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.

中文翻译：

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使用重组Alg1，Alg2和Alg11蛋白的化学酶法合成脂质连接的GlcNAc ₂ Man ₅寡糖

用作真核蛋白N-糖基化供体底物的真核脂质连接寡糖（LLOs）的生物合成始于内质网的胞质侧，并包括向五聚-焦磷酸-GlcNAc ₂依次添加五个甘露糖单元。这些反应由Alg1，Alg2和Alg11基因产物催化，产生Dol-PP-GlcNAc ₂ Man ₅，一种LLO中间体，随后被翻转至内质网腔。尽管先前已经描述了活性Alg1的纯化，但是Alg11和Alg2的研究主要是在体内进行的。我们在此描述功能性全长Alg2蛋白的表达和纯化。与纯化的可溶性结构域Alg1和Alg11一起，我们使用Alg2从合成的LLO开始化学酶促生成Dol-PP-GlcNAc ₂ Man ₅类似物，该LLO包含与不同长度的寡聚异戊二烯载体偶联的壳二糖部分（C ₁₀，C ₁₅，C ₂₀和C ₂₅）。我们发现，虽然除了通过ALG1第一甘露糖单元的成功与所有的LLO分子中，ALG2催化反应是唯一有效的，如果受体的脂质连接寡糖含有四个或五个异戊二烯单元（C足够长的脂质尾₂₀和C ₂₅）。用Alg11转化后，将所得的含C ₂₀或C ₂₅的GlcNAc ₂ Man ₅ LLO类似物成功用作布鲁氏锥虫纯化的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促方法，用于产生真核LLO类似物，并且是对神秘的Alg2反应机理进行后续机理研究的基础。