由尿液純化而來的Tamm-Horsfall glycoprotein 活化中性白血球的分子機制研究

Abstract

Tamm-Horsfall glycoprotein (THP) 是由亨利式管(Henle’s loop)中的上行端及遠曲小管所分泌的醣蛋白，分子量約有80-90 KD，其醣分子佔全部重量25-35%，大部分由sialic acid組成。THP 被認具有保護泌尿道表皮細胞免於被病原菌入侵的功能。在先前的研究指出，THP 可以和不同的蛋白質結合: 包括免疫球蛋白輕鏈、第一介白素(IL-1), 補體(complement component) 1q,以及腫瘤壞死因子-α(TNF-α)；然而對於THP如何影響免疫系統其詳細的機制至今依然不清楚，因此我們將THP以各種不同的酵素處理，試圖去了解究竟是THP分子外圍的醣側鏈或是其核心蛋白質結構才具有關鍵性的功能；我們發現THP經不同醣分解酵素處理之後依然具可以刺激多核型白血球的活性，但是THP經蛋白分解酵素處理之後便會失去其功能，所以我們認為THP的核心蛋白結構之重要性是大於側鏈的醣分子。接下來我們發現多核型白血球在p38 的抑制劑(SB 203580)處之後便會失去其吞噬能力，這表示THP和脂多糖(lipopolysaccharide)對於多核型白血球吞噬能力的刺激都是需要p38路徑參與其中。最後我們進一步發現，THP 具有類似表皮生長因子(EGF)刺激多核型白血球的功能，THP 和表皮生長因子都會活化多核型白血球吞噬能力、導致細胞骨架的變化以及ERK1/2的表現；然而一旦我們利用表皮生長因子受體的抑制劑(GW2974)便可以抑制THP和表皮生長因子對多核型白血球的活化能力，但卻不會影響脂多糖對多核型白血球的活化。總結,在論文中我們證明了THP會活化多核型白血球，推測可能藉由THP中EGF-like domain來活化p38以及ERK1/2的訊息傳遞路徑來達成。

Tamm-Horsfall glycoprotein (THP) or uromodulin is produced by renal tubular cells of the ascending limp of Henle’s loop. Tamm-Horsfall glycoprotein is a 80-90KDa GPI-anchored protein and contains approximately 25-35% of carbohydrate-side chain in weight with abundant sialic acid. THP is an important defense molecule in protecting urinary tract epithelial cells from microbial invasion. Our previous data has shown that THP can bind to diverse proteins including immunoglobulin light chains, complement component 1q, interleukin-1(IL-1), and tumor necrosis factor-α (TNF-α).In addition, we found that THP could enhance PMN phagocytosis. However, the mechanism phagocytosis-enhancing activity of THP remained unclear. For further elucidating the mechanism for this activity by THP, we purified THP from normal human urine. At first, THP was cleaved by different carbohydrate-and protein-degrading enzymes and the data demonstrated that the protein-core structure was more important for activating PMN phagocytosis than carbohydrate-side chains. Next, we found that THP could bind to the surface membrane of PMN and induced phosphorylation of MAP kinase (p38),ERK1/2 and NF-кB signaling pathways. Furthermore, p38 inhibitor SB203580 could abolish LPS and THP induced-PMN phagocytosis. Finally, we found THP and EGF (Epidermal growth factor) exhibited a similar function on PMN and HL-60(human promyelocytic leukemia cells). Our results demonstrated that EGF and THP could induce PMN phagocytosis via rearrangement of cytoskeletal molecules by increasing expression of cdc42, RhoA and Rac. GW2974 (EGFR inhibitor) could reduce the THP- and EGF-induced PMN phagocytosis and ERK1/2 expression in HL-60. In contrast, GW2974 had no effect on LPS-induced PMN phagocytosis. Putting these results together, we concluded that THP used EGF-like domain to stimulate PMN via EGF singling pathway to phosphorylated p38 and ERK1/2. The EGF-like domain in THP molecule may play an important role in PMN phagocytosis-enhancing activity.