古细菌细胞通常被糖基化的S层蛋白包膜。古细菌蛋白糖基化不仅提供了对其生态位的适应,而且还提供了对其进化轨迹的宝贵见解。值得注意的是,嗜热Thermoproteota修饰蛋白质与N-聚糖,包括两个GlcNAc单位在还原端,类似于保存在真核生物中的“核心结构”。最近,阿斯加德古细菌,现在被归类为普罗米修斯考古门的成员,为了解古细菌在真核发生中的作用提供了前所未有的机会。尽管在这个古细菌组中存在指示蛋白质N-糖基化的基因,这些还没有经过实验研究。在这里,我们对第一个分离的Asgard古细菌普罗米修斯古菌进行了糖蛋白组分析。通过高分辨率LC-MS/MS分析鉴定了700多种不同的蛋白质。然而,没有证据表明推定的S层蛋白存在或糖基化.相反,该古细菌中的N-糖基化主要在细胞外溶质结合蛋白中观察到,可能与寡肽的化学接受或跨膜转运有关。聚糖修饰发生在位于保守的N-X-S/T序列子内的天冬酰胺残基上,与在其他古细菌中发现的模式一致,细菌,和真核生物。出乎意料的是,在这个古细菌中发现了三种结构不同的N-聚糖缺乏传统的核心结构,呈现包含非典型糖的独特成分。值得注意的是,这些糖之一可能是用苏氨酸残基修饰的HexNAc,类似于先前在甲烷细菌中温产甲烷菌中观察到的修饰。我们的发现促进了我们对阿斯加德古细菌生理学和进化动力学的理解。
Archaeal cells are typically enveloped by glycosylated S-layer proteins. Archaeal protein glycosylation provides valuable insights not only into their adaptation to their niches but also into their evolutionary trajectory. Notably, thermophilic Thermoproteota modify proteins with N-glycans that include two GlcNAc units at the reducing end, resembling the \"core structure\" preserved across eukaryotes. Recently, Asgard archaea, now classified as members of the phylum Promethearchaeota, have offered unprecedented opportunities for understanding the role of archaea in
eukaryogenesis. Despite the presence of genes indicative of protein N-glycosylation in this archaeal group, these have not been experimentally investigated. Here we performed a glycoproteome analysis of the firstly isolated Asgard archaeon Promethearchaeum syntrophicum. Over 700 different proteins were identified through high-resolution LC-MS/MS analysis, however, there was no evidence of either the presence or glycosylation of putative S-layer proteins. Instead, N-glycosylation in this archaeon was primarily observed in an extracellular solute-binding protein, possibly related to chemoreception or transmembrane transport of oligopeptides. The glycan modification occurred on an asparagine residue located within the conserved N-X-S/T sequon, consistent with the pattern found in other archaea, bacteria, and eukaryotes. Unexpectedly, three structurally different N-glycans lacking the conventional core structure were identified in this archaeon, presenting unique compositions that included atypical sugars. Notably, one of these sugars was likely HexNAc modified with a threonine residue, similar to modifications previously observed in mesophilic methanogens within the Methanobacteriati. Our findings advance our understanding of Asgard archaea physiology and evolutionary dynamics.