Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells.
Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells.
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挽救 UGGT1 敲除哺乳动物细胞中与罕见疾病相关的错误折叠突变糖蛋白的分泌。
DOI：
10.1101/2023.05.30.542711
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Trerotola,M
中科院分区：
文献类型：
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作者：
Tax,Gábor;Guay,KevinP;Soldà,Tatiana;Hitchman,CharlieJ;Hill,JohanC;Vasiljević,Snežana;Lia,Andrea;Modenutti,CarlosP;Straatman,KeesR;Santino,Angelo;Molinari,Maurizio;Zitzmann,Nicole;Hebert,DanielN;Roversi,Pietro;Trerotola,M
内质网(ER)对错误折叠的糖蛋白的保留是由ER定位的真核糖蛋白分泌检查点UDP-葡萄糖糖蛋白葡萄糖转移酶(UGGT)介导的。该酶识别错误折叠的糖蛋白，并通过重新糖化其N-连接的多糖之一来标记它的内质网滞留。在分泌型糖蛋白基因先天突变的背景下，UGGT介导的内质网滞留可以导致罕见的疾病，即使突变的糖蛋白保持活性(“响应性突变”)。利用激光共聚焦扫描显微镜，我们研究了引起凝胶样滴状角膜营养不良(GDLD)的人trop-2-Q118E、E227K和L186P突变体的亚细胞定位。与正确定位于质膜的野生型trop-2相比，这些trop-2突变体保留在内质网中。我们在CRISPR/Cas9介导的UGGT1和/或UGGT2基因抑制的哺乳动物细胞中研究了trop-2 Q118E、E227K和L186P突变体的荧光嵌合体。TROP-2突变体Q118E、E227K和L186P在UGGT1−/−细胞中的膜定位被成功挽救。UGGT1还能有效地使trop-2-Q118E-EYFPin Cellula重新糖化。这项研究支持这样一种假设，即UGGT1调制将构成一种新的治疗策略，用于治疗与错误折叠的膜糖蛋白相关的病理情况(只要突变损害但不会消除功能)，并鼓励测试ER糖蛋白折叠调节剂作为广谱解救分泌物的质量控制药物，用于由反应性分泌糖蛋白突变引起的罕见疾病。
Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER‐localized eukaryotic glycoprotein secretion checkpoint, UDP‐glucose glycoprotein glucosyl‐transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re‐glucosylating one of itsN‐linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT‐mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity (“responsive mutant”). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop‐2‐Q118E, E227K and L186P mutants, which cause gelatinous drop‐like corneal dystrophy (GDLD). Compared with the wild‐type Trop‐2, which is correctly localized at the plasma membrane, these Trop‐2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop‐2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9‐mediated inhibition of theUGGT1and/orUGGT2genes. The membrane localization of the Trop‐2 Q118E, E227K and L186P mutants was successfully rescued inUGGT1−/−cells. UGGT1 also efficiently reglucosylated Trop‐2‐Q118E‐EYFPin cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad‐spectrum rescue‐of‐secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.