title: N-Glycosylation Is Important for Proper Haloferax volcanii S-Layer Stability and Function.
abstract: N-Glycosylation, the covalent linkage of glycans to select Asn residues of target proteins, is an almost universal posttranslational modification in archaea. However, whereas roles for N-glycosylation have been defined in eukarya and bacteria, the function of archaeal N-glycosylation remains unclear. Here, the impact of perturbed N-glycosylation on the structure and physiology of the haloarchaeonHaloferax volcaniiwas considered. Cryo-electron microscopy was used to examine right-side-out membrane vesicles prepared from cells of a parent strain and from strains lacking genes encoding glycosyltransferases involved in assembling the N-linked pentasaccharide decorating the surface layer (S-layer) glycoprotein, the sole component of the S-layer surroundingH. volcaniicells. Whereas a regularly repeating S-layer covered the entire surface of vesicles prepared from parent strain cells, vesicles from the mutant cells were only partially covered. To determine whether such N-glycosylation-related effects on S-layer assembly also affected cell function, the secretion of a reporter protein was addressed in the parent and N-glycosylation mutant strains. Compromised S-layer glycoprotein N-glycosylation resulted in impaired transfer of the reporter past the S-layer and into the growth medium. Finally, an assessment of S-layer glycoprotein susceptibility to added proteases in the mutants revealed that in cells lacking AglD, which is involved in adding the final pentasaccharide sugar, a distinct S-layer glycoprotein conformation was assumed in which the N-terminal region was readily degraded. Perturbed N-glycosylation thus affects S-layer glycoprotein folding. These findings suggest thatH. volcaniicould adapt to changes in its surroundings by modulating N-glycosylation so as to affect S-layer architecture and function.IMPORTANCELong held to be a process unique to eukaryotes, it is now accepted that bacteria and archaea also perform N-glycosylation, namely, the covalent attachment of sugars to select asparagine residues of target proteins. Yet, while information on the importance of N-glycosylation in eukaryotes and bacteria is available, the role of this posttranslational modification in archaea remains unclear. Here, insight into the purpose of archaeal N-glycosylation was gained by addressing the surface layer (S-layer) surrounding cells of the halophilic speciesHaloferax volcaniiRelying on mutant strains defective in N-glycosylation, such efforts revealed that compromised N-glycosylation affected S-layer integrity and the transfer of a secreted reporter protein across the S-layer into the growth medium, as well as the conformation of the S-layer glycoprotein, the sole component of the S-layer. Thus, by modifying N-glycosylation,H. volcaniicells can change how they interact with their surroundings.
authors.affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, Israel.
authors.forename: Adi
authors.initials: A
authors.lastname: Tamir
authors.affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, Israel jeichler@bgu.ac.il.
authors.forename: Jerry
authors.initials: J
authors.lastname: Eichler
mesh_terms: D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics*; D019622:Haloferax volcanii / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D008562:Membrane Glycoproteins; D009844:Oligosaccharides; C055764:S-layer proteins; D016695:Glycosyltransferases
keywords: Archaea; N-glycosylation; S-layer; S-layer glycoprotein; halophile