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      Complementation of an aglB Mutant of Methanococcus maripaludis with Heterologous Oligosaccharyltransferases

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          Abstract

          The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis Δ aglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis Δ aglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis Δ aglB strain were unsuccessful.

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          Most cited references58

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          Mechanisms and principles of N-linked protein glycosylation.

          N-linked glycosylation, a protein modification system present in all domains of life, is characterized by a high structural diversity of N-linked glycans found among different species and by a large number of proteins that are glycosylated. Based on structural, functional, and phylogenetic approaches, this review discusses the highly conserved processes that are at the basis of this unique general protein modification system. Copyright © 2011 Elsevier Ltd. All rights reserved.
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            Methanogens: reevaluation of a unique biological group.

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              An evolving view of the eukaryotic oligosaccharyltransferase.

              Asparagine-linked glycosylation (ALG) is one of the most common protein modification reactions in eukaryotic cells, as many proteins that are translocated across or integrated into the rough endoplasmic reticulum (RER) carry N-linked oligosaccharides. Although the primary focus of this review will be the structure and function of the eukaryotic oligosaccharyltransferase (OST), key findings provided by the analysis of the archaebacterial and eubacterial OST homologues will be reviewed, particularly those that provide insight into the recognition of donor and acceptor substrates. Selection of the fully assembled donor substrate will be considered in the context of the family of human diseases known as congenital disorders of glycosylation (CDG). The yeast and vertebrate OST are surprisingly complex hetero-oligomeric proteins consisting of seven or eight subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Stt3p, Wbp1p, and Swp1p in yeast; ribophorin I, DAD1, N33/IAP, OST4, STT3A/STT3B, Ost48, and ribophorin II in mammals). Recent findings from several laboratories have provided overwhelming evidence that the STT3 subunit is critical for catalytic activity. Here, we will consider the evolution and assembly of the eukaryotic OST in light of recent genomic evidence concerning the subunit composition of the enzyme in diverse eukaryotes.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                1 December 2016
                2016
                : 11
                : 12
                : e0167611
                Affiliations
                [1 ]Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada
                [2 ]Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
                Tulane University Health Sciences Center, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: YD HAV KJ.

                • Funding acquisition: KFJ CMK.

                • Investigation: YD HAV JS AS CMK KFJ.

                • Methodology: YD HAV KFJ AB CMK.

                • Resources: CMK KFJ.

                • Supervision: KFJ CMK.

                • Visualization: YD KFJ AB CMK.

                • Writing – original draft: YD KFJ.

                • Writing – review & editing: YD HAV JS AB CMK KFJ.

                Article
                PONE-D-16-40924
                10.1371/journal.pone.0167611
                5131992
                27907170
                d15a1747-c046-44f2-93dc-e08d82dffdea
                © 2016 Ding et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 October 2016
                : 17 November 2016
                Page count
                Figures: 6, Tables: 3, Pages: 19
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000038, Natural Sciences and Engineering Research Council of Canada;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000038, Natural Sciences and Engineering Research Council of Canada;
                Award Recipient :
                Funding for the project was supplied by two separate grants from Natural Sciences and Engineering Research Council of Canada (NSERC), one to KFJ and the other to CMK. NSERC website http://www.nserc-crsng.gc.ca/index_eng.asp. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Motif Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Motif Analysis
                Biology and Life Sciences
                Microbiology
                Archaean Biology
                Biology and Life Sciences
                Biochemistry
                Lipids
                Ecology and Environmental Sciences
                Extremophiles
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Amino Acids
                Aliphatic Amino Acids
                Alanine
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Amino Acids
                Aliphatic Amino Acids
                Alanine
                Biology and Life Sciences
                Biochemistry
                Proteins
                Amino Acids
                Aliphatic Amino Acids
                Alanine
                Biology and Life Sciences
                Organisms
                Archaeans
                Methanogens
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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                Uncategorized

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