Definition of the bacterial N-glycosylation site consensus sequence
The Campylobacter jejuni pgl locus encodes an N ‐linked protein glycosylation machinery that can be functionally transferred into Escherichia coli . In this system, we analyzed the elements in the C. jejuni N ‐glycoprotein AcrA required for accepting an N ‐glycan. We found that the eukaryotic primar...
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| Veröffentlicht in: | The EMBO journal 2006-05, Vol.25 (9), p.1957-1966 |
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| container_end_page | 1966 |
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| container_issue | 9 |
| container_start_page | 1957 |
| container_title | The EMBO journal |
| container_volume | 25 |
| creator | Kowarik, Michael Young, N Martin Numao, Shin Schulz, Benjamin L Hug, Isabelle Callewaert, Nico Mills, Dominic C Watson, David C Hernandez, Marcela Kelly, John F Wacker, Michael Aebi, Markus |
| description | The
Campylobacter jejuni pgl
locus encodes an
N
‐linked protein glycosylation machinery that can be functionally transferred into
Escherichia coli
. In this system, we analyzed the elements in the
C. jejuni N
‐glycoprotein AcrA required for accepting an
N
‐glycan. We found that the eukaryotic primary consensus sequence for
N
‐glycosylation is
N
terminally extended to D/E‐Y‐N‐X‐S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were
N
‐glycosylated when they were either artificially introduced or when they were present in non‐
C. jejuni
proteins. We were able to produce recombinant glycoproteins with engineered
N
‐glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in
C. jejuni N
‐glycoproteins.
N
‐glycosylation of AcrA by the eukaryotic OST in
Saccharomyces cerevisiae
occurred independent of the acidic residue at the −2 position. Thus, bacterial
N
‐glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence. |
| doi_str_mv | 10.1038/sj.emboj.7601087 |
| format | Article |
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Campylobacter jejuni pgl
locus encodes an
N
‐linked protein glycosylation machinery that can be functionally transferred into
Escherichia coli
. In this system, we analyzed the elements in the
C. jejuni N
‐glycoprotein AcrA required for accepting an
N
‐glycan. We found that the eukaryotic primary consensus sequence for
N
‐glycosylation is
N
terminally extended to D/E‐Y‐N‐X‐S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were
N
‐glycosylated when they were either artificially introduced or when they were present in non‐
C. jejuni
proteins. We were able to produce recombinant glycoproteins with engineered
N
‐glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in
C. jejuni N
‐glycoproteins.
N
‐glycosylation of AcrA by the eukaryotic OST in
Saccharomyces cerevisiae
occurred independent of the acidic residue at the −2 position. Thus, bacterial
N
‐glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.