title: Structural characterization of the N-linked pentasaccharide decorating glycoproteins of the halophilic archaeon Haloferax volcanii.
abstract: N-Glycosylation is a post-translational modification performed in all three domains of life. In the halophilic archaea Haloferax volcanii, glycoproteins such as the S-layer glycoprotein are modified by an N-linked pentasaccharide assembled by a series of Agl (archaeal glycosylation) proteins. In the present study, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy were used to define the structure of this glycan attached to at least four of the seven putative S-layer glycoprotein N-glycosylation sites, namely Asn-13, Asn-83, Asn-274 and Asn-279. Such approaches detected a trisaccharide corresponding to glucuronic acid (GlcA)-β1,4-GlcA-β1,4-glucose-β1-Asn, a tetrasaccharide corresponding to methyl-O-4-GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, and a pentasaccharide corresponding to hexose-1,2-[methyl-O-4-]GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, with previous MS and radiolabeling experiments showing the hexose at the non-reducing end of the pentasaccharide to be mannose. The present analysis thus corrects the earlier assignment of the penultimate sugar as a methyl ester of a hexuronic acid, instead revealing this sugar to be a methylated GlcA. The assignments made here are in good agreement with what was already known of the Hfx. volcanii N-glycosylation pathway from previous genetic and biochemical efforts while providing new insight into the process.
authors: Department of Life Sciences, Ben Gurion University of the Negev, PO Box 653, Beersheva 84105, Israel.
authors: Lina
authors: L
authors: Kandiba
authors: Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland.
authors: Chia-Wei
authors: CW
authors: Lin
authors: Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland.
authors: Markus
authors: M
authors: Aebi
authors: Department of Life Sciences, Ben Gurion University of the Negev, PO Box 653, Beersheva 84105, Israel.
authors: Jerry
authors: J
authors: Eichler
authors: Université de Lille, CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F 59000 Lille, France.
authors: Yann
authors: Y
authors: Guerardel
mesh_terms: D006031:Glycosylation; D019622:Haloferax volcanii / Q000737:chemistry*; D008358:Mannose / Q000737:chemistry; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D008562:Membrane Glycoproteins; D009844:Oligosaccharides; C055764:S-layer proteins; D008358:Mannose
keywords: N-linked glycosylation; S-layer glycoprotein; archaea; mass spectrometry; nuclear magnetic resonance