Abstract
N-glycosylation is a post-translational modification that occurs in all three domains. In Archaea, however, N-linked glycans present a degree of compositional diversity not observed in either Eukarya or Bacteria. As such, it is surprising that nonulosonic acids (NulOs), nine-carbon sugars that include sialic acids, pseudaminic acids, and legionaminic acids, are routinely detected as components of protein-linked glycans in Eukarya and Bacteria but not in Archaea. In the following, we report that the N-linked glycan attached to the S-layer glycoprotein of the haloarchaea Halorubrum sp. PV6 includes an N-formylated legionaminic acid. Analysis of the Halorubrum sp. PV6 genome led to the identification of sequences predicted to comprise the legionaminic acid biosynthesis pathway. The transcription of pathway genes was confirmed, as was the co-transcription of several of these genes. In addition, the activities of LegI, which catalyzes the condensation of 2,4-di-N-acetyl-6-deoxymannose and phosphoenolpyruvate to generate legionaminic acid, and LegF, which catalyzes the addition of cytidine monophosphate (CMP) to legionaminic acid, both heterologously expressed in Haloferax volcanii, were demonstrated. Further genome analysis predicts that the genes encoding enzymes of the legionaminic acid biosynthetic pathway are clustered together with sequences seemingly encoding components of the N-glycosylation pathway in this organism. In defining the first example of a legionaminic acid biosynthesis pathway in Archaea, the findings reported here expand our insight into archaeal N-glycosylation, an almost universal post-translational modification in this domain of life.
Highlights
Post-translational modifications represent a major source of proteomic expansion
We reported that VP4, the major structural protein of the haloarchaeal pleomorphic virus Halorubrum pleomorphic virus 1 (HRPV-1), is N-glycosylated by a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid, and a terminal 5-N-formyl-legionaminic acid residue, when directly isolated from virions produced by the host strain, Halorubrum sp
No N-acetylneuraminic acid was generated when either N-acetylmannosamine or phosphoenolpyruvate were omitted from the reaction, or when cellulose beads pre-incubated with a lysate prepared from Hfx. volcanii cells not transformed to express cellulose-binding domain (CBD)-tagged legionaminic acid synthase (LegI) were tested
Summary
Of the numerous processing events that can modify a protein, N-glycosylation, or the covalent attachment of a glycan to selected Asn residues of a target protein, is the most complex. Long held to be specific to Eukarya, it is clear that Archaea and Bacteria perform this post-translational modification. While N-glycosylation in Bacteria is believed to solely occur in the delta/epsilon proteobacteria (Nothaft and Szymanski, 2010), such protein processing appears to be an almost universal trait in Archaea (Kaminski et al, 2013a). Domain-related diversity in the sugar composition of N-linked glycans serves to distinguish eukaryal, bacterial, and archaeal N-glycosylation (Schwarz and Aebi, 2011; Eichler, 2013)
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