Identification of Genes Involved in the Assembly and Biosynthesis of the N-linked Flagellin Glycan in the Archaeon, Methanococcus maripaludis
MetadataShow full item record
N-glycosylation is a metabolic process found in all three domains of life. It is the attachment of a polysaccharide glycan to asparagine (Asn) residues within the amino acid motif, Asn-Xaa-Ser/Thr. In the archaeon, Methanococcus maripaludis, a tetrasaccharide glycan was isolated from purified flagella and its structure determined by mass spectrometry analysis. The linking sugar to the protein is surprisingly, N-acetylgalactosamine (β-GalNAc), with the next proximal sugar a derivative of N-acetylglucosamine (β-GlcNAc), being named β-GlcNAc3Ac, and the third sugar a derivative of N-acetylmannosamine (β-ManNAc), with an attached threonine residue on the C6 carbon (β-ManNAc3NAm). The terminal sugar is an unusual diglycoside of aldulose ((5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose). Previous genetic analyses identified the glycosyltransferases (GTs) responsible for the transfer of the second and third sugars of the glycan, as well as the oligosaccharyltransferase (OST) which attaches the glycan to protein. Left unidentified were the first and fourth GTs, the flippase as well as any genes involved in glycan sugar biosynthesis and modification. In this work, genes suspected to be involved in the biosynthesis of N-linked sugars, as well as those that might encode the missing GTs and flippase were targeted for in-frame deletion. Mutants with a deleted annotated GT gene (MMP1088) had a small decrease in flagellin molecular weight as determined by immunoblotting. Mass spectrometry (MS) analysis confirmed that the N-linked glycan was missing the terminal sugar as well as the threonine found on the third sugar of wildtype cells. Mutants with a deleted gene annotated to be involved in acetamidino synthesis (a functional group that is present on the third sugar), also had a decrease in flagellin molecular weight. MS analysis determined that the N-linked glycan was missing the acetamidino group on the third sugar as well as its attached threonine, along with the terminal sugar. Both mutants were able to assemble functional flagella but had impaired motility compared to wildtype cells in mini-swarm agar. Deletions were also constructed in four other GT genes considered candidates in assembly of the linking sugar. However, none of these mutants had the expected decrease in flagellin molecular weight. With the work done in this study, the glycosyl transferase that attaches the last sugar of the M. maripaludis N-linked assembly pathway has been identified as well as a gene involved in the biosynthesis and modification of the glycan sugars.