A Metabolic Labeling Approach toward Proteomic Analysis of Mucin-Type O-Linked Glycosylation
Mucin-type O-linked glycoproteins are involved in a variety of biological interactions in higher eukaryotes. The biosynthesis of these glycoproteins is initiated by a family of polypeptide N-acetyl-α -galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2003-12, Vol.100 (25), p.14846-14851 |
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| creator | Hang, Howard C. Yu, Chong Kato, Darryl L. Bertozzi, Carolyn R. |
| description | Mucin-type O-linked glycoproteins are involved in a variety of biological interactions in higher eukaryotes. The biosynthesis of these glycoproteins is initiated by a family of polypeptide N-acetyl-α -galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. From a panel of synthetic derivatives, we identified an azido GalNAc analog (N-azidoacetylgalactosamine, GalNAz) that is metabolized by numerous cell types and installed on mucin-type O-linked glycoproteins by the ppGalNAcTs. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. The approach was validated by labeling a recombinant glycoprotein that is known to possess O-linked glycans with GalNAz. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. The ability to label mucin-type O-linked glycoproteins with chemical tags should facilitate their identification by proteomic strategies. |
| doi_str_mv | 10.1073/pnas.2335201100 |
| format | Article |
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The biosynthesis of these glycoproteins is initiated by a family of polypeptide N-acetyl-α -galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. From a panel of synthetic derivatives, we identified an azido GalNAc analog (N-azidoacetylgalactosamine, GalNAz) that is metabolized by numerous cell types and installed on mucin-type O-linked glycoproteins by the ppGalNAcTs. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. The approach was validated by labeling a recombinant glycoprotein that is known to possess O-linked glycans with GalNAz. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. The ability to label mucin-type O-linked glycoproteins with chemical tags should facilitate their identification by proteomic strategies.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.2335201100</identifier><identifier>PMID: 14657396</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Animals ; Azides ; Biological Sciences ; Biosynthesis ; Blotting, Western ; Cellular biology ; Cellular metabolism ; Chemistry ; CHO Cells ; COS Cells ; Cricetinae ; Cytometry ; Dose-Response Relationship, Drug ; Flow Cytometry ; Glycoproteins ; Glycoproteins - chemistry ; Glycosylation ; HeLa Cells ; Humans ; Jurkat Cells ; Lectins - chemistry ; Mice ; Models, Biological ; Models, Chemical ; Monosaccharides - chemistry ; Mucins - chemistry ; NIH 3T3 Cells ; Physical Sciences ; Polysaccharides ; Proteins ; Proteome ; Proteomics ; Recombinant Proteins - chemistry ; T lymphocytes</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2003-12, Vol.100 (25), p.14846-14851</ispartof><rights>Copyright 1993-2003 National Academy of Sciences of the United States of America</rights><rights>Copyright National Academy of Sciences Dec 9, 2003</rights><rights>Copyright © 2003, The National Academy of Sciences 2003</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c561t-b1578b1a6c8d8b22676827d25eff78ce7ed42e8b63f690846367c5f4346836863</citedby><cites>FETCH-LOGICAL-c561t-b1578b1a6c8d8b22676827d25eff78ce7ed42e8b63f690846367c5f4346836863</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/100/25.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/3148523$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/3148523$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27924,27925,53791,53793,58017,58250</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/14657396$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hang, Howard C.</creatorcontrib><creatorcontrib>Yu, Chong</creatorcontrib><creatorcontrib>Kato, Darryl L.</creatorcontrib><creatorcontrib>Bertozzi, Carolyn R.</creatorcontrib><title>A Metabolic Labeling Approach toward Proteomic Analysis of Mucin-Type O-Linked Glycosylation</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Mucin-type O-linked glycoproteins are involved in a variety of biological interactions in higher eukaryotes. The biosynthesis of these glycoproteins is initiated by a family of polypeptide N-acetyl-α -galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. From a panel of synthetic derivatives, we identified an azido GalNAc analog (N-azidoacetylgalactosamine, GalNAz) that is metabolized by numerous cell types and installed on mucin-type O-linked glycoproteins by the ppGalNAcTs. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. The approach was validated by labeling a recombinant glycoprotein that is known to possess O-linked glycans with GalNAz. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. The ability to label mucin-type O-linked glycoproteins with chemical tags should facilitate their identification by proteomic strategies.