Pleiotropic roles of N-glycans for enzyme activities and stabilities of MIPC synthases, Csh1 and Sur1/Csg1, in Saccharomyces cerevisiae
Pleiotropic roles of N-glycans for enzyme activities and stabilities of MIPC synthases, Csh1 and Sur1/Csg1, in Saccharomyces cerevisiae
复制标题
N-聚糖对酿酒酵母中 MIPC 合酶 Csh1 和 Sur1/Csg1 的酶活性和稳定性的多效性作用
DOI：
10.1093/glycob/cwac035
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发表时间：
2022
期刊：
影响因子：
4.3
通讯作者：
Moriguchi Takashi
中科院分区：
文献类型：
--
作者：
Uemura Satoshi;Moriguchi Takashi
甘露糖基磷酸神经酰胺 (MIPC) 是一种膜脂，属于酿酒酵母中的复合鞘脂。 MIPC 由高尔基体腔中的 2 个冗余酶 Sur1/Csg1 和 Csh1 合成。根据神经酰胺部分上羟基的位置和数量，MIPC 由 5 个亚型（A、B'、B、C 和 D 型）组成。 Sur1对MIPC-B和MIPC-C合成的影响比Csh1更大。在这项研究中，我们阐明了 Sur1 和 Csh1 上附着的 N-聚糖所发挥的作用，并剖析了这 2 种酶底物识别的机制。 Sur1 在 Asn-224 上携带 N 聚糖，而 Csh1 在 Asn-51 和 Asn-247 上携带 N 聚糖。虽然细胞内蛋白质通常含有核心型 N-聚糖，但 Csh1 的 Asn-51 上的 N-聚糖表现出独特的甘露聚糖样结构，含有甘露糖长骨架。 Sur1 N224Q 和 Csh1 N51Q 突变体表现出合成每种酶的特定 MIPC 亚型的活性降低，表明这些 N-聚糖通过其催化结构域在底物识别中发挥作用。此外，在 Sur1 (Sur1-NST51) 密码子 51 处异位插入 N-糖基化共有序列 (NST) 会导致甘露聚糖的人工修饰，从而显着降低蛋白质稳定性。我们的结果表明 Sur1-NST51 突变蛋白稳定性的降低可能归因于甘露聚糖的潜在结构改变。总的来说，本研究揭示了 Sur1 和 Csh1 的重要管腔结构域，这些结构域通过甘露聚糖修饰决定底物特异性和/或蛋白质稳定性。
Mannosyl phosphorylceramide (MIPC) is a membrane lipid classified as a complex sphingolipid inSaccharomyces cerevisiae. MIPC is synthesized by 2 redundant enzymes, Sur1/Csg1 and Csh1, in the Golgi lumen. MIPC consists of 5 subtypes (A, B′, B, C, and D-type) according to the position and number of hydroxyl groups on the ceramide moiety. Sur1 exerts higher impact on synthesis of MIPC-B and MIPC-C than Csh1. In this study, we elucidated the roles played by N-glycans attached to Sur1 and Csh1, and dissected the mechanisms underlying substrate recognition by these 2 enzymes. Sur1 carries an N-glycan on Asn-224, whereas Csh1 has N-glycans on Asn-51 and Asn-247. Although intracellular proteins usually harbor core-type N-glycans, the N-glycan on Asn-51 of Csh1 exhibited a unique mannan-like structure containing a long backbone of mannose. Sur1 N224Q and Csh1 N51Q mutants exhibited a decrease in the activity to synthesize specific MIPC subtypes for each enzyme, suggesting that these N-glycans play a role in substrate recognition through their catalytic domains. Moreover, ectopic insertion of an N-glycosylation consensus sequence (NST) at codon 51 of Sur1 (Sur1-NST51) resulted in an artificial modification with mannan, which markedly decreased protein stability. Our results suggest that the diminished stability of the Sur1-NST51 mutant protein could be attributable to potential structural alterations by the mannan. Collectively, the present study reveals essential luminal domains of Sur1 and Csh1 that dictate substrate specificity and/or the protein stabilities via mannan modification.