TY - JOUR
T1 - Asn-linked oligosaccharide chain of a crenarchaeon, Pyrobaculum calidifontis, is reminiscent of the eukaryotic high-mannose-type glycan
AU - Fujinami, Daisuke
AU - Taguchi, Yuya
AU - Kohda, Daisuke
N1 - Funding Information:
We dedicate this paper to our mentor, the late Professor Fuyuhiko Inagaki, Hokkaido University, Japan, for his fundamental contribution to the relayed TOCSY (alias, relayed HOHAHA) experiments and his continuous support of our research activities. We thank Ms. Mizuho Oda and Dr. Takenori Nitta (Laboratory for Technical Support, Medical Institute of Bioregulation, Kyushu University) for the proteomic analysis. We also thank Prof. Kenji Sonomoto and Dr. Takuya Noguchi (Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University) for assistance with the gas– liquid chromatography experiment.
Funding Information:
This work was supported by Japan Society for the Promotion of Science [JP26119002 to D.K.].
Publisher Copyright:
© The Author 2017.
PY - 2017/8/1
Y1 - 2017/8/1
N2 - 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.
AB - 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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U2 - 10.1093/glycob/cwx044
DO - 10.1093/glycob/cwx044
M3 - Article
C2 - 28510654
AN - SCOPUS:85028351863
SN - 0959-6658
VL - 27
SP - 701
EP - 712
JO - Glycobiology
JF - Glycobiology
IS - 8
ER -