</description><identifier>ISSN: 0261-4189</identifier><identifier>EISSN: 1460-2075</identifier><identifier>DOI: 10.1038/sj.emboj.7601087</identifier><identifier>PMID: 16619027</identifier><identifier>CODEN: EMJODG</identifier><language>eng</language><publisher>Chichester, UK: John Wiley & Sons, Ltd</publisher><subject>Amino Acid Sequence - genetics ; Amino Acid Substitution - genetics ; Amino Acids - chemistry ; Amino Acids - genetics ; Amino Acids - metabolism ; Bacteria ; Bacterial Outer Membrane Proteins - chemistry ; Bacterial Outer Membrane Proteins - genetics ; Bacterial Outer Membrane Proteins - metabolism ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Campylobacter jejuni ; Campylobacter jejuni - metabolism ; consensus sequence ; Consensus Sequence - genetics ; E coli ; EMBO23 ; EMBO31 ; Escherichia coli ; Eukaryotes ; Glycoproteins ; Glycoproteins - chemistry ; Glycoproteins - genetics ; Glycoproteins - metabolism ; Glycosylation ; Hexosyltransferases - metabolism ; Lipoproteins - chemistry ; Lipoproteins - genetics ; Lipoproteins - metabolism ; Membrane Proteins - metabolism ; Molecular Sequence Data ; Mutation ; N-glycosylation ; oligosaccharyltransferase ; periplasm ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae - metabolism ; Site selection</subject><ispartof>The EMBO journal, 2006-05, Vol.25 (9), p.1957-1966</ispartof><rights>European Molecular Biology Organization 2006</rights><rights>Copyright © 2006 European Molecular Biology Organization</rights><rights>Copyright Nature Publishing Group May 3, 2006</rights><rights>Copyright © 2006, European Molecular Biology Organization 2006</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c6707-93778442ab488aba77e021d01c8d8ad1eb26cfa4c38192e83b5a354f38872b703</citedby><cites>FETCH-LOGICAL-c6707-93778442ab488aba77e021d01c8d8ad1eb26cfa4c38192e83b5a354f38872b703</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1456941/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1456941/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,725,778,782,883,1414,1430,27907,27908,41103,42172,45557,45558,46392,46816,51559,53774,53776</link.rule.ids><linktorsrc>$$Uhttps://doi.org/10.1038/sj.emboj.7601087$$EView_record_in_Springer_Nature$$FView_record_in_$$GSpringer_Nature</linktorsrc><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16619027$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kowarik, Michael</creatorcontrib><creatorcontrib>Young, N Martin</creatorcontrib><creatorcontrib>Numao, Shin</creatorcontrib><creatorcontrib>Schulz, Benjamin L</creatorcontrib><creatorcontrib>Hug, Isabelle</creatorcontrib><creatorcontrib>Callewaert, Nico</creatorcontrib><creatorcontrib>Mills, Dominic C</creatorcontrib><creatorcontrib>Watson, David C</creatorcontrib><creatorcontrib>Hernandez, Marcela</creatorcontrib><creatorcontrib>Kelly, John F</creatorcontrib><creatorcontrib>Wacker, Michael</creatorcontrib><creatorcontrib>Aebi, Markus</creatorcontrib><title>Definition of the bacterial N-glycosylation site consensus sequence</title><title>The EMBO journal</title><addtitle>EMBO J</addtitle><addtitle>EMBO J</addtitle><description>The
Campylobacter jejuni pgl
locus encodes an
N
‐linked protein glycosylation machinery that can be functionally transferred into
Escherichia coli
. In this system, we analyzed the elements in the
C. jejuni N
‐glycoprotein AcrA required for accepting an
N
‐glycan. We found that the eukaryotic primary consensus sequence for
N
‐glycosylation is
N
terminally extended to D/E‐Y‐N‐X‐S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were
N
‐glycosylated when they were either artificially introduced or when they were present in non‐
C. jejuni
proteins. We were able to produce recombinant glycoproteins with engineered
N
‐glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in
C. jejuni N
‐glycoproteins.
