Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, China.
Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark.
Rapid Commun Mass Spectrom. 2022 Nov 15;36(21):e9376. doi: 10.1002/rcm.9376.
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.
分析糖蛋白和比较不同真核起源的蛋白质 N-糖基化需要无偏且稳健的分析工作流程。脊椎动物蛋白质 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-葡糖胺基)天冬酰胺酰胺酶（PNGases），这些酶是从糖蛋白中释放天冬酰胺连接的寡糖（N-聚糖）所必需的酶工具。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-聚糖的分析受到缺乏合适的 PNGase 的阻碍。此外，液相色谱-质谱（LC-MS）工作流程，如氢氘交换质谱（HDX-MS），需要在低 pH 值下高效酶解 N-聚糖，以促进糖蛋白的全面结构分析。在此，我们描述了一种以前未研究过的源自土壤细菌鲁达氏纤维素分解菌（Rc）的超强酸性细菌 N-聚糖酶（PNGase H），与以前描述的 PNGase H 变体相比，它具有显著改善的酶学性质。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 的表达蛋白水平更高（提高了 3.8 倍），且比活更高（增加了约 56%）。与 PNGase Dj 相比，重组 PNGase Rc 能够更快地使辣根过氧化物酶和牛乳铁蛋白糖蛋白去糖基化（10 分钟与 6 小时）。PNGase Rc 的多功能性通过从各种样品（如桃果实、大王花菇、鼠血清和土壤线虫秀丽隐杆线虫）中释放 N-聚糖得到证明。在 AlphaFold 蛋白质模型中显示的只有两个二硫键的存在（到目前为止，所有其他超强酸性 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 PNGase Rc 的改进重组表达产量提供了可能的解释。
