Assembling Glycan-Charged Dolichol Phosphates: Chemoenzymatic Synthesis of a Haloferax volcanii N-Glycosylation Pathway Intermediate.
Assembling Glycan-Charged Dolichol Phosphates: Chemoenzymatic Synthesis of a Haloferax volcanii N-Glycosylation Pathway Intermediate.
复制标题
组装带聚糖的多羟基磷酸酯：化学酶法合成 Haloferax volcanii N-糖基化途径中间体。
DOI：
10.1021/acs.bioconjchem.7b00436
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发表时间：
2017
影响因子：
4.7
通讯作者：
Eichler,Jerry
中科院分区：
文献类型：
--
作者：
Elharar,Yifat;Podilapu,AnandaRao;Guan,Ziqiang;Kulkarni,SuvarnS;Eichler,Jerry
N-糖基化是糖链的共价连接以选择蛋白质靶标ASN残基，是一种翻译后修饰，由生命的三个结构域执行。在嗜盐考古中，对这一普遍的蛋白质加工事件的了解相对较深入，已经确定了编码古生菌糖基化(AGL)途径组件的基因，这些基因负责N-连接的五糖的组装和附着。与其他地方一样，N-连接的葡聚糖在转移到目标ASN残基之前组装在磷酸二醇载体上。然而，由于目前对Hfx知之甚少。VolcaniiAgl途径在蛋白质水平上，古生菌似乎唯一的能力是使用Dolp作为葡聚糖脂载体，而不是真核生物使用的Dolicol焦磷酸，仍然知之甚少。考虑到这一点，采用化学酶方法对AglG进行生化研究，AglG是该途径的五种糖基转移酶之一。因此，一种新的区域和立体选择性地还原天然分离的聚戊烯醇，使得在非常温和的条件下通过不对称转移氢化容易地获得S-多利醇。该化合物被用来生成N-连接的五糖的前体--葡萄糖的DolP，以及DolP-葡萄糖-葡萄糖糖醛酸和DolP-葡萄糖糖醛酸。AglG，从Hfx中提纯。在高盐度条件下的火山膜，就像在现场遇到的那样，随后与尿苷二磷酸(UDP)-葡萄糖醛酸和DolP-葡萄糖结合，生成DolP-葡萄糖-葡萄糖酸。本文开发的用于研究AglG活性的体外系统是研究嗜盐糖基转移酶的第一个这样的工具，并将允许详细了解古代N-糖基化。
N-glycosylation, the covalent attachment of glycans to select protein target Asn residues, is a post-translational modification performed by all three domains of life. In the halophilic archaeaHaloferax volcanii, in which understanding of this universal protein-processing event is relatively well-advanced, genes encoding the components of the archaeal glycosylation (Agl) pathway responsible for the assembly and attachment of an N-linked pentasaccharide have been identified. As elsewhere, the N-linked glycan is assembled on phosphodolichol carriers before transfer to target Asn residues. However, as little is presently known of theHfx. volcaniiAgl pathway at the protein level, the seemingly unique ability of Archaea to use dolichol phosphate (DolP) as the glycan lipid carrier, rather than dolichol pyrophosphate used by eukaryotes, remains poorly understood. With this in mind, a chemoenzymatic approach was taken to biochemically study AglG, one of the five glycosyltransferases of the pathway. Accordingly, a novel regio- and stereoselective reduction of naturally isolated polyprenol gave facile access toS-dolichol via asymmetric transfer hydrogenation under very mild conditions. This compound was used to generate glucose-charged DolP, a precursor of the N-linked pentasaccharide, as well as DolP–glucose–glucuronic acid and DolP–glucuronic acid. AglG, purified fromHfx. volcaniimembranes in hypersaline conditions, like those encountered in situ, was subsequently combined with uridine diphosphate (UDP)–glucuronic acid and DolP–glucose to yield DolP–glucose–glucuronic acid. The in vitro system for the study of AglG activity developed here represents the first such tool for studying halophilic glycosyltransferases and will allow for a detailed understanding of archaeal N-glycosylation.