N-linked glycosylation is a posttranslational modification involving the attachment of a carbohydrate to an asparagine (Asn) residue located within an Asn-X-Ser/Thr consensus sequence. This process is well understood in Eucarya and, more recently, in Bacteria. However, information about the equivalent process in Archaea is limited. This lack of knowledge includes data on glycan structures, assembly, and attachment. Assembly is believed to involve the sequential addition of monosaccharides to a membrane-embedded lipid carrier using a series of glycosyltransferases. Once the glycan is completed, it is transported across the membrane via a flippase and transferred to the protein using an oligosaccharyltransferase (Stt3p homolog). Working with the archaeon Methanococcus maripaludis, a novel glycan N-linked to the flagellin proteins of this organism was identified. Mass spectrometry has identified the glycan to have a mass of 1036.4 Da and secondary fragmentation pattern analysis of the intact glycan indicates that in addition to an N-acetylglucosamine residue (203 Da) and a di-N-acetylhexosamine (258 Da), there is an additional terminal mass of 575 Da suggesting that the glycan may be a tetrasaccharide. Genes suspected to be involved in flagellin glycosylation were targeted for in-frame deletion. The deletion of a number of these genes resulted in mutants that had a downward shift in flagellin molecular mass as evidenced by immunoblotting with anti-flagellin antisera. The annotation of these genes indicates that they are involved in the assembly of the glycan and its subsequent transfer to the target protein. Mutants that carried a deletion of an oligosaccharyltransferase (MMP1424) had flagellins that appeared completely unmodified by immunoblotting. Furthermore, mutants that had deletions in one of two glycosyltransferase genes (MMP1080, MMP1079) produced flagellins of intermediate molecular mass. Subsequent complementation of these glycosyltransferase mutations partially or fully restored flagellins to wild-type mass. Examination by electron microscopy showed that two mutants (ΔMMP1424, ΔMMP1079) were unable to assemble flagellar filaments while one glycosyltransferase mutant (ΔMMP1080) still produced flagellar filaments. Collectively, these data begin to reveal the pathway and requirement of N-linked glycosylation in archaea using M. maripaludis as a model. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-09-27 11:14:57.497
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/738 |
| Date | 01 October 2007 |
| Creators | VanDyke, David Jonathan |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | 2097107 bytes, application/pdf |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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