Glycosylation status of nicastrin influences catalytic activity and substrate preference of γ-secretase.
Glycosylation status of nicastrin influences catalytic activity and substrate preference of γ-secretase.
复制标题
尼卡斯特林的糖基化状态影响γ-分泌酶的催化活性和底物偏好。
DOI：
10.1016/j.bbrc.2018.05.126
复制
发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Funamoto S.
中科院分区：
文献类型：
--
作者：
Moniruzzaman M;Ishihara S;Nobuhara M;Higashide H;Funamoto S.
γ-分泌酶复合物是由nicastrin（NCT）、早老素（PS）、早老素增强子-2（PEN-2）和前咽缺陷1（Aph-1）组装而成的，催化淀粉样前体蛋白裂解生成淀粉样β蛋白（Aβ），是阿尔茨海默病的主要致病因子。NCT通过复杂的糖基化而成熟，并通过与催化亚基PS相互作用在γ-分泌酶活性中发挥重要作用。然而，NCT糖基化对γ-分泌酶活性和底物特异性的作用仍然未知。本研究旨在研究NCT糖基化对一组糖基化突变凝集素抗性CHO（Lec）细胞中γ-分泌酶活性和底物特异性的影响。CHO Lec-1细胞缺乏糖基转移酶-I、GnT-I，因此NCT上的N-聚糖均为寡甘露糖型，而CHO Lec-2细胞由于胞苷5′-单磷酸-唾液酸转运蛋白受损，合成含唾液酸缺陷型寡糖的NCT。在此，我们报告了突变体CHO Lec-1和Lec-2在基于细胞的试验和生化试验中均降低γ-分泌酶活性，并且CHO Lec-1优先降低Aβ生成。γ-分泌酶复合物的内源水平、γ-分泌酶亚基的亚细胞分布和功能性γ-分泌酶复合物的水平在突变体中保持不变。有趣的是，免疫共沉淀研究显示突变的γ-分泌酶可以识别底物和亲本γ-分泌酶一样。我们的数据表明，NCT的彻底糖基化对γ-分泌酶的酶活性和底物偏好性至关重要。
γ-Secretase complex, the assembly of nicastrin (NCT), Presenilin (PS), Presenilin Enhancer-2 (PEN-2) and Anterior pharynx defective 1 (Aph-1), catalyzes the cleavage of amyloid precursor protein to generate amyloid-β protein (Aβ), the main culprit of Alzheimer's disease. NCT becomes matured through complex glycosylation and play important role in γ-secretase activity by interacting with catalytic subunit PS. However, the role of NCT glycosylation on γ-secretase activity and substrate specificity is still unknown. The purpose of this study is to investigate the effect of NCT glycosylation on γ-secretase activity and substrate specificity in a group of glycosylation mutant lectin resistant CHO (Lec) cells. CHO Lec-1 cells lack glycosyltransferase-I, GnT-I, thusN-glycan on NCT are all oligomannose type, whereas CHO Lec-2 cells synthesize NCT containing sialic acid deficient oligosaccharides due to the impairment of cytidine 5′-monophosphate-sialic acid transporter. Here, we reported that mutant CHO Lec-1 and Lec-2 reduced γ-secretase activity in both cell-based and biochemical assays, and that CHO Lec-1 preferentially reduced Aβ generation. Endogenous level of γ-secretase complex, subcellular distribution of γ-secretase subunits and the level of functional γ-secretase complex remained unchanged in mutants. Interestingly, Coimmunoprecipitation study revealed that mutant γ-secretase could recognize substrate as well as parental γ-secretase. Our data suggests that thorough glycosylation of NCT is critical for enzymatic activity and substrate preference of γ-secretase.