Revisiting N-glycosylation in Halobacterium salinarum: Characterizing a dolichol phosphate- and glycoprotein-bound tetrasaccharide

Abstract

Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic Archaea remains either unclear or unknown. The composition of an N-linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40 years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N-linked glycan as corresponding to a tetrasacchride comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasacchride. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Halobacterium salinarum, will allow delineation of the N-glycosylation pathway in this organism.

Identifying Components of a Halobacterium salinarum N-Glycosylation Pathway

“…This is apparently in contrast to what is thought to occur in other Archaea, specifically S. acidocaldarius, where Agl3 was shown to be a UDPsulfoquinovose synthase and important for N-glycosylation, suggesting that in this species, sulfation occurs at the nucleotidelinked sugar level (Meyer et al, 2011). The absence of N-linked glycan sulfation, as well as the detection of precursors/breakdown products of the DolP-and Asn-bound disulfated tetrasaccharide presenting a single sulfate group on either the second or the fourth hexuronic acid, respectively, reported in our earlier efforts (Zaretsky et al, 2019;Vershinin et al, 2021) could reflect lability of sugar-bound sulfate groups. However, since sulfated breakdown products were detected by MS/MS of the tetrasaccharide attached to either DolP or protein targets (see Figure 4A and Supplementary Figure 2A), whereas no sulfation of the precursor glycans in the mutant strains was seen, despite the identical processing of parent and mutant strain samples, it is unlikely that sulfate group loss occurred in the present study.…”

“…haloarchaeon (Vershinin et al, 2021). For this, the composition of the N-linked tetrasaccharide attached to modified Asn residues was examined by LC-ESI MS following proteolytic digestion of Hbt.…”

“…salinarum performs this posttranslational modification. Most recently, mass spectrometry was used to define the composition of the N-linked glycan, shown to be a tetrasaccharide comprising Hex-HexA(SO 3 )-HexA-HexA(SO 3 ) (Vershinin et al, 2021). These efforts thus clarified earlier and often contradictory descriptions of this moiety (Wieland et al, 1980(Wieland et al, , 1983(Wieland et al, , 1985Lechner et al, 1985b).…”

“…In each panel, circles correspond to hexoses, split diamonds correspond to hexuronic acids and the N corresponds to the Asn-479-containing peptide. Vershinin et al, 2021). To assess the involvement of VNG1062G, VNG1066C, and VNG1067G in the assembly of this lipid-linked tetrasaccharide, the DolP pool was isolated from parent strain and vng1062G, vng1066C, and vng1067G strain cells and examined by LC-ESI MS.…”

“…Very recently, mass spectrometry was used to revisit the composition of the N-linked tetrasaccharide decorating the Hbt. salinarum S-layer glycoprotein and archaellins, showing it to comprise a hexose as the linking sugar, a sulfated hexuronic acid at position two, a hexuronic acid at position three and a second sulfated hexuronic acid at position four ( Vershinin et al, 2021 ). In the same study, methylation of the fourth sugar of the tetrasaccharide at the DolP-linked stage was confirmed.…”