AglR is required for addition of the final mannose residue of the N-linked glycan decorating the Haloferax volcanii S-layer glycoprotein

• Published in: Biochimica et biophysica acta Vol. 1820; no. 10; pp. 1664 - 1670
• Main Authors: Kaminski, Lina, Guan, Ziqiang, Abu-Qarn, Mehtap, Konrad, Zvia, Eichler, Jerry
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2012
• Subjects: Amino Acid Sequence; Archaea; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Archaeal Proteins - physiology; biogenesis; bioinformatics; Chromatography, Liquid; Dolichylphosphate-mannose; gene deletion; glycoproteins; Glycosylation; Haloferax volcanii; Haloferax volcanii - genetics; Haloferax volcanii - metabolism; lipopolysaccharides; mannose; Mannose - metabolism; mass spectrometry; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Membrane Glycoproteins - physiology; N-glycosylation; oligosaccharides; Organisms, Genetically Modified; phosphates; plasma membrane; Polysaccharides - chemistry; Polysaccharides - metabolism; post-translational modification; Protein Processing, Post-Translational - genetics; radiolabeling; S-layer glycoprotein; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; ABC; CBB; Haloferax volcanii; DolPP-Man5; Archaea; MS/MS; ER; N-glycosylation; LLO; DolP; DDW; RT-PCR; Dolichylphosphate-mannose; S-layer glycoprotein; LC-ESI/MS; Man; EIC
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2012.06.014

Recent studies of Haloferax volcanii have begun to elucidate the steps of N-glycosylation in Archaea, where this universal post-translational modification remains poorly described. In Hfx. volcanii, a series of Agl proteins catalyzes the assembly and attachment of a N-linked pentasaccharide to the S-layer glycoprotein. Although roles have been assigned to the majority of Agl proteins, others await description. In the following, the contribution of AglR to N-glycosylation was addressed. A combination of bioinformatics, gene deletion, mass spectrometry and metabolic radiolabeling served to show a role for AglR in archaeal N-glycosylation at both the dolichol phosphate and reporter glycoprotein levels. The modified behavior of the S-layer glycoprotein isolated from cells lacking AglR points to an involvement of this protein in N-glycosylation. In cells lacking AglR, glycan-charged dolichol phosphate, including mannose-charged dolichol phosphate, accumulates. At the same time, the S-layer glycoprotein does not incorporate mannose, the final subunit of the N-linked pentasaccharide decorating this protein. AglR is a homologue of Wzx proteins, annotated as flippases responsible for delivering lipid-linked O-antigen precursor oligosaccharides across the bacterial plasma membrane during lipopolysaccharide biogenesis. The effects resulting from aglR deletion are consistent with AglR interacting with dolichol phosphate-mannose, possibly acting as a dolichol phosphate-mannose flippase or contributing to such activity. Little is known of how lipid-linked oligosaccharides are translocated across membrane during N-glycosylation. The possibility of Hfx. volcanii AglR mediating or contributing to flippase activity could help address this situation. ► In cells lacking AglR, glycan-charged dolichol phosphates accumulate. ► In ΔaglR cells, the final subunit of the N-linked pentasaccharide is absent. ► AglR is a homologue of Wzx, thought to ‘flip’ lipid-linked glycans in bacteria.