Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins
The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. C...
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| Veröffentlicht in: | Glycobiology (Oxford) 2014-01, Vol.24 (1), p.26-38 |
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| creator | Maria Cherian, Reeja Gaunitz, Stefan Nilsson, Anki Liu, Jining Karlsson, Niclas G Holgersson, Jan |
| description | The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG2b fusion protein, the pigeon α1,4-galactosyltransferase (α4Gal-T) and the core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG2b and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG2b. In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG2b and a P(k)-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG2b and P(k)-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon α4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG2b bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins. |
| doi_str_mv | 10.1093/glycob/cwt086 |
| format | Article |
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Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG2b fusion protein, the pigeon α1,4-galactosyltransferase (α4Gal-T) and the core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG2b and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG2b. In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG2b and a P(k)-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG2b and P(k)-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon α4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG2b bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.</description><identifier>ISSN: 0959-6658</identifier><identifier>ISSN: 1460-2423</identifier><identifier>EISSN: 1460-2423</identifier><identifier>DOI: 10.1093/glycob/cwt086</identifier><identifier>PMID: 24082034</identifier><language>eng</language><publisher>England</publisher><subject>Animals ; blood group P1 ; CHO Cells ; Clinical Medicine ; COLONIZATION ; Columbidae ; Cricetinae ; Cricetulus ; CRYSTAL-STRUCTURE ; Globosides - chemistry ; Globosides - genetics ; Globosides - metabolism ; GROUP-A ; HELICOBACTER-PYLORI ; HEMOLYTIC-UREMIC SYNDROME ; Humans ; Immunoglobulin G - chemistry ; Immunoglobulin G - genetics ; Immunoglobulin G - metabolism ; Klinisk medicin ; MAJOR GLYCOPROTEINS ; mass spectrometry ; Medicin och hälsovetenskap ; Membrane Glycoproteins - chemistry ; Membrane Glycoproteins - genetics ; Membrane Glycoproteins - metabolism ; Mice ; mucin ; N-Acetylglucosaminyltransferases - chemistry ; N-Acetylglucosaminyltransferases - genetics ; N-Acetylglucosaminyltransferases - metabolism ; N-GLYCAN STRUCTURES ; PIGEON EGG-WHITE ; Polysaccharides - chemistry ; Polysaccharides - genetics ; Polysaccharides - metabolism ; Protein Binding ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Shiga Toxin 1 - chemistry ; Shiga Toxin 1 - genetics ; Shiga Toxin 1 - metabolism ; Shiga Toxin 2 - chemistry ; Shiga Toxin 2 - genetics ; Shiga Toxin 2 - metabolism ; Shiga-like toxin ; Shiga-Toxigenic Escherichia coli - chemistry ; Shiga-Toxigenic Escherichia coli - genetics ; Shiga-Toxigenic Escherichia coli - metabolism ; SHIGELLA-DYSENTERIAE ; SPR ; STREPTOCOCCUS-SUIS ; UROPATHOGENIC ESCHERICHIA-COLI</subject><ispartof>Glycobiology (Oxford), 2014-01, Vol.24 (1), p.26-38</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c458t-49744c1fbcb2f95993897b9662e83d983f702393deecca089db9be01d5b294ad3</citedby><cites>FETCH-LOGICAL-c458t-49744c1fbcb2f95993897b9662e83d983f702393deecca089db9be01d5b294ad3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,4024,27923,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24082034$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://gup.ub.gu.se/publication/195026$$DView record from Swedish Publication Index$$Hfree_for_read</backlink><backlink>$$Uhttp://kipublications.ki.se/Default.aspx?queryparsed=id:128289222$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><creatorcontrib>Maria Cherian, Reeja</creatorcontrib><creatorcontrib>Gaunitz, Stefan</creatorcontrib><creatorcontrib>Nilsson, Anki</creatorcontrib><creatorcontrib>Liu, Jining</creatorcontrib><creatorcontrib>Karlsson, Niclas G</creatorcontrib><creatorcontrib>Holgersson, Jan</creatorcontrib><title>Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins</title><title>Glycobiology (Oxford)</title><addtitle>Glycobiology</addtitle><description>The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG2b fusion protein, the pigeon α1,4-galactosyltransferase (α4Gal-T) and the core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG2b and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG2b. In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG2b and a P(k)-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG2b and P(k)-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon α4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG2b bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.