Abstract
Despite providing the first example of archaeal N-glycosylation almost 50 years ago, detailed insight into the pathway used by Halobacterium salinarum to assemble and attach an N-linked tetrasaccharide decorating glycoproteins in this haloarchaea has only recently appeared. Still, numerous components of this pathway remain to be identified, including sulfotransferase(s), which modify the third and fourth tetrasaccharide sugars. In the present report, a series of bioinformatics, genetic, biochemical, and structural approaches served to reveal how membrane-associated VNG1056C and soluble VNG1057C respectively sulfate the iduronic acid at tetrasaccharide position three and the terminal glucuronic acid, seemingly independent of each other. Deletion of VNG1056C but not of VNG1057C reduced cell motility to a minor degree and did not cause archaellum filament bundling. Finally, transcription of VNG1056C or VNG1057C was augmented upon deletion of the other when cells were grown in low but not high salinity conditions possibly in an attempt to compensate for the loss of sugar sulfation resulting from the deletion. This augmented transcription, however, had no effect on the extent of tetrasaccharide sulfation. With demonstrated roles in Hbt. salinarum N-glycosylation, VNG1056C and VNG1057C were respectively re-annotated as Agl30 and Agl31, employing the nomenclature used to define archaeal N-glycosylation pathway components. IMPORTANCE: Like essentially all Archaea, the halophile Halobacterium salinarum performs N-glycosylation, namely, the covalent attachment of a glycan moiety to select asparagine residues in a target protein. Moreover, Hbt. salinarum represents one of the few current archaeal examples in which the pathway of N-glycosylation has been largely defined. Still, several components of this pathway remain to be defined, including the sulfotransferase(s) responsible for modifying the iduronic acid and glucuronic acid corresponding to the third and final sugars of the N-linked tetrasaccharide that decorates glycoproteins in this haloarchaeon. In the present report, a series of bioinformatics, genetic, biochemical, and structural approaches served to reveal how membrane-associated VNG1056C and soluble VNG1057C respectively sulfate the iduronic acid at tetrasaccharide position three and the terminal glucuronic acid, seemingly independent of each other. The need for two different enzymes reflects the sulfation of these sugars at distinct positions.