Yuan Chong, Smith William L
From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109.
From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109
J Biol Chem. 2015 Feb 27;290(9):5606-20. doi: 10.1074/jbc.M114.632463. Epub 2014 Dec 29.
Cyclooxygenases (COXs) catalyze the committed step in prostaglandin (PG) biosynthesis. COX-1 is constitutively expressed and stable, whereas COX-2 is inducible and short lived. COX-2 is degraded via endoplasmic reticulum (ER)-associated degradation (ERAD) following post-translational glycosylation of Asn-594. COX-1 and COX-2 are found in abundance on the luminal surfaces of the ER and inner membrane of the nuclear envelope. Using confocal immunocytofluorescence, we detected both COX-2 and microsomal PGE synthase-1 (mPGES-1) but not COX-1 in the Golgi apparatus. Inhibition of trafficking between the ER and Golgi retarded COX-2 ERAD. COX-2 has a C-terminal STEL sequence, which is an inefficient ER retention signal. Substituting this sequence with KDEL, a robust ER retention signal, concentrated COX-2 in the ER where it was stable and slowly glycosylated on Asn-594. Native COX-2 and a recombinant COX-2 having a Golgi targeting signal but not native COX-1 exhibited efficient catalytic coupling to mPGES-1. We conclude that N-glycosylation of Asn-594 of COX-2 occurs in the ER, leading to anterograde movement of COX-2 to the Golgi where the Asn-594-linked glycan is trimmed prior to retrograde COX-2 transport to the ER for ERAD. Having an inefficient ER retention signal leads to sluggish Golgi to ER transit of COX-2. This permits significant Golgi residence time during which COX-2 can function catalytically. Cytosolic phospholipase A2α, which mobilizes arachidonic acid for PG synthesis, preferentially translocates to the Golgi in response to physiologic Ca(2+) mobilization. We propose that cytosolic phospholipase A2α, COX-2, and mPGES-1 in the Golgi comprise a dedicated system for COX-2-dependent PGE2 biosynthesis.
环氧化酶（COXs）催化前列腺素（PG）生物合成的关键步骤。COX-1组成性表达且稳定，而COX-2是可诱导的且寿命短暂。COX-2在天冬酰胺-594进行翻译后糖基化后通过内质网（ER）相关降解（ERAD）途径被降解。COX-1和COX-2大量存在于内质网的腔表面和核膜的内膜上。利用共聚焦免疫细胞荧光技术，我们在高尔基体中检测到了COX-2和微粒体前列腺素E合酶-1（mPGES-1），但未检测到COX-1。内质网和高尔基体之间运输的抑制延缓了COX-2的ERAD。COX-2有一个C末端STEL序列，这是一个低效的内质网滞留信号。用一个强大的内质网滞留信号KDEL替换该序列，使COX-2在内质网中聚集，在那里它是稳定的并且天冬酰胺-594上的糖基化缓慢。天然COX-2和具有高尔基体靶向信号的重组COX-2（但不是天然COX-1）表现出与mPGES-1的有效催化偶联。我们得出结论，COX-2的天冬酰胺-594的N-糖基化在内质网中发生，导致COX-2向高尔基体的顺向移动，在那里与天冬酰胺-594相连的聚糖在COX-2逆向转运回内质网进行ERAD之前被修剪。具有低效的内质网滞留信号导致COX-2从高尔基体到内质网的转运迟缓。这使得COX-2在高尔基体中有显著的停留时间，在此期间COX-2可以发挥催化功能。胞质磷脂酶A2α将花生四烯酸动员用于PG合成，响应生理Ca(2+)动员优先转运到高尔基体。我们提出，高尔基体中的胞质磷脂酶A2α、COX-2和mPGES-1构成了一个用于COX-2依赖性前列腺素E2生物合成的专用系统。
