抑制C1GALT1調控O-醣基化造成LC3脂質化的累積

Abstract

細胞自噬在癌症發生中扮演無可或缺的腳色，而轉譯後修飾亦參與在起始細胞自噬的條件中。前人研究指出，醣基化會藉由調控內質網與高基氏體的平衡進而導致細胞自噬的發生。O-醣基化中，GalNAc 類 O-醣基化為最常見的修飾，其中C1GALT1 為延長醣類鍵結的關鍵酵素。O-醣基化修飾與細胞自噬皆已知有潛力做為癌症治療的對象，但研究尚未指出 O-醣基化與細胞自噬之間的關聯性。此研究分別以基因層面調控 C1GALT1 表現、利用市售藥物 itraconazole 與 benzyl-α-GalNAc 降解 C1GALT1 與抑制 O-醣基化去探討轉錄與轉譯層面對於細胞自噬的影響，再進一步利用流式細胞儀分析報告基因 LC3 去量化自噬小泡的變化。我們發現當抑制C1GALT1 的時候，會使 LC3 的表現量上升並且造成 LC3B 脂質化的累積；且以藥物(itraconazole benzyl-α-GalNAc) 調控 O-醣基化修飾的時候亦有相同的效果。以 pEGFP-LC3 感染細胞觀察到抑制 C1GALT1 會造成自噬小泡的累積，但以mRFP-GFP tandem fluorescent LC3 載體 (ptfLC3) 感染細胞並無觀察到剃除C1GALT1 的細胞之自噬通量上升。我們認為，抑制 C1GALT1，改變細胞中O-醣基化修飾會使 LC3B 脂質化，進而造成自噬小泡的累積。

Autophagy plays an essential role during cancer progression, and post-translational modification is crucial in regulating the initiation of autophagy. Of which, glycosylation has been discovered to impact autophagy via the maintenance of membrane flux between ER and Golgi. GalNAc-type O-glycosylation is the most common type of O-link glycosylation, with core 1 β1,3-galactosyltransferase (C1GALT1) responsible for the elongation of O-glycosylation. As previous studies have shown that C1GALT1 is a potential target for cancer therapeutic, little do we understand the impact of Oglycosylation on autophagy. With multiple approaches, we observed a dynamic induction of LC3 lipidation upon down-regulation of C1GALT1 by genetic manipulation or supplement of O-glycosylation inhibition drugs, such as itraconazole and benzyl-α-GalNAc, suggesting an activation of autophagy. Moreover, the flow cytometry analysis of autophagy reporter system suggested an accumulation of autophagosomes and no increase of autophagic flux. In conclusion, we propose that by modulating C1GALT1, truncated forms of O-glycosylation on surface proteins activate autophagy by triggering lipidated LC3 accumulation.