Asn-linked oligosaccharide chain of a crenarchaeon, Pyrobaculum calidifontis, is reminiscent of the eukaryotic high-mannose-type glycan

Daisuke Fujinami, Yuya Taguchi, Daisuke Kohda

    Research output: Contribution to journalArticle

    2 Citations (Scopus)

    Abstract

    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. calidifontis N-glycan has a biantennary, high-mannose-type structure consisting of up to 11 monosaccharide residues. The base portion of the P. calidifontis N-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. calidifontis N-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. calidifontis N-glycan are underlined. In spite of these differences, the structure of the P. calidifontis N-glycan is the most similar to the eukaryotic counterparts, among all archaeal N-glycans reported to date.

    Original languageEnglish
    Pages (from-to)701-712
    Number of pages12
    JournalGlycobiology
    Volume27
    Issue number8
    DOIs
    Publication statusPublished - Aug 1 2017

    Fingerprint

    Pyrobaculum
    Mannose
    Oligosaccharides
    Polysaccharides
    Crenarchaeota
    Glycosylation
    Glycopeptides
    Euryarchaeota
    Sulfolobus
    P-Glycoproteins
    Monosaccharides
    Archaea
    Tandem Mass Spectrometry
    Chromatography
    Lectins
    Labeling
    Mass spectrometry
    Cultured Cells
    Magnetic Resonance Spectroscopy
    Nuclear magnetic resonance

    All Science Journal Classification (ASJC) codes

    • Biochemistry

    Cite this

    Asn-linked oligosaccharide chain of a crenarchaeon, Pyrobaculum calidifontis, is reminiscent of the eukaryotic high-mannose-type glycan. / Fujinami, Daisuke; Taguchi, Yuya; Kohda, Daisuke.

    In: Glycobiology, Vol. 27, No. 8, 01.08.2017, p. 701-712.

    Research output: Contribution to journalArticle

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    abstract = "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. calidifontis N-glycan has a biantennary, high-mannose-type structure consisting of up to 11 monosaccharide residues. The base portion of the P. calidifontis N-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. calidifontis N-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. calidifontis N-glycan are underlined. In spite of these differences, the structure of the P. calidifontis N-glycan is the most similar to the eukaryotic counterparts, among all archaeal N-glycans reported to date.",
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