The analysis of glycoproteins and the comparison of protein N-glycosylation from different eukaryotic origins require unbiased and robust analytical workflows. The structural and functional analysis of vertebrate protein N-glycosylation currently depends extensively on bacterial peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases (PNGases), which are indispensable enzymatic tools in releasing asparagine-linked oligosaccharides (N-glycans) from glycoproteins. So far, only limited PNGase candidates are available for N-glycans analysis, and particularly the analysis of plant and invertebrate N-glycans is hampered by the lack of suitable PNGases. Furthermore, liquid chromatography–mass spectrometry (LC–MS) workflows, such as hydrogen deuterium exchange mass spectrometry (HDX-MS), require a highly efficient enzymatic release of N-glycans at low pH values to facilitate the comprehensive structural analysis of glycoproteins. Herein, we describe a previously unstudied superacidic bacterial N-glycanase (PNGase H⁺) originating from the soil bacterium Rudaea cellulosilytica (Rc), which has significantly improved enzymatic properties compared to previously described PNGase H⁺ variants. Active and soluble recombinant PNGase Rc was expressed at a higher protein level (3.8-fold) and with higher specific activity (~56% increase) compared to the currently used PNGase H⁺ variant from Dyella japonicum (Dj). Recombinant PNGase Rc was able to deglycosylate the glycoproteins horseradish peroxidase and bovine lactoferrin significantly faster than PNGase Dj (10 min vs. 6 h). The versatility of PNGase Rc was demonstrated by releasing N-glycans from a diverse array of samples such as peach fruit, king trumpet mushroom, mouse serum, and the soil nematode Caenorhabditis elegans. The presence of only two disulfide bonds shown in the AlphaFold protein model (so far all other superacidic PNGases possess more disulfide bonds) could be corroborated by intact mass- and peptide mapping analysis and provides a possible explanation for the improved recombinant expression yield of PNGase Rc.

中文翻译：

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来自 Rudaea cellulosilytica 的 PNGase H + 变体，具有改进的去糖基化效率，用于快速分析真核生物 N-聚糖和糖蛋白的氢氘交换质谱分析

糖蛋白的分析和来自不同真核来源的蛋白质N-糖基化的比较需要公正且稳健的分析工作流程。脊椎动物蛋白N-糖基化的结构和功能分析目前广泛依赖于细菌肽-N4-( N-乙酰-β-氨基葡萄糖)天冬酰胺酰胺酶(PNGases)，这是释放天冬酰胺连接寡糖( N-聚糖)必不可少的酶促工具) 来自糖蛋白。到目前为止，只有有限的 PNGase 候选物可用于N-聚糖分析，特别是植物和无脊椎动物N的分析-聚糖因缺乏合适的PNG酶而受到阻碍。此外，液相色谱-质谱 (LC-MS) 工作流程，例如氢氘交换质谱 (HDX-MS)，需要在低 pH 值下高效酶促释放N-聚糖，以促进糖蛋白的全面结构分析。在这里，我们描述了一种以前未研究过的超酸性细菌N-聚糖酶 (PNGase H ⁺ )，它来源于土壤细菌Rudaea cellulosilytica (Rc)，与之前描述的 PNGase H ^{+相比，它具有显着改善的酶特性}变体。与目前使用的来自Dyella japonicum (Dj)的 PNGase H ⁺变体相比，活性和可溶性重组 PNGase Rc 以更高的蛋白质水平（3.8 倍）和更高的比活性（增加约 56% ）表达。重组 PNGase Rc 能够比 PNGase Dj 更快地使糖蛋白辣根过氧化物酶和牛乳铁蛋白去糖基化（10 分钟对 6 小时）。PNGase Rc 的多功能性通过从各种样品中释放N-聚糖来证明，这些样品包括桃果实、小号王蘑菇、小鼠血清和土壤线虫秀丽隐杆线虫. AlphaFold 蛋白质模型中仅显示两个二硫键（到目前为止，所有其他超酸性 PNGase 都具有更多的二硫键）可以通过完整的质量和肽图分析得到证实，并为提高 PNGase Rc 的重组表达产量提供了可能的解释.