、抑制細胞凋亡、血管新生、代謝和抗藥性等等反應。目前已知人類EGFR的突變和非小型細胞肺癌的形成有密切關係，因此我們推測人類表皮生長因子受體的硫酸化可能和肺癌的致病機制有關，是很值得深入探討的研究題目。本研究合成EGFR的蛋白質序列運用本實驗室已建立耦合酶分析法，以及從A549細胞株的免疫沉澱實驗，實驗證實EGFR極有可能為蛋白質酪胺酸硫酸化之受質，並且找出硫酸化的位置。未來將研究硫酸化對EGFR在生理功能的影響及肺癌相關疾病機制，以供探討癌症的形成與設計藥物的參考。

Tyrosine sulfation is a widespread post-translational modification found in secreted proteins and cellular surface receptors. It has been identified as a key modulator of protein-protein interactions. Tyrosine sulfation has an important role in virus infection, inflammatory responses and many cancer or other diseases. Activation of epidermal growth factor receptor (EGFR) triggers anti-apoptotic signaling, proliferation, angiogenesis, and drug resistance, which leads to the development and progression of human epithelial cancers. We examined the sequence and structure of human EGFR and found four potential tyrosine sulfation sites. Four peptides that contains the potential tyrosine sulfation site were synthesized and used as substrates of tyrosylprotein sulfotransferase (TPST). Native EGFR from A549 cell lysates was examined for potential post-translational sulfation. Immunoprecipitation following SDS-PAGE and Western blotting with anti-sulfotyrosine and anti-EGFR antibodies were used to identify native sulfated EGFR. In this research, we demonstrate that only the extracellular portion of EGFR is likely to be sulfated. Analysis on the extracts of lung cancer cells also clearly indicates that human EGFR is sulfated. It is intriguing how sulfation on EGFR may affect the functions of this important receptor. In the future, we would like to examine the possible effects of EGFR sulfation on lung cancer signaling pathway and hopefully contribute to drug design and the treatment of cancer disease.