Deciphering a pathway of Halobacterium salinarum N-glycosylation.

Lina Kandiba,Jerry Eichler

doi:10.1002/mbo3.215

Abstract

Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis of one of the two N-linked oligosaccharides described in this species. As in Hfx. volcanii, where agl (archaeal glycosylation) genes that encode proteins responsible for the assembly and attachment of a pentasaccharide to target protein Asn residues are clustered in the genome, Hbt. salinarum also contains a group of clustered homologous genes (VNG1048G-VNG1068G). Introduction of these Hbt. salinarum genes into Hfx. volcanii mutant strains deleted of the homologous sequence restored the lost activity. Moreover, transcription of the Hbt. salinarum genes in the native host, as well as in vitro biochemical confirmation of the predicted functions of several of the products of these genes provided further support for assignments made following bioinformatics and genetic experiments. Based on the results obtained in this study, the first description of an N-glycosylation pathway in Hbt. salinarum is offered.

Highlights

Once thought restricted to Eukarya, it is clear that Nglycosylation is a posttranslational modification performed across evolution (Larkin and Imperiali 2011; Aebi 2013; Eichler 2013; Nothaft and Szymanski 2013; Jarrell et al 2014)
MicrobiologyOpen published by John Wiley & Sons Ltd
Deleted Hfx. volcanii agl genes can be functionally replaced by their Hbt. salinarum homologues In Hfx. volcanii, all but one of the agl genes involved in the assembly and attachment of the N-linked pentasaccharide decorating glycoproteins in this species are found within an aglB-based gene cluster (Yurist-Doutsch and Eichler 2009; Yurist-Doutsch et al 2010)

Summary

Introduction

Once thought restricted to Eukarya, it is clear that Nglycosylation is a posttranslational modification performed across evolution (Larkin and Imperiali 2011; Aebi 2013; Eichler 2013; Nothaft and Szymanski 2013; Jarrell et al 2014). In each of the three domains, N-glycosylation involves the assembly of lipid-linked oligosaccharides that are subsequently transferred to target Asn resides It is the archaeal version of this universal proteinprocessing event that generates an unparalleled degree of diversity, relative to what is seen in Eukarya or Bacteria. The lipid-charged glycans are translocated across the membrane by an unknown mechanism, AglR is apparently involved in the process (Kaminski et al 2012) At this point, the oligosaccharyltransferase AglB (AbuQarn and Eichler 2006; Abu-Qarn et al 2007) delivers the translocated tetrasaccharide and its precursors from the lipid carrier to select Asn residues of the glycoprotein. By exploiting the predicted similarities between Hfx. volcanii Agl proteins and their Hbt. salinarum counterparts and confirming those predictions experimentally, the present study provides the first description of a pathway responsible for N-glycosylation in Hbt. salinarum

Experimental Procedures

Results

Discussion