O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA
The monosaccharide O‐linked N‐acetyl glucosamine (O‐GlcNAc) is an essential and dynamic post‐translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O‐GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently rep...
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| Veröffentlicht in: | Current protocols 2021-05, Vol.1 (5), p.e117-n/a |
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| creator | Ramirez, Daniel H. Ge, Yun Woo, Christina M. |
| description | The monosaccharide O‐linked N‐acetyl glucosamine (O‐GlcNAc) is an essential and dynamic post‐translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O‐GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O‐GlcNAc on a target protein in cells using an engineered O‐GlcNAc transferase (OGT) or split O‐GlcNAcase (OGA) fused to a nanobody. Target protein O‐GlcNAcylation and de‐O‐GlcNAcylation complements methods to interrogate the role of O‐GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody‐OGT and nanobody‐splitOGA systems to screen for O‐GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O‐GlcNAc and cloning procedures to adapt the method for the user's target protein of interest. © 2021 Wiley Periodicals LLC.
Basic Protocol 1: Target protein O‐GlcNAcylation of JunB using nanobody‐OGT
Basic Protocol 2: Target protein deglycosylation of Nup62 using nanobody‐splitOGA
Alternate Protocol: Verification of the O‐GlcNAc state of a tagged target protein through chemoenzymatic labeling
Support Protocol: Cloning of new nanobody‐OGT/nanobody‐splitOGA and target protein pairs |
| doi_str_mv | 10.1002/cpz1.117 |
| format | Article |
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Basic Protocol 1: Target protein O‐GlcNAcylation of JunB using nanobody‐OGT
Basic Protocol 2: Target protein deglycosylation of Nup62 using nanobody‐splitOGA
Alternate Protocol: Verification of the O‐GlcNAc state of a tagged target protein through chemoenzymatic labeling
Support Protocol: Cloning of new nanobody‐OGT/nanobody‐splitOGA and target protein pairs</description><identifier>ISSN: 2691-1299</identifier><identifier>EISSN: 2691-1299</identifier><identifier>DOI: 10.1002/cpz1.117</identifier><identifier>PMID: 33950562</identifier><language>eng</language><publisher>United States</publisher><subject>Acetylglucosamine ; deglycosylation ; Glucosamine ; glycosylation ; N-Acetylglucosaminyltransferases - genetics ; nanobodies ; O‐GlcNAc ; post‐translational modification ; Protein Processing, Post-Translational ; Proteins ; proximity‐direction</subject><ispartof>Current protocols, 2021-05, Vol.1 (5), p.e117-n/a</ispartof><rights>2021 Wiley Periodicals LLC</rights><rights>2021 Wiley Periodicals LLC.</rights><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c2707-23f361c49241ee0610fcdc69f39a93e7d5b044a59797fb03c1f0af0be5f250603</citedby><cites>FETCH-LOGICAL-c2707-23f361c49241ee0610fcdc69f39a93e7d5b044a59797fb03c1f0af0be5f250603</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fcpz1.117$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fcpz1.117$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1417,27924,27925,45574,45575</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33950562$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ramirez, Daniel H.</creatorcontrib><creatorcontrib>Ge, Yun</creatorcontrib><creatorcontrib>Woo, Christina M.</creatorcontrib><title>O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA</title><title>Current protocols</title><addtitle>Curr Protoc</addtitle><description>The monosaccharide O‐linked N‐acetyl glucosamine (O‐GlcNAc) is an essential and dynamic post‐translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O‐GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O‐GlcNAc on a target protein in cells using an engineered O‐GlcNAc transferase (OGT) or split O‐GlcNAcase (OGA) fused to a nanobody. Target protein O‐GlcNAcylation and de‐O‐GlcNAcylation complements methods to interrogate the role of O‐GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody‐OGT and nanobody‐splitOGA systems to screen for O‐GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O‐GlcNAc and cloning procedures to adapt the method for the user's target protein of interest. © 2021 Wiley Periodicals LLC.
