Identifying Components of a Halobacterium salinarum N-Glycosylation Pathway.

Zlata Vershinin,Marianna Zaretsky,Ziqiang Guan,Jerry Eichler

doi:10.3389/fmicb.2021.779599

Zlata Vershinin, Marianna Zaretsky + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2021.779599

Copy DOI

Abstract

Whereas N-glycosylation is a seemingly universal process in Archaea, pathways of N-glycosylation have only been experimentally verified in a mere handful of species. Toward expanding the number of delineated archaeal N-glycosylation pathways, the involvement of the putative Halobacterium salinarum glycosyltransferases VNG1067G, VNG1066C, and VNG1062G in the assembly of an N-linked tetrasaccharide decorating glycoproteins in this species was addressed. Following deletion of each encoding gene, the impact on N-glycosylation of the S-layer glycoprotein and archaellins, major glycoproteins in this organism, was assessed by mass spectrometry. Likewise, the pool of dolichol phosphate, the lipid upon which this glycan is assembled, was also considered in each deletion strain. Finally, the impacts of such deletions were characterized in a series of biochemical, structural and physiological assays. The results revealed that VNG1067G, VNG1066C, and VNG1062G, renamed Agl25, Agl26, and Agl27 according to the nomenclature used for archaeal N-glycosylation pathway components, are responsible for adding the second, third and fourth sugars of the N-linked tetrasaccharide decorating Hbt. salinarum glycoproteins. Moreover, this study demonstrated how compromised N-glycosylation affects various facets of Hbt. salinarum cell behavior, including the transcription of archaellin-encoding genes.

Highlights

N-glycosylation, a post-translational modification in which glycans are assembled on lipid carriers and transferred to selected Asn residues in target proteins, is performed in all three domains of life (Aebi, 2013; Nothaft and Szymanski, 2013; Jarrell et al, 2014; Eichler, 2019)
The only protein shown to participate in Hbt. salinarum N-glycosylation is VNG1068G, which corresponds to the archaeal oligosaccharyltransferase AglB (Zaretsky et al, 2019)
As a first step in determining whether VNG1062G, VNG1066C or VNG1067G contribute to Hbt. salinarum N-glycosylation, cells lacking the encoding genes were assessed in assays in which cells lacking AglB, and unable to N-glycosylate, behaved differently than did parent strain cells (Zaretsky et al, 2019)

Summary

Introduction

N-glycosylation, a post-translational modification in which glycans are assembled on lipid carriers and transferred to selected Asn residues in target proteins, is performed in all three domains of life (Aebi, 2013; Nothaft and Szymanski, 2013; Jarrell et al, 2014; Eichler, 2019). In the case of archaeal N-glycosylation, one finds degrees of diversity in terms of the lipid carrier upon which the N-linked glycan is assembled, the steps involved in N-linked glycan assembly and glycan composition and architecture not seen in the other two domains (Schwarz and Aebi, 2011; Eichler, 2013, 2020; Eichler and Guan, 2017). Such diversity is exemplified by N-glycosylation in Halobacterium salinarum, a halophilic archaeon that grow in NaCl concentrations near or at saturation (Grant et al, 2001). Archaellins, the building blocks of the archaellar swimming device, the archaellum (Jarrell and Albers, 2012), were shown to be N-glycosylated (Wieland et al, 1985)

Methods

Results

Conclusion