</description><subject>Animals</subject><subject>blood group P1</subject><subject>CHO Cells</subject><subject>Clinical Medicine</subject><subject>COLONIZATION</subject><subject>Columbidae</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>CRYSTAL-STRUCTURE</subject><subject>Globosides - chemistry</subject><subject>Globosides - genetics</subject><subject>Globosides - metabolism</subject><subject>GROUP-A</subject><subject>HELICOBACTER-PYLORI</subject><subject>HEMOLYTIC-UREMIC SYNDROME</subject><subject>Humans</subject><subject>Immunoglobulin G - chemistry</subject><subject>Immunoglobulin G - genetics</subject><subject>Immunoglobulin G - metabolism</subject><subject>Klinisk medicin</subject><subject>MAJOR GLYCOPROTEINS</subject><subject>mass spectrometry</subject><subject>Medicin och hälsovetenskap</subject><subject>Membrane Glycoproteins - chemistry</subject><subject>Membrane Glycoproteins - genetics</subject><subject>Membrane Glycoproteins - metabolism</subject><subject>Mice</subject><subject>mucin</subject><subject>N-Acetylglucosaminyltransferases - chemistry</subject><subject>N-Acetylglucosaminyltransferases - genetics</subject><subject>N-Acetylglucosaminyltransferases - metabolism</subject><subject>N-GLYCAN STRUCTURES</subject><subject>PIGEON EGG-WHITE</subject><subject>Polysaccharides - chemistry</subject><subject>Polysaccharides - genetics</subject><subject>Polysaccharides - metabolism</subject><subject>Protein Binding</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Shiga Toxin 1 - chemistry</subject><subject>Shiga Toxin 1 - genetics</subject><subject>Shiga Toxin 1 - metabolism</subject><subject>Shiga Toxin 2 - chemistry</subject><subject>Shiga Toxin 2 - genetics</subject><subject>Shiga Toxin 2 - metabolism</subject><subject>Shiga-like toxin</subject><subject>Shiga-Toxigenic Escherichia coli - chemistry</subject><subject>Shiga-Toxigenic Escherichia coli - genetics</subject><subject>Shiga-Toxigenic Escherichia coli - metabolism</subject><subject>SHIGELLA-DYSENTERIAE</subject><subject>SPR</subject><subject>STREPTOCOCCUS-SUIS</subject><subject>UROPATHOGENIC ESCHERICHIA-COLI</subject><issn>0959-6658</issn><issn>1460-2423</issn><issn>1460-2423</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kktv1DAUhS0EokNhyRZ5ySbUrzj2ElVAkSoVCVhbfiVjJrFD7HQYfn0NMy1sYGXr3u-ca-seAF5i9AYjSS-G8WCTubD7ggR_BDaYcdQQRuhjsEGylQ3nrTgDz3L-hhDmWLRPwRlhSBBE2Qb8_LwNg27GsPOwpB8hQhOiy3AfyhbW1hbq2-BCOdQunNaxhFs9-ljgTdXEnXfQjCk5OCxpneEnDJ0vfplC1LFkmGLV2BCbcpg97NccamVeUvEh5ufgSa_H7F-cznPw9f27L5dXzfXNh4-Xb68by1pRGiY7xizujTWkrx-SVMjOSM6JF9RJQfsOESqp895ajYR0RhqPsGsNkUw7eg6ao2_e-3k1al7CpJeDSjqoU2lXb161hLddV3n5T76-3f0R3QsxEURIQsh_Zw3rrGppWH9LZIsIr_zrI1-Nv68-FzWFbP046ujTmlXdJqcdRfQva7uknBffP5hjpH5FQR2joI5RqPyrk_VqJu8e6Pvd0ztlvrWj</recordid><startdate>201401</startdate><enddate>201401</enddate><creator>Maria Cherian, Reeja</creator><creator>Gaunitz, Stefan</creator><creator>Nilsson, Anki</creator><creator>Liu, Jining</creator><creator>Karlsson, Niclas G</creator><creator>Holgersson, Jan</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><scope>ADTPV</scope><scope>AOWAS</scope><scope>F1U</scope></search><sort><creationdate>201401</creationdate><title>Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins</title><author>Maria Cherian, Reeja ; Gaunitz, Stefan ; Nilsson, Anki ; Liu, Jining ; Karlsson, Niclas G ; Holgersson, Jan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c458t-49744c1fbcb2f95993897b9662e83d983f702393deecca089db9be01d5b294ad3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Animals</topic><topic>blood group P1</topic><topic>CHO Cells</topic><topic>Clinical Medicine</topic><topic>COLONIZATION</topic><topic>Columbidae</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>CRYSTAL-STRUCTURE</topic><topic>Globosides - chemistry</topic><topic>Globosides - genetics</topic><topic>Globosides - metabolism</topic><topic>GROUP-A</topic><topic>HELICOBACTER-PYLORI</topic><topic>HEMOLYTIC-UREMIC SYNDROME</topic><topic>Humans</topic><topic>Immunoglobulin G - chemistry</topic><topic>Immunoglobulin