Pyrobaculum calidifontis is a hyperthermophilic archaeon that belongs to the phylum Crenarchaeota. In contrast to the phylum Euryarchaeota, only the N-glycan structure of the genus Sulfolobus is known in Crenarchaeota. Here, we enriched glycoproteins from cultured P. calidifontis cells, by ConA lectin chromatography. The MASCOT search identified proteins with at least one potential N-glycosylation site. The tandem mass spectrometry (MS/MS) analysis of 12 small tryptic glycopeptides confirmed the canonical N-glycosylation consensus in P. calidifontis. We determined the N-linked oligosaccharide structure produced by an in vitro enzymatic oligosaccharyl transfer reaction. Pyrobaculum calidifontis cells were cultured in rich medium supplemented with ¹³C-glucose, for the metabolic labeling of N-oligosaccharide donors. An incubation with a synthetic peptide substrate produced glycopeptides with isotopically labeled oligosaccharide moieties. The MS and nuclear magnetic resonance analyses revealed that the P. calidifontisN-glycan has a biantennary, high-mannose-type structure consisting of up to 11 monosaccharide residues. The base portion of the P. calidifontisN-glycan strongly resembles the eukaryotic core structure, α-Man-(1-3)-(α-Man-(1-6)-)β-Man-(1-4)-β-GlcNAc-(1-4)-β-GlcNAc-Asn. Structural differences exist in the anomeric configuration between Man and GlcNAc, and the chitobiose structure is chemically modified: one GlcNAc residue is oxidized to glucoronate, and the GlcNAc residues are both modified with an additional acetamido group at the C-3 position. As a result, the core structure of the P. calidifontisN-glycan is α-Man-(1-3)-(α-Man-(1-6)-)α-Man-(1-4)-β-GlcANAc3NAc-(1-4)-β-GlcNAc3NAc-Asn, in which the unique features of the P. calidifontisN-glycan are underlined. In spite of these differences, the structure of the P. calidifontisN-glycan is the most similar to the eukaryotic counterparts, among all archaeal N-glycans reported to date.

中文翻译：

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Crnarchaeon的Pyrobaculum calidifontis的Asn连接寡糖链让人想起真核生物高甘露糖型聚糖

Pyrobaculum calidifontis是属于Crenarchaeota门的超高温古细菌。与真人门相反，在Crenarchaeota中只有Sulfolobus属的N-聚糖结构是已知的。在这里，我们通过ConA凝集素层析从培养的P. calidifontis细胞中富集了糖蛋白。MASCOT搜索确定了具有至少一个潜在的N-糖基化位点的蛋白质。串联质谱（MS / MS）分析的12种小胰蛋白酶糖肽证实了拟南芥中的典型N-糖基化共识。我们确定了由体外酶促寡糖基转移反应产生的N-连接的寡糖结构。拟南芥将细胞在补充有¹³ C-葡萄糖的丰富培养基中培养，以进行N-寡糖供体的代谢标记。与合成肽底物的孵育产生了具有同位素标记的寡糖部分的糖肽。MS和核磁共振分析表明，P。calidifontis N-聚糖具有双触角，高甘露糖型结构，最多包含11个单糖残基。P. calidifontis N的基部-聚糖非常类似于真核核心结构，α-Man-（1-3）-（α-Man-（1-6）-）β-Man-（1-4）-β-GlcNAc-（1-4） -β-GlcNAc-Asn。Man和GlcNAc之间的异头构型存在结构差异，并且化学修饰了壳二糖结构：一个GlcNAc残基被氧化成葡萄糖酸，而GlcNAc残基都被C-3处的另一个乙酰氨基基团修饰。结果，P。calidifontis N-聚糖的核心结构为α-Man-（1-3）-（α-Man-（1-6）-）α -Man- （1-4）-β- Glc A NAc 3NAc-（1-4）-β- GlcNAc3NAc -Asn，其中P. calidifontis N的独特特征-聚糖带有下划线。尽管存在这些差异，但是在迄今为止报道的所有古细菌N-聚糖中，P。calidifontis N-聚糖的结构与真核对应物最相似。