Determination of Carbohydrate Structures N-Linked to Soluble CD154 and Characterization of the Interactions of CD40 with CD154 Expressed in Pichia pastoris and Chinese Hamster Ovary Cells
CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohy...
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| Veröffentlicht in: | Protein expression and purification 2001-11, Vol.23 (2), p.301-310 |
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| creator | Khandekar, Sanjay S. Silverman, Carol Wells-Marani, Jennifer Bacon, Alicia M. Birrell, Helen Brigham-Burke, Michael DeMarini, Douglas J. Jonak, Zdenka L. Camilleri, Patrick Fishman-Lobell, Jacqueline |
| description | CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10–30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions. |
| doi_str_mv | 10.1006/prep.2001.1501 |
| format | Article |
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Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10–30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. 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Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10–30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.</description><subject>Animals</subject><subject>Asparagine - chemistry</subject><subject>B-Lymphocytes - immunology</subject><subject>Carbohydrate Conformation</subject><subject>Carbohydrate Metabolism</subject><subject>Carbohydrates - chemistry</subject><subject>CD40 Antigens - chemistry</subject><subject>CD40 Antigens - genetics</subject><subject>CD40 Antigens - immunology</subject><subject>CD40 Antigens - metabolism</subject><subject>CD40 Ligand - chemistry</subject><subject>CD40 Ligand - genetics</subject><subject>CD40 Ligand - immunology</subject><subject>CD40 Ligand - metabolism</subject><subject>Cells, Cultured</subject><subject>CHO Cells</subject><subject>Cloning, Molecular</subject><subject>Cricetinae</subject><subject>Humans</subject><subject>Mannose - chemistry</subject><subject>Mannose - metabolism</subject><subject>Pichia - genetics</subject><subject>Protein Binding</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Solubility</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU1v1DAQhi0Eoh9w5Yh84pbF9iZOfETZQiutKFLhbDn2RDEkdrCdQvlr_DmcbgQnTmONn3k0oxehV5TsKCH87Rxg3jFC6I5WhD5B55QIXhBWi6fru-RFJVhzhi5i_Jopykn1HJ3lWnNO-Dn6fYAEYbJOJesd9j1uVej88GCCSoDvUlh0WgJE_LE4WvcNDE4e3_lx6UbA7YFWJVbO4HZQQemssr_-mtIA-MblXv7IrfhoP5QE_7Bp2GavfuYLYsxa6_Anqwer8Kxi8sHGTWwdRMDXaopZhW_vVXjALYxjfIGe9WqM8HKrl-jL-6vP7XVxvP1w0747FnpfklSUujGVEbwTPVNG7RmrAUrS8wo6xkmp6rrpKyFMww01Yl8KSjuTC6Fd32vYX6I3J-8c_PcFYpKTjTpvoBz4JcqaMcZryjO4O4E6-BgD9HIOdsr7SkrkGpdc45JrXHKNKw-83sxLN4H5h2_5ZKA5AZDvu7cQZNQWnAZjA-gkjbf_c_8BQ-ml0w</recordid><startdate>20011101</startdate><enddate>20011101</enddate><creator>Khandekar, Sanjay S.</creator><creator>Silverman, Carol</creator><creator>Wells-Marani, Jennifer</creator><creator>Bacon, Alicia M.</creator><creator>Birrell, Helen</creator><creator>Brigham-Burke, Michael</creator><creator>DeMarini, Douglas J.</creator><creator>Jonak, Zdenka L.</creator><creator>Camilleri, Patrick</creator><creator>Fishman-Lobell, Jacqueline</creator><general>Elsevier Inc</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>7X8</scope></search><sort><creationdate>20011101</creationdate><title>Determination of Carbohydrate Structures N-Linked to Soluble CD154 and Characterization of the Interactions of CD40 with CD154 Expressed in Pichia pastoris and Chinese Hamster Ovary Cells</title><author>Khandekar, Sanjay S. ; Silverman, Carol ; Wells-Marani, Jennifer ; Bacon, Alicia M. ; Birrell, Helen ; Brigham-Burke, Michael ; DeMarini, Douglas J. ; Jonak, Zdenka L. ; Camilleri, Patrick ; Fishman-Lobell, Jacqueline</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c340t-4c8d5d96b9f2ada3227ee40f65eb2604a778f599d86d1d934911bd34901bffce3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Animals</topic><topic>Asparagine - chemistry</topic><topic>B-Lymphocytes - immunology</topic><topic>Carbohydrate Conformation</topic><topic>Carbohydrate Metabolism</topic><topic>Carbohydrates - chemistry</topic><topic>CD40 Antigens - chemistry</topic><topic>CD40 Antigens - genetics</topic><topic>CD40 Antigens - immunology</topic><topic>CD40 Antigens - metabolism</topic><topic>CD40 Ligand - chemistry</topic><topic>CD40 Ligand - genetics</topic><topic>CD40 Ligand - immunology</topic><topic>CD40 Ligand - metabolism</topic><topic>Cells, Cultured</topic><topic>CHO Cells</topic><topic>Cloning, Molecular</topic><topic>Cricetinae</topic><topic>Humans</topic><topic>Mannose - chemistry</topic><topic>Mannose - metabolism</topic><topic>Pichia - genetics</topic><topic>Protein Binding</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Solubility</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Khandekar, Sanjay S.</creatorcontrib><creatorcontrib>Silverman, Carol</creatorcontrib><creatorcontrib>Wells-Marani, Jennifer</creatorcontrib><creatorcontrib>Bacon, Alicia M.</creatorcontrib><creatorcontrib>Birrell, Helen</creatorcontrib><creatorcontrib>Brigham-Burke, Michael</creatorcontrib><creatorcontrib>DeMarini, Douglas J.</creatorcontrib><creatorcontrib>Jonak, Zdenka L.</creatorcontrib><creatorcontrib>Camilleri, Patrick</creatorcontrib><creatorcontrib>Fishman-Lobell, Jacqueline</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>Protein expression and purification</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Khandekar, Sanjay S.</au><au>Silverman, Carol</au><au>Wells-Marani, Jennifer</au><au>Bacon, Alicia M.</au><au>Birrell, Helen</au><au>Brigham-Burke, Michael</au><au>DeMarini, Douglas J.</au><au>Jonak, Zdenka L.</au><au>Camilleri, Patrick</au><au>Fishman-Lobell, Jacqueline</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Determination of Carbohydrate Structures N-Linked to Soluble CD154 and Characterization of the Interactions of CD40 with CD154 Expressed in Pichia pastoris and Chinese Hamster Ovary Cells</atitle><jtitle>Protein expression and purification</jtitle><addtitle>Protein Expr Purif</addtitle><date>2001-11-01</date><risdate>2001</risdate><volume>23</volume><issue>2</issue><spage>301</spage><epage>310</epage><pages>301-310</pages><issn>1046-5928</issn><eissn>1096-0279</eissn><abstract>CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10–30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>11676606</pmid><doi>10.1006/prep.2001.1501</doi><tpages>10</tpages></addata></record> |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Animals Asparagine - chemistry B-Lymphocytes - immunology Carbohydrate Conformation Carbohydrate Metabolism Carbohydrates - chemistry CD40 Antigens - chemistry CD40 Antigens - genetics CD40 Antigens - immunology CD40 Antigens - metabolism CD40 Ligand - chemistry CD40 Ligand - genetics CD40 Ligand - immunology CD40 Ligand - metabolism Cells, Cultured CHO Cells Cloning, Molecular Cricetinae Humans Mannose - chemistry Mannose - metabolism Pichia - genetics Protein Binding Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Solubility |
| title | Determination of Carbohydrate Structures N-Linked to Soluble CD154 and Characterization of the Interactions of CD40 with CD154 Expressed in Pichia pastoris and Chinese Hamster Ovary Cells |
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