G - genetics</topic><topic>Immunoglobulin G - metabolism</topic><topic>Klinisk medicin</topic><topic>MAJOR GLYCOPROTEINS</topic><topic>mass spectrometry</topic><topic>Medicin och hälsovetenskap</topic><topic>Membrane Glycoproteins - chemistry</topic><topic>Membrane Glycoproteins - genetics</topic><topic>Membrane Glycoproteins - metabolism</topic><topic>Mice</topic><topic>mucin</topic><topic>N-Acetylglucosaminyltransferases - chemistry</topic><topic>N-Acetylglucosaminyltransferases - genetics</topic><topic>N-Acetylglucosaminyltransferases - metabolism</topic><topic>N-GLYCAN STRUCTURES</topic><topic>PIGEON EGG-WHITE</topic><topic>Polysaccharides - chemistry</topic><topic>Polysaccharides - genetics</topic><topic>Polysaccharides - metabolism</topic><topic>Protein Binding</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Shiga Toxin 1 - chemistry</topic><topic>Shiga Toxin 1 - genetics</topic><topic>Shiga Toxin 1 - metabolism</topic><topic>Shiga Toxin 2 - chemistry</topic><topic>Shiga Toxin 2 - genetics</topic><topic>Shiga Toxin 2 - metabolism</topic><topic>Shiga-like toxin</topic><topic>Shiga-Toxigenic Escherichia coli - chemistry</topic><topic>Shiga-Toxigenic Escherichia coli - genetics</topic><topic>Shiga-Toxigenic Escherichia coli - metabolism</topic><topic>SHIGELLA-DYSENTERIAE</topic><topic>SPR</topic><topic>STREPTOCOCCUS-SUIS</topic><topic>UROPATHOGENIC ESCHERICHIA-COLI</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Maria Cherian, Reeja</creatorcontrib><creatorcontrib>Gaunitz, Stefan</creatorcontrib><creatorcontrib>Nilsson, Anki</creatorcontrib><creatorcontrib>Liu, Jining</creatorcontrib><creatorcontrib>Karlsson, Niclas G</creatorcontrib><creatorcontrib>Holgersson, Jan</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><collection>SwePub</collection><collection>SwePub Articles</collection><collection>SWEPUB Göteborgs universitet</collection><jtitle>Glycobiology (Oxford)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Maria Cherian, Reeja</au><au>Gaunitz, Stefan</au><au>Nilsson, Anki</au><au>Liu, Jining</au><au>Karlsson, Niclas G</au><au>Holgersson, Jan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins</atitle><jtitle>Glycobiology (Oxford)</jtitle><addtitle>Glycobiology</addtitle><date>2014-01</date><risdate>2014</risdate><volume>24</volume><issue>1</issue><spage>26</spage><epage>38</epage><pages>26-38</pages><issn>0959-6658</issn><issn>1460-2423</issn><eissn>1460-2423</eissn><abstract>The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG2b fusion protein, the pigeon α1,4-galactosyltransferase (α4Gal-T) and the core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG2b and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG2b. In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG2b and a P(k)-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG2b and P(k)-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon α4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG2b bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.</abstract><cop>England</cop><pmid>24082034</pmid><doi>10.1093/glycob/cwt086</doi><tpages>13</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals blood group P1 CHO Cells Clinical Medicine COLONIZATION Columbidae Cricetinae Cricetulus CRYSTAL-STRUCTURE Globosides - chemistry Globosides - genetics Globosides - metabolism GROUP-A HELICOBACTER-PYLORI HEMOLYTIC-UREMIC SYNDROME Humans Immunoglobulin G - chemistry Immunoglobulin G - genetics Immunoglobulin G - metabolism Klinisk medicin MAJOR GLYCOPROTEINS mass spectrometry Medicin och hälsovetenskap Membrane Glycoproteins - chemistry Membrane Glycoproteins - genetics Membrane Glycoproteins - metabolism Mice mucin N-Acetylglucosaminyltransferases - chemistry N-Acetylglucosaminyltransferases - genetics N-Acetylglucosaminyltransferases - metabolism N-GLYCAN STRUCTURES PIGEON EGG-WHITE Polysaccharides - chemistry Polysaccharides - genetics Polysaccharides - metabolism Protein Binding Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Shiga Toxin 1 - chemistry Shiga Toxin 1 - genetics Shiga Toxin 1 - metabolism Shiga Toxin 2 - chemistry Shiga Toxin 2 - genetics Shiga Toxin 2 - metabolism Shiga-like toxin Shiga-Toxigenic Escherichia coli - chemistry Shiga-Toxigenic Escherichia coli - genetics Shiga-Toxigenic Escherichia coli - metabolism SHIGELLA-DYSENTERIAE SPR STREPTOCOCCUS-SUIS UROPATHOGENIC ESCHERICHIA-COLI |
| title | Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins |
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