Vasudevan Deepika, Takeuchi Hideyuki, Johar Sumreet Singh, Majerus Elaine, Haltiwanger Robert S
Department of Biochemistry and Cell Biology, 450 Life Sciences Building, Stony Brook University, Stony Brook, NY 11794-5215, USA.
Department of Internal Medicine, Division of Hematology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Curr Biol. 2015 Feb 2;25(3):286-295. doi: 10.1016/j.cub.2014.11.049. Epub 2014 Dec 24.
O-fucose is added to cysteine-rich domains called thrombospondin type 1 repeats (TSRs) by protein O-fucosyltransferase 2 (POFUT2) and is elongated with glucose by β3-glucosyltransferase (B3GLCT). Mutations in B3GLCT result in Peters plus syndrome (PPS), an autosomal recessive disorder characterized by eye and other developmental defects. Although 49 putative targets are known, the function of the disaccharide and its role in PPS remain unexplored.
Here we show that while POFUT2 is required for secretion of all targets tested, B3GLCT only affects the secretion of a subset, consistent with the observation that B3GLCT mutant phenotypes in PPS patients are less severe than embryonic lethal phenotypes of Pofut2-null mice. O-glycosylation occurs cotranslationally, as TSRs fold. Mass spectral analysis reveals that TSRs from mature, secreted protein are stoichiometrically modified with the disaccharide, whereas TSRs from protein still folding in the ER are partially modified, suggesting that O-glycosylation marks folded TSRs and promotes ER exit. In vitro unfolding assays demonstrate that fucose and glucose stabilize folded TSRs in an additive manner. In vitro refolding assays under redox conditions showed that POFUT2 recognizes, glycosylates, and stabilizes the folded form of TSRs, resulting in a net acceleration of folding.
While known ER quality-control machinery rely on identifying and tagging unfolded proteins, we find that POFUT2 and B3GLCT mediate a noncanonical ER quality-control mechanism that recognizes folded TSRs and stabilizes them by glycosylation. Our findings provide a molecular basis for the defects observed in PPS and potential targets that contribute to the pathology.
蛋白O-岩藻糖基转移酶2（POFUT2）将O-岩藻糖添加到富含半胱氨酸的结构域，即血小板反应蛋白I型重复序列（TSR），并由β3-葡萄糖基转移酶（B3GLCT）将其与葡萄糖进行延伸。B3GLCT突变导致彼得斯综合征（PPS），这是一种常染色体隐性疾病，其特征为眼部及其他发育缺陷。尽管已知49个推定靶点，但二糖的功能及其在PPS中的作用仍未得到探索。
我们在此表明，虽然POFUT2是所有测试靶点分泌所必需的，但B3GLCT仅影响一部分靶点的分泌，这与PPS患者中B3GLCT突变表型不如Pofut2基因敲除小鼠胚胎致死表型严重的观察结果一致。O-糖基化在共翻译过程中发生，此时TSR折叠。质谱分析表明，来自成熟分泌蛋白的TSR以化学计量方式被二糖修饰，而来自内质网中仍在折叠的蛋白的TSR则被部分修饰，这表明O-糖基化标记折叠的TSR并促进其从内质网输出。体外解折叠试验表明，岩藻糖和葡萄糖以累加方式稳定折叠的TSR。氧化还原条件下的体外重折叠试验表明POFUT2识别、糖基化并稳定TSR的折叠形式，从而导致折叠的净加速。
虽然已知的内质网质量控制机制依赖于识别和标记未折叠蛋白，但我们发现POFUT2和B3GLCT介导一种非经典的内质网质量控制机制，该机制识别折叠的TSR并通过糖基化使其稳定。我们的研究结果为PPS中观察到的缺陷以及导致病理的潜在靶点提供了分子基础。