</description><subject>Animals</subject><subject>Azides</subject><subject>Biological Sciences</subject><subject>Biosynthesis</subject><subject>Blotting, Western</subject><subject>Cellular biology</subject><subject>Cellular metabolism</subject><subject>Chemistry</subject><subject>CHO Cells</subject><subject>COS Cells</subject><subject>Cricetinae</subject><subject>Cytometry</subject><subject>Dose-Response Relationship, Drug</subject><subject>Flow Cytometry</subject><subject>Glycoproteins</subject><subject>Glycoproteins - chemistry</subject><subject>Glycosylation</subject><subject>HeLa Cells</subject><subject>Humans</subject><subject>Jurkat Cells</subject><subject>Lectins - chemistry</subject><subject>Mice</subject><subject>Models, Biological</subject><subject>Models, Chemical</subject><subject>Monosaccharides - chemistry</subject><subject>Mucins - chemistry</subject><subject>NIH 3T3 Cells</subject><subject>Physical Sciences</subject><subject>Polysaccharides</subject><subject>Proteins</subject><subject>Proteome</subject><subject>Proteomics</subject><subject>Recombinant Proteins - chemistry</subject><subject>T lymphocytes</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0b9v1DAUB3ALgehxMLMgZDEgdUjrX7GdgeFUQYt0VRnKhmQ5jtP68MWp7QD57_HpTj1gYfLwPu_56X0BeI3RGUaCno-DTmeE0pogjBF6AhYYNbjirEFPwQIhIirJCDsBL1LaIISaWqLn4AQzXgva8AX4toLXNus2eGfgWrfWu-EOrsYxBm3uYQ4_dezglxiyDdtCVoP2c3IJhh5eT8YN1e08WnhTrd3w3Xbw0s8mpNnr7MLwEjzrtU_21eFdgq-fPt5eXFXrm8vPF6t1ZWqOc9XiWsgWa25kJ1tCuOCSiI7Utu-FNFbYjhErW0573iDJOOXC1D2jjEvKJadL8GE_d5zare2MHXLUXo3RbXWcVdBO_V0Z3L26Cz8UaRpZrrcE7w_9MTxMNmW1dclY7_Vgw5SUwDXa_VTgu3_gJkyxnCSpEgBlgnFR0PkemRhSirZ_XAQjtUtN7VJTx9RKx9s_9z_6Q0wFnB7ArvM4DilSF1VOovrJ-2x_5WLhf2whb_Zkk3KIj4aWal3W-g0qwrUb</recordid><startdate>20031209</startdate><enddate>20031209</enddate><creator>Hang, Howard C.</creator><creator>Yu, Chong</creator><creator>Kato, Darryl L.</creator><creator>Bertozzi, Carolyn R.</creator><general>National Academy of Sciences</general><general>National Acad Sciences</general><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>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20031209</creationdate><title>A Metabolic Labeling Approach toward Proteomic Analysis of Mucin-Type O-Linked Glycosylation</title><author>Hang, Howard C. ; Yu, Chong ; Kato, Darryl L. ; Bertozzi, Carolyn R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c561t-b1578b1a6c8d8b22676827d25eff78ce7ed42e8b63f690846367c5f4346836863</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Animals</topic><topic>Azides</topic><topic>Biological Sciences</topic><topic>Biosynthesis</topic><topic>Blotting, Western</topic><topic>Cellular biology</topic><topic>Cellular metabolism</topic><topic>Chemistry</topic><topic>CHO Cells</topic><topic>COS Cells</topic><topic>Cricetinae</topic><topic>Cytometry</topic><topic>Dose-Response Relationship, Drug</topic><topic>Flow Cytometry</topic><topic>Glycoproteins</topic><topic>Glycoproteins - chemistry</topic><topic>Glycosylation</topic><topic>HeLa Cells</topic><topic>Humans</topic><topic>Jurkat Cells</topic><topic>Lectins - chemistry</topic><topic>Mice</topic><topic>Models, Biological</topic><topic>Models, Chemical</topic><topic>Monosaccharides - chemistry</topic><topic>Mucins - chemistry</topic><topic>NIH 3T3 Cells</topic><topic>Physical Sciences</topic><topic>Polysaccharides</topic><topic>Proteins</topic><topic>Proteome</topic><topic>Proteomics</topic><topic>Recombinant Proteins - chemistry</topic><topic>T lymphocytes</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hang, Howard C.</creatorcontrib><creatorcontrib>Yu, Chong</creatorcontrib><creatorcontrib>Kato, Darryl L.</creatorcontrib><creatorcontrib>Bertozzi, Carolyn R.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hang, Howard C.</au><au>Yu, Chong</au><au>Kato, Darryl L.</au><au>Bertozzi, Carolyn R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A Metabolic Labeling Approach toward Proteomic Analysis of Mucin-Type O-Linked Glycosylation</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>2003-12-09</date><risdate>2003</risdate><volume>100</volume><issue>25</issue><spage>14846</spage><epage>14851</epage><pages>14846-14851</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>Mucin-type O-linked glycoproteins are involved in a variety of biological interactions in higher eukaryotes. The biosynthesis of these glycoproteins is initiated by a family of polypeptide N-acetyl-α -galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. From a panel of synthetic derivatives, we identified an azido GalNAc analog (N-azidoacetylgalactosamine, GalNAz) that is metabolized by numerous cell types and installed on mucin-type O-linked glycoproteins by the ppGalNAcTs. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. The approach was validated by labeling a recombinant glycoprotein that is known to possess O-linked glycans with GalNAz. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. The ability to label mucin-type O-linked glycoproteins with chemical tags should facilitate their identification by proteomic strategies.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>14657396</pmid><doi>10.1073/pnas.2335201100</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Azides Biological Sciences Biosynthesis Blotting, Western Cellular biology Cellular metabolism Chemistry CHO Cells COS Cells Cricetinae Cytometry Dose-Response Relationship, Drug Flow Cytometry Glycoproteins Glycoproteins - chemistry Glycosylation HeLa Cells Humans Jurkat Cells Lectins - chemistry Mice Models, Biological Models, Chemical Monosaccharides - chemistry Mucins - chemistry NIH 3T3 Cells Physical Sciences Polysaccharides Proteins Proteome Proteomics Recombinant Proteins - chemistry T lymphocytes |
| title | A Metabolic Labeling Approach toward Proteomic Analysis of Mucin-Type O-Linked Glycosylation |
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