Ramírez Ana S, Boilevin Jérémy, Lin Chia-Wei, Ha Gan Bee, Janser Daniel, Aebi Markus, Darbre Tamis, Reymond Jean-Louis, Locher Kaspar P
Department of Biology, Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH), Schafmattstrasse 20, CH-8093 Zürich, Switzerland.
Department of Chemistry and Biochemistry, University of Berne, CH-3012 Berne, Switzerland.
Glycobiology. 2017 Aug 1;27(8):726-733. doi: 10.1093/glycob/cwx045.
The biosynthesis of eukaryotic lipid-linked oligosaccharides (LLOs) that act as donor substrates in eukaryotic protein N-glycosylation starts on the cytoplasmic side of the endoplasmic reticulum and includes the sequential addition of five mannose units to dolichol-pyrophosphate-GlcNAc2. These reactions are catalyzed by the Alg1, Alg2 and Alg11 gene products and yield Dol-PP-GlcNAc2Man5, an LLO intermediate that is subsequently flipped to the lumen of the endoplasmic reticulum. While the purification of active Alg1 has previously been described, Alg11 and Alg2 have been mostly studied in vivo. We here describe the expression and purification of functional, full length Alg2 protein. Along with the purified soluble domains Alg1 and Alg11, we used Alg2 to chemo-enzymatically generate Dol-PP-GlcNAc2Man5 analogs starting from synthetic LLOs containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C10, C15, C20 and C25). We found that while the addition of the first mannose unit by Alg1 was successful with all of the LLO molecules, the Alg2-catalyzed reaction was only efficient if the acceptor LLOs contained a sufficiently long lipid tail of four or five isoprenyl units (C20 and C25). Following conversion with Alg11, the resulting C20 or C25 -containing GlcNAc2Man5 LLO analogs were successfully used as donor substrates of purified single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei. Our results provide a chemo-enzymatic method for the generation of eukaryotic LLO analogs and are the basis of subsequent mechanistic studies of the enigmatic Alg2 reaction mechanism.
真核生物脂质连接寡糖（LLO）作为真核生物蛋白质N-糖基化中的供体底物，其生物合成始于内质网的细胞质侧，包括将五个甘露糖单元依次添加到焦磷酸多萜醇-GlcNAc2上。这些反应由Alg1、Alg2和Alg11基因产物催化，生成Dol-PP-GlcNAc2Man5，这是一种LLO中间体，随后翻转到内质网腔中。虽然之前已经描述了活性Alg1的纯化，但Alg11和Alg2大多是在体内进行研究的。我们在此描述了功能性全长Alg2蛋白的表达和纯化。连同纯化的可溶性结构域Alg1和Alg11，我们使用Alg2从含有与不同长度（C10、C15、C20和C25）的寡聚异戊二烯载体偶联的壳二糖部分的合成LLO开始，化学酶法生成Dol-PP-GlcNAc2Man5类似物。我们发现，虽然Alg1添加第一个甘露糖单元对所有LLO分子都成功，但只有当受体LLO含有足够长的由四个或五个异戊二烯单元组成的脂质尾（C20和C25）时，Alg2催化的反应才有效。在用Alg11转化后，所得含C20或C25的GlcNAc2Man5 LLO类似物成功用作来自布氏锥虫的纯化单亚基寡糖基转移酶STT3A的供体底物。我们的结果提供了一种化学酶法来生成真核生物LLO类似物，并且是后续对神秘的Alg2反应机制进行机理研究的基础。