N
‐glycosylation of AcrA by the eukaryotic OST in
Saccharomyces cerevisiae
occurred independent of the acidic residue at the −2 position. Thus, bacterial
N
‐glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.</description><subject>Amino Acid Sequence - genetics</subject><subject>Amino Acid Substitution - genetics</subject><subject>Amino Acids - chemistry</subject><subject>Amino Acids - genetics</subject><subject>Amino Acids - metabolism</subject><subject>Bacteria</subject><subject>Bacterial Outer Membrane Proteins - chemistry</subject><subject>Bacterial Outer Membrane Proteins - genetics</subject><subject>Bacterial Outer Membrane Proteins - metabolism</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Campylobacter jejuni</subject><subject>Campylobacter jejuni - metabolism</subject><subject>consensus sequence</subject><subject>Consensus Sequence - genetics</subject><subject>E coli</subject><subject>EMBO23</subject><subject>EMBO31</subject><subject>Escherichia coli</subject><subject>Eukaryotes</subject><subject>Glycoproteins</subject><subject>Glycoproteins - chemistry</subject><subject>Glycoproteins - genetics</subject><subject>Glycoproteins - metabolism</subject><subject>Glycosylation</subject><subject>Hexosyltransferases - metabolism</subject><subject>Lipoproteins - chemistry</subject><subject>Lipoproteins - genetics</subject><subject>Lipoproteins - metabolism</subject><subject>Membrane Proteins - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Mutation</subject><subject>N-glycosylation</subject><subject>oligosaccharyltransferase</subject><subject>periplasm</subject><subject>Saccharomyces cerevisiae</subject><subject>Saccharomyces cerevisiae - metabolism</subject><subject>Site selection</subject><issn>0261-4189</issn><issn>1460-2075</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNqFkUFv1DAUhC0EotuWOxdQxIFbFttxbOeC1Ia2gEq5FDhajvdl65C1i50U9t_XbVa7BQn15MN8M56nQeglwXOCC_kudnNYNb6bC44JluIJmhHGcU6xKJ-iGaac5IzIag_tx9hhjEspyHO0RzgnFaZihuoP0FpnB-td5ttsuIKs0WaAYHWfXeTLfm18XPf6Hoh2gMx4F8HFMWYRfo3gDByiZ63uI7zYvAfo2-nJZf0xP_969qk-Os8NF1jkVSGEZIzqhkmpGy0EYEoWmBi5kHpBoKHctJqZQpKKgiyaUhclawspBW0ELg7Q-yn3emxWsDDghqB7dR3sSoe18tqqvxVnr9TS3yjCSl4xkgLebgKCT9XjoFY2Guh77cCPUZEq9ZNMJPDNP2Dnx-DScYkpKU-N7tLwBJngYwzQbpsQrO7mUbFT9_OozTzJ8vrhBTvDZo8EVBPw2_awfjRQnXw5_rwLJ5M3JptbQnhQ-v-FXk0ep4cxwPbDnZ5Puo0D_NnKOvxUXBSiVD8uzhStj_FlLb-rqrgF_5LIwA</recordid><startdate>20060503</startdate><enddate>20060503</enddate><creator>Kowarik, Michael</creator><creator>Young, N Martin</creator><creator>Numao, Shin</creator><creator>Schulz, Benjamin L</creator><creator>Hug, Isabelle</creator><creator>Callewaert, Nico</creator><creator>Mills, Dominic C</creator><creator>Watson, David C</creator><creator>Hernandez, Marcela</creator><creator>Kelly, John F</creator><creator>Wacker, Michael</creator><creator>Aebi, Markus</creator><general>John Wiley & Sons, Ltd</general><general>Nature Publishing Group UK</general><general>Blackwell Publishing 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of the bacterial N-glycosylation site consensus sequence</title><author>Kowarik, Michael ; Young, N Martin ; Numao, Shin ; Schulz, Benjamin L ; Hug, Isabelle ; Callewaert, Nico ; Mills, Dominic C ; Watson, David C ; Hernandez, Marcela ; Kelly, John F ; Wacker, Michael ; Aebi, Markus</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c6707-93778442ab488aba77e021d01c8d8ad1eb26cfa4c38192e83b5a354f38872b703</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Amino Acid Sequence - genetics</topic><topic>Amino Acid Substitution - genetics</topic><topic>Amino Acids - chemistry</topic><topic>Amino Acids - genetics</topic><topic>Amino Acids - metabolism</topic><topic>Bacteria</topic><topic>Bacterial Outer Membrane Proteins - chemistry</topic><topic>Bacterial Outer Membrane Proteins - genetics</topic><topic>Bacterial Outer Membrane Proteins - metabolism</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Campylobacter jejuni</topic><topic>Campylobacter jejuni - metabolism</topic><topic>consensus sequence</topic><topic>Consensus Sequence - genetics</topic><topic>E coli</topic><topic>EMBO23</topic><topic>EMBO31</topic><topic>Escherichia coli</topic><topic>Eukaryotes</topic><topic>Glycoproteins</topic><topic>Glycoproteins - chemistry</topic><topic>Glycoproteins - genetics</topic><topic>Glycoproteins - metabolism</topic><topic>Glycosylation</topic><topic>Hexosyltransferases - metabolism</topic><topic>Lipoproteins - chemistry</topic><topic>Lipoproteins - genetics</topic><topic>Lipoproteins - metabolism</topic><topic>Membrane Proteins - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Mutation</topic><topic>N-glycosylation</topic><topic>oligosaccharyltransferase</topic><topic>periplasm</topic><topic>Saccharomyces cerevisiae</topic><topic>Saccharomyces cerevisiae - metabolism</topic><topic>Site selection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kowarik, Michael</creatorcontrib><creatorcontrib>Young, N Martin</creatorcontrib><creatorcontrib>Numao, Shin</creatorcontrib><creatorcontrib>Schulz, Benjamin L</creatorcontrib><creatorcontrib>Hug, Isabelle</creatorcontrib><creatorcontrib>Callewaert, Nico</creatorcontrib><creatorcontrib>Mills, Dominic C</creatorcontrib><creatorcontrib>Watson, David C</creatorcontrib><creatorcontrib>Hernandez, Marcela</creatorcontrib><creatorcontrib>Kelly, John F</creatorcontrib><creatorcontrib>Wacker, Michael</creatorcontrib><creatorcontrib>Aebi, Markus</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central 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J</addtitle><date>2006-05-03</date><risdate>2006</risdate><volume>25</volume><issue>9</issue><spage>1957</spage><epage>1966</epage><pages>1957-1966</pages><issn>0261-4189</issn><eissn>1460-2075</eissn><coden>EMJODG</coden><abstract>The
Campylobacter jejuni pgl
locus encodes an
N
‐linked protein glycosylation machinery that can be functionally transferred into
Escherichia coli
. In this system, we analyzed the elements in the
C. jejuni N
‐glycoprotein AcrA required for accepting an
N
‐glycan. We found that the eukaryotic primary consensus sequence for
N
‐glycosylation is
N
terminally extended to D/E‐Y‐N‐X‐S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were
N
‐glycosylated when they were either artificially introduced or when they were present in non‐
C. jejuni
proteins. We were able to produce recombinant glycoproteins with engineered
N
‐glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in
C. jejuni N
‐glycoproteins.
N
‐glycosylation of AcrA by the eukaryotic OST in
Saccharomyces cerevisiae
occurred independent of the acidic residue at the −2 position. Thus, bacterial
N
‐glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.</abstract><cop>Chichester, UK</cop><pub>John Wiley & Sons, Ltd</pub><pmid>16619027</pmid><doi>10.1038/sj.emboj.7601087</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 0261-4189 |
| ispartof | The EMBO journal, 2006-05, Vol.25 (9), p.1957-1966 |
| issn | 0261-4189 1460-2075 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_1456941 |
| source | Springer Nature OA Free Journals |
| subjects | Amino Acid Sequence - genetics Amino Acid Substitution - genetics Amino Acids - chemistry Amino Acids - genetics Amino Acids - metabolism Bacteria Bacterial Outer Membrane Proteins - chemistry Bacterial Outer Membrane Proteins - genetics Bacterial Outer Membrane Proteins - metabolism Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Campylobacter jejuni Campylobacter jejuni - metabolism consensus sequence Consensus Sequence - genetics E coli EMBO23 EMBO31 Escherichia coli Eukaryotes Glycoproteins Glycoproteins - chemistry Glycoproteins - genetics Glycoproteins - metabolism Glycosylation Hexosyltransferases - metabolism Lipoproteins - chemistry Lipoproteins - genetics Lipoproteins - metabolism Membrane Proteins - metabolism Molecular Sequence Data Mutation N-glycosylation oligosaccharyltransferase periplasm Saccharomyces cerevisiae Saccharomyces cerevisiae - metabolism Site selection |
| title | Definition of the bacterial N-glycosylation site consensus sequence |
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