Basic Protocol 1: Target protein O‐GlcNAcylation of JunB using nanobody‐OGT
Basic Protocol 2: Target protein deglycosylation of Nup62 using nanobody‐splitOGA
Alternate Protocol: Verification of the O‐GlcNAc state of a tagged target protein through chemoenzymatic labeling
Support Protocol: Cloning of new nanobody‐OGT/nanobody‐splitOGA and target protein pairs</description><subject>Acetylglucosamine</subject><subject>deglycosylation</subject><subject>Glucosamine</subject><subject>glycosylation</subject><subject>N-Acetylglucosaminyltransferases - genetics</subject><subject>nanobodies</subject><subject>O‐GlcNAc</subject><subject>post‐translational modification</subject><subject>Protein Processing, Post-Translational</subject><subject>Proteins</subject><subject>proximity‐direction</subject><issn>2691-1299</issn><issn>2691-1299</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kM9KAzEQh4MottSCTyA5etmaP5tdcixLrULp9lAvXkI2m9TINrsmW0o9-Qg-o0_illbtRRiYYfjmY_gBcI3RCCNE7lTzjkcYp2egTxKOI0w4Pz-Ze2AYwivqUIYpjskl6FHKGWIJ6QORf318Tis1Hys4cSvrtPbWrWDtoIRL6Ve6hQtft9o62FWmqyrArW1f4Fy6uqjLXXefT5dQuvJ0FZrKtvl0fAUujKyCHh77ADzdT5bZQzTLp4_ZeBYpkqI0ItTQBKuYkxhrjRKMjCpVwg3lklOdlqxAcSwZT3lqCkQVNkgaVGhmCEMJogNwe_A2vn7b6NCKtQ2q-1Y6XW-CIIwQyhOC4j9U-ToEr41ovF1LvxMYiX2iYp-o6BLt0JujdVOsdfkL_uTXAdEB2NpK7_4ViWzxjPfCb1qHgDY</recordid><startdate>202105</startdate><enddate>202105</enddate><creator>Ramirez, Daniel H.</creator><creator>Ge, Yun</creator><creator>Woo, Christina M.</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>202105</creationdate><title>O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA</title><author>Ramirez, Daniel H. ; Ge, Yun ; Woo, Christina M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c2707-23f361c49241ee0610fcdc69f39a93e7d5b044a59797fb03c1f0af0be5f250603</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Acetylglucosamine</topic><topic>deglycosylation</topic><topic>Glucosamine</topic><topic>glycosylation</topic><topic>N-Acetylglucosaminyltransferases - genetics</topic><topic>nanobodies</topic><topic>O‐GlcNAc</topic><topic>post‐translational modification</topic><topic>Protein Processing, Post-Translational</topic><topic>Proteins</topic><topic>proximity‐direction</topic><toplevel>online_resources</toplevel><creatorcontrib>Ramirez, Daniel H.</creatorcontrib><creatorcontrib>Ge, Yun</creatorcontrib><creatorcontrib>Woo, Christina M.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Current protocols</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ramirez, Daniel H.</au><au>Ge, Yun</au><au>Woo, Christina M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA</atitle><jtitle>Current protocols</jtitle><addtitle>Curr Protoc</addtitle><date>2021-05</date><risdate>2021</risdate><volume>1</volume><issue>5</issue><spage>e117</spage><epage>n/a</epage><pages>e117-n/a</pages><issn>2691-1299</issn><eissn>2691-1299</eissn><abstract>The monosaccharide O‐linked N‐acetyl glucosamine (O‐GlcNAc) is an essential and dynamic post‐translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O‐GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O‐GlcNAc on a target protein in cells using an engineered O‐GlcNAc transferase (OGT) or split O‐GlcNAcase (OGA) fused to a nanobody. Target protein O‐GlcNAcylation and de‐O‐GlcNAcylation complements methods to interrogate the role of O‐GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody‐OGT and nanobody‐splitOGA systems to screen for O‐GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O‐GlcNAc and cloning procedures to adapt the method for the user's target protein of interest. © 2021 Wiley Periodicals LLC.
Basic Protocol 1: Target protein O‐GlcNAcylation of JunB using nanobody‐OGT
Basic Protocol 2: Target protein deglycosylation of Nup62 using nanobody‐splitOGA
Alternate Protocol: Verification of the O‐GlcNAc state of a tagged target protein through chemoenzymatic labeling
Support Protocol: Cloning of new nanobody‐OGT/nanobody‐splitOGA and target protein pairs</abstract><cop>United States</cop><pmid>33950562</pmid><doi>10.1002/cpz1.117</doi><tpages>28</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Access via Wiley Online Library |
| subjects | Acetylglucosamine deglycosylation Glucosamine glycosylation N-Acetylglucosaminyltransferases - genetics nanobodies O‐GlcNAc post‐translational modification Protein Processing, Post-Translational Proteins proximity‐direction |
| title | O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA |
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