Identification of genes involved in the biosynthesis of the third and fourth sugars of the Methanococcus maripaludis archaellin N-linked tetrasaccharide

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DOIResolve DOI: http://doi.org/10.1128/JB.00668-13
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TypeArticle
Journal titleJournal of Bacteriology
ISSN0021-9193
Volume195
Issue18
Pages40944104; # of pages: 11
Subjectcarbohydrate; methyltransferase; article; bioinformatics; biosynthesis; controlled study; gene; gene deletion; gene identification; glycosylation; mass spectrometry; Methanococcus maripaludis; mmp1084 gene; mmp1085 gene; mmp1086 gene; mmp1087 gene; nonhuman; priority journal; Archaeal Proteins; Carbohydrate Sequence; Computational Biology; Gene Deletion; Genes, Archaeal; Glycosylation; Mass Spectrometry; Membrane Proteins; Methanococcus; Methyltransferases; Multigene Family; Polysaccharides; Protein Processing, Post-Translational; Reverse Transcriptase Polymerase Chain Reaction; Threonine; Transferases
AbstractN-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is the unique sugar (5S)-2-acetamido-2,4-dideoxy-5-Omethyl-α-L-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes-mmp1084, mmp1085, mmp1086, and mmp1087-were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a Δmmp1086Δmmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in Δmmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the Δmmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087, either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV. © 2013, American Society for Microbiology.
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LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); Human Health Therapeutics (HHT-TSH)
Peer reviewedYes
NPARC number21269868
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Record identifier064506c3-1ae4-416c-8195-7a16be705625
Record created2013-12-13
Record modified2016-05-09
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