Two Distinct N-Glycosylation Pathways Process the Haloferax volcanii S-Layer Glycoprotein upon Changes in Environmental Salinity

Abstract

N-glycosylation in Archaea presents aspects of this posttranslational modification not seen in either Eukarya or Bacteria. In the haloarchaeon Haloferax volcanii, the surface (S)-layer glycoprotein can be simultaneously modified by two different N-glycans. Asn-13 and Asn-83 are modified by a pentasaccharide, whereas Asn-498 is modified by a tetrasaccharide of distinct composition, with N-glycosylation at this position being related to environmental conditions. Specifically, N-glycosylation of Asn-498 is detected when cells are grown in the presence of 1.75 but not 3.4 M NaCl. While deletion of genes encoding components of the pentasaccharide assembly pathway had no effect on the biosynthesis of the tetrasaccharide bound to Asn-498, deletion of genes within the cluster spanning HVO_2046 to HVO_2061 interfered with the assembly and attachment of the Asn-498-linked tetrasaccharide. Transfer of the “low-salt” tetrasaccharide from the dolichol phosphate carrier upon which it is assembled to S-layer glycoprotein Asn-498 did not require AglB, the oligosaccharyltransferase responsible for pentasaccharide attachment to Asn-13 and Asn-83. Finally, although biogenesis of the low-salt tetrasaccharide is barely discernible upon growth at the elevated salinity, this glycan was readily detected under such conditions in strains deleted of pentasaccharide biosynthesis pathway genes, indicative of cross talk between the two N-glycosylation pathways.

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…While the absence of aglG, aglI, and aglE compromised pentasaccharide assembly, as expected, such deletions had no effect on the appearance of the low-salt tetrasaccharide attached to S-layer glycoprotein Asn-498 when cells were grown in medium with reduced salinity (95). It can thus be concluded that a novel N-glycosylation pathway is involved in the assembly of the tetrasaccharide N-linked to S-layer glycoprotein Asn-498.…”

“…To determine whether AglB is also involved in delivering the low-salt tetrasaccharide to S-layer glycoprotein Asn-498 when cells are grown under low-salinity conditions, the N-glycosylation profile of the S-layer glycoprotein in a strain lacking AglB was assessed by mass spectrometry. Such analysis revealed peaks corresponding to the S-layer glycoprotein-derived Asn-498-containing peptide to be modified by the low-salt tetrasaccharide as well as by its precursors (95), indicating that AglB is not the OST responsible for transferring this glycan from DolP to S-layer glycoprotein Asn-498. Consequently, a currently unidentified OST seems to serve this role.…”

Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation

“…sibly as-yet unidentified AglB genes with very low sequence homology, or even functionally homologous enzymes (41). Archaea produce multiple LLOs containing a variety of oligosaccharide and dolichol structures in one organism, and multiple AglB enzymes expressed in either a constitutive or inducible manner to use these LLOs.…”

A Rhomboid Protease Gene Deletion Affects a Novel Oligosaccharide N-Linked to the S-layer Glycoprotein of Haloferax volcanii

“…This could account for the differences in intensity, observed in the protein gels. In order to test this hypothesis, we compared the glycans associated with the S-layer glycoprotein, a very abundant membrane protein that has been extensively used as a model to study protein glycosylation (20) in H. volcanii. We show, for the first time, the presence of N-linked oligosaccharides hydrolyzed by PNGase F in the S-layer glycoprotein of H. volcanii, because after the enzymatic treatment, the HPAEC-PAD profile clearly showed the presence of released oligosaccharides.…”

“…Thus, in response to changes in environmental salinity, H. volcanii modulates not only the N-linked glycans decorating the S-layer glycoprotein but also the sites of such posttranslational modification (19). Recently, it has been reported that two different glycosylation pathways participate in the synthesis of the tetra-and pentasaccharide (20).…”