Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, China.
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 6, Higashi, Tsukuba, Japan.
Glycobiology. 2018 Oct 1;28(10):741-753. doi: 10.1093/glycob/cwy060.
In eukaryotes, the biosynthesis of a highly conserved dolichol-linked oligosaccharide (DLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) begins on the cytoplasmic face of the endoplasmic reticulum (ER) and ends within the lumen. Two functionally distinguished heteromeric glycosyltransferase (GTase) complexes are responsible for the cytosolic DLO assembly. Alg1, a β-1, 4 mannosyltransferase (MTase) physically interacts with Alg2 and Alg11 proteins to form the multienzyme complex which catalyzes the addition of all five mannose to generate the Man5GlcNAc2-PP-Dol intermediate. Despite the fact that Alg1 plays a central role in the formation of the multi-MTase has been confirmed, the topological information of Alg1 including the molecular mechanism of membrane association are still poorly understood. Using a combination of bioinformatics and biological approaches, we have undertaken a structural and functional study on Alg1 protein, in which the enzymatic activities of Alg1 and its variants were monitored by a complementation assay using the GALpr-ALG1 yeast strain, and further confirmed by a liquid chromatography-mass spectrometry-based in vitro quantitative assay. Computational and experimental evidence confirmed Alg1 shares structure similarity with Alg13/14 complex, which has been defined as a membrane-associated GT-B GTase. Particularly, we provide clear evidence that the N-terminal transmembrane domain including the following positively charged amino acids and an N-terminal amphiphilic-like α helix domain exposed on the protein surface strictly coordinate the Alg1 orientation on the ER membrane. This work provides detailed membrane topology of Alg1 and further reveals its biological importance at the spatial aspect in coordination of cytosolic DLO biosynthesis.
在真核生物中，高度保守的多萜醇连接寡糖 (DLO) 前体 Glc3Man9GlcNAc2-焦磷酸化多萜醇 (PP-Dol) 的生物合成始于内质网 (ER) 的细胞质侧，在内腔中结束。两个功能上有区别的异源糖基转移酶 (GTase) 复合物负责细胞质 DLO 的组装。Alg1 是一种 β-1,4 甘露糖基转移酶 (MTase)，与 Alg2 和 Alg11 蛋白物理相互作用，形成多酶复合物，催化所有五个甘露糖的添加，生成 Man5GlcNAc2-PP-Dol 中间体。尽管 Alg1 在多 MTase 的形成中起着核心作用已得到证实，但 Alg1 的拓扑信息，包括膜结合的分子机制，仍知之甚少。我们结合生物信息学和生物学方法，对 Alg1 蛋白进行了结构和功能研究，通过使用 GALpr-ALG1 酵母菌株的互补测定法监测 Alg1 及其变体的酶活性，并通过基于液相色谱-质谱的体外定量测定法进一步证实。计算和实验证据证实 Alg1 与 Alg13/14 复合物具有结构相似性，该复合物被定义为膜相关 GT-B GTase。特别是，我们提供了明确的证据，表明包括带正电荷氨基酸的 N 端跨膜结构域和暴露在蛋白质表面的 N 端两亲性α螺旋结构域严格协调 Alg1 在 ER 膜上的取向。这项工作提供了 Alg1 的详细膜拓扑结构，并进一步揭示了其在协调细胞质 DLO 生物合成方面的空间生物学重要性。
