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Link to original content: https://pubmed.ncbi.nlm.nih.gov/30245679
Sialic Acid-Like Sugars in Archaea: Legionaminic Acid Biosynthesis in the Halophile Halorubrum sp. PV6 - PubMed Skip to main page content
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. 2018 Sep 7:9:2133.
doi: 10.3389/fmicb.2018.02133. eCollection 2018.

Sialic Acid-Like Sugars in Archaea: Legionaminic Acid Biosynthesis in the Halophile Halorubrum sp. PV6

Marianna Zaretsky  1 , Elina Roine  2 , Jerry Eichler  1
Affiliations

Sialic Acid-Like Sugars in Archaea: Legionaminic Acid Biosynthesis in the Halophile Halorubrum sp. PV6

Marianna Zaretsky et al. Front Microbiol. .

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.

Keywords: Halorubrum; N-formylation; N-glycosylation; S-layer glycoprotein; archaea; halophile; legionaminic acid.

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Figures

FIGURE 1
FIGURE 1
N-glycosylation profile of Halorubrum sp. PV6 S-layer glycoprotein Asn-94. LC-ESI/MS analysis of the Asn-94-containing peptide derived from the Halorubrum sp. PV6 S-layer glycoprotein following digestion with trypsin and GluC protease was performed. Shown are doubly charged [M+2H]2+ ion peaks corresponding to (A) the 78TGSYAIGGPDAADGAFNVTVVTPR101 peptide (m/z 1168.58), and the same peptide successively modified by (B) a hexose (m/z 1249.61), (C) a hexuronic acid (m/z 1337.62), (D) a hexose (m/z 1418.65), (E) a sulfated/phosphorylated hexuronic acid (m/z 1546.65), and (F) a 5-N-formyl-legionaminic acid (m/z 1697.70). In each panel, the N-glycosylation status of the peptide is schematically depicted, where “N” corresponds to Asn-94. Employing symbol nomenclature for glycans guidelines (Varki et al., 2015), open circles correspond to hexoses, open squares containing a diagonal correspond to hexuronic acids, and the yellow diamond corresponds to 5-N-formyl-legionaminic acid.
FIGURE 2
FIGURE 2
MS/MS verification of Halorubrum sp. PV6 S-layer glycoprotein Asn-94 glycosylation. To verify that glycosylation of the hexose-modified S-layer glycoprotein-derived peptide, 78TGSYAIGGPDAADGAFNVTVVTPR101, occurs on Asn-94, the MS spectrum of the [M+2H]2+ base peak of this peptide, observed at m/z 1249.61, was obtained via mass-dependent acquisition. The y-ion series includes fragments that contain the hexose-modified Asn-94 residue. The inset shows the fragmentation scheme, with the Asn94-bound hexose being represented by the open circle. Peaks corresponding to y-ion series fragments are indicated by stars.
FIGURE 3
FIGURE 3
The predicted pathway of Halorubrum sp. PV6 legionaminic acid biosynthesis. The pathway (Glaze et al., 2008; Schoenhofen et al., 2009) starts with the dehydratase LegB that generates NDP-4-keto-6-deoxy-N-acetylglucosamine (GlcNAc) from NDP-GlcNAc. The aminotransferase LegC next produces the amino sugar NDP-4-amino-6-deoxy-GlcNAc. The acetyltransferase LegH subsequently generates NDP-N,N-diacetamido-6-deoxy-glucose (NDP-diacetamido-basillosamine), which is then converted into 2,4-diacetamido-6-deoxy-mannose by the hydrolyzing 2-epimerase LegG. The legionaminic synthase LegI now condenses this sugar with pyruvate to yield legionaminic acid. Finally, the sugar is activated by the actions of LegF, the CMP-legionaminic acid synthase. In the diagram, the enzymes that catalyze each pathway step, as well as the products formed, are listed.
FIGURE 4
FIGURE 4
Schematic depiction of the region of the Halorubrum sp. PV6 genome containing ORFs encoding putative components involved in 5-N-formyl-legionaminic acid biosynthesis and N-glycosylation. The scheme presents the orientation but not the actual length of each ORF. Sequence information can be found at GenBank accession number MH673034.
FIGURE 5
FIGURE 5
Genes putatively involved in legionaminic acid biosynthesis are co-transcribed. (A) RT-PCR products obtained using primer pairs designed to amplify the indicated ORF and cDNA (with reverse transcriptase; upper panel) or RNA pre-treated with DNase but not with reverse transcriptase (without reverse transcriptase; lower panel) as template. (B) Schematic depiction of the Halorubrum sp. PV6 genome containing genes encoding putative components involved in legionaminic acid biosynthesis suspected of being co-transcribed. Primer pairs beginning within the upstream sequence and ending within the downstream sequence are schematically depicted. Primer pairs a, b, c, d, and e are designed to amplify products of the listed sizes. (C) RT-PCR products obtained using each primer pair and cDNA (with reverse transcriptase; upper panel) or RNA pre-treated with DNase but not with reverse transcriptase (without reverse transcriptase; lower panel) as template. In (A,C), the positions of bp markers are provided on the left of each panel and values are listed on the left of the upper profile.
FIGURE 6
FIGURE 6
Halorubrum sp. PV6 LegI and LegF contribute to legionaminic acid biosynthesis. (A) Halorubrum sp. PV6 LegI and (B) LegF were purified from total lysates of Hfx. volcanii expressing each CBD-tagged protein (applied) on cellulose (bound). Aliquots of each pool were separated by 12% SDS-PAGE and Coomassie stained. In each panel, the positions of molecular weight markers are indicated on the left, while the position of the purified protein is indicated on the right. (C) LegI activity was confirmed in reactions containing cellulose-bound CBD-LegI, N-acetylmannosamine, and phosphoenolpyruvate (black circles) or in which the N-acetylmannosamine (gray circles) or phosphoenolpyruvate (gray squares) were omitted, or where cellulose incubated with the lysate of Hfx. volcanii not expressing the CBD-tagged protein was added instead of CBD-LegI-bearing beads (gray triangles). Each point is the average of three repeats ± SEM. (D) LegF activity was confirmed in reactions containing cellulose-bound CBD-LegF, N-acetylneuraminic acid and CTP (black circles) or in which the N-acetylneuraminic acid (gray circles) or CTP (gray squares) were omitted, or where cellulose incubated with the lysate of Hfx. volcanii not expressing the CBD-tagged protein was added instead of CBD-LegF-bearing beads (gray triangles). The amount of N-acetylneuraminic acid produced in each reaction was determined every 30 min over a 2 h interval, except in the latter two controls, when measurements were only taken at the start and the end of the experiment. Each point is the average of three repeats ± SEM.
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