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Functional analysis of recombinant calreticulin fragment 39-272: implications for immunobiological activities of calreticulin in health and disease
Although calreticulin (CRT) is a major Ca(2+)-binding luminal resident protein, it can also appear on the surface of various types of cells and it functions as an immunopotentiating molecule. However, molecular mechanisms underlying the potent immunobiological activity of cell surface CRT are still...
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Published in: | The Journal of immunology (1950) 2010-10, Vol.185 (8), p.4561-4569 |
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container_title | The Journal of immunology (1950) |
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creator | Hong, Chao Qiu, Xiang Li, Yue Huang, Qianrong Zhong, Zhaoyan Zhang, Yan Liu, Xiangyuan Sun, Lin Lv, Ping Gao, Xiao-Ming |
description | Although calreticulin (CRT) is a major Ca(2+)-binding luminal resident protein, it can also appear on the surface of various types of cells and it functions as an immunopotentiating molecule. However, molecular mechanisms underlying the potent immunobiological activity of cell surface CRT are still unclear. In the present study, a recombinant fragment (rCRT/39-272) covering the lectin-like N domain and partial P domain of murine CRT has been expressed in Escherichia coli. The affinity-purified rCRT/39-272 assembles into homodimers and oligomers in solution and exhibits high binding affinity to various glycans, including carrageenan, alginic acids, and hyaluronic acids. Functionally, rCRT/39-272 is capable of driving the activation and maturation of B cells and cytokine production by macrophages in a TLR-4-dependent manner in vitro. It specifically binds recombinant mouse CD14, but not BAFFR and CD40. It is also able to trigger Ig class switching by B cells in the absence of T cell help both in vitro and in vivo. Furthermore, this fragment of CRT exhibits strong adjuvanticity when conjugated to polysaccharides or expressed as part of a fusion protein. Soluble CRT can be detected in the sera of patients with rheumatoid arthritis or systemic lupus erythematosus, but not in healthy subjects. We argue that CRT, either on the membrane surface of cells or in soluble form, is a potent stimulatory molecule to B cells and macrophages via the TLR-4/CD14 pathway and plays important roles in the pathogenesis of autoimmune diseases. |
doi_str_mv | 10.4049/jimmunol.1000536 |
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However, molecular mechanisms underlying the potent immunobiological activity of cell surface CRT are still unclear. In the present study, a recombinant fragment (rCRT/39-272) covering the lectin-like N domain and partial P domain of murine CRT has been expressed in Escherichia coli. The affinity-purified rCRT/39-272 assembles into homodimers and oligomers in solution and exhibits high binding affinity to various glycans, including carrageenan, alginic acids, and hyaluronic acids. Functionally, rCRT/39-272 is capable of driving the activation and maturation of B cells and cytokine production by macrophages in a TLR-4-dependent manner in vitro. It specifically binds recombinant mouse CD14, but not BAFFR and CD40. It is also able to trigger Ig class switching by B cells in the absence of T cell help both in vitro and in vivo. Furthermore, this fragment of CRT exhibits strong adjuvanticity when conjugated to polysaccharides or expressed as part of a fusion protein. Soluble CRT can be detected in the sera of patients with rheumatoid arthritis or systemic lupus erythematosus, but not in healthy subjects. We argue that CRT, either on the membrane surface of cells or in soluble form, is a potent stimulatory molecule to B cells and macrophages via the TLR-4/CD14 pathway and plays important roles in the pathogenesis of autoimmune diseases.</description><identifier>ISSN: 0022-1767</identifier><identifier>EISSN: 1550-6606</identifier><identifier>DOI: 10.4049/jimmunol.1000536</identifier><identifier>PMID: 20855873</identifier><language>eng</language><publisher>United States</publisher><subject>Adolescent ; Adult ; Aged ; Aged, 80 and over ; Amino Acid Sequence ; Animals ; Arthritis, Rheumatoid - immunology ; Autoimmunity - immunology ; B-Lymphocytes - immunology ; B-Lymphocytes - metabolism ; Blotting, Western ; Calreticulin - chemistry ; Calreticulin - immunology ; Calreticulin - metabolism ; Cell Separation ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Female ; Flow Cytometry ; Humans ; Lipopolysaccharide Receptors - immunology ; Lipopolysaccharide Receptors - metabolism ; Lupus Erythematosus, Systemic - immunology ; Lymphocyte Activation - immunology ; Macrophage Activation - immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Nude ; Middle Aged ; Molecular Sequence Data ; Polymerase Chain Reaction ; Recombinant Proteins - chemistry ; Recombinant Proteins - immunology ; Recombinant Proteins - metabolism ; Signal Transduction - immunology ; Toll-Like Receptor 4 - immunology ; Toll-Like Receptor 4 - metabolism ; Young Adult</subject><ispartof>The Journal of immunology (1950), 2010-10, Vol.185 (8), p.4561-4569</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c438t-5ab09efc45843084b06528ae5867de55cfca5b392a4e682efb0d024d5ce58de33</citedby><cites>FETCH-LOGICAL-c438t-5ab09efc45843084b06528ae5867de55cfca5b392a4e682efb0d024d5ce58de33</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20855873$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hong, Chao</creatorcontrib><creatorcontrib>Qiu, Xiang</creatorcontrib><creatorcontrib>Li, Yue</creatorcontrib><creatorcontrib>Huang, Qianrong</creatorcontrib><creatorcontrib>Zhong, Zhaoyan</creatorcontrib><creatorcontrib>Zhang, Yan</creatorcontrib><creatorcontrib>Liu, Xiangyuan</creatorcontrib><creatorcontrib>Sun, Lin</creatorcontrib><creatorcontrib>Lv, Ping</creatorcontrib><creatorcontrib>Gao, Xiao-Ming</creatorcontrib><title>Functional analysis of recombinant calreticulin fragment 39-272: implications for immunobiological activities of calreticulin in health and disease</title><title>The Journal of immunology (1950)</title><addtitle>J Immunol</addtitle><description>Although calreticulin (CRT) is a major Ca(2+)-binding luminal resident protein, it can also appear on the surface of various types of cells and it functions as an immunopotentiating molecule. However, molecular mechanisms underlying the potent immunobiological activity of cell surface CRT are still unclear. In the present study, a recombinant fragment (rCRT/39-272) covering the lectin-like N domain and partial P domain of murine CRT has been expressed in Escherichia coli. The affinity-purified rCRT/39-272 assembles into homodimers and oligomers in solution and exhibits high binding affinity to various glycans, including carrageenan, alginic acids, and hyaluronic acids. Functionally, rCRT/39-272 is capable of driving the activation and maturation of B cells and cytokine production by macrophages in a TLR-4-dependent manner in vitro. It specifically binds recombinant mouse CD14, but not BAFFR and CD40. It is also able to trigger Ig class switching by B cells in the absence of T cell help both in vitro and in vivo. Furthermore, this fragment of CRT exhibits strong adjuvanticity when conjugated to polysaccharides or expressed as part of a fusion protein. Soluble CRT can be detected in the sera of patients with rheumatoid arthritis or systemic lupus erythematosus, but not in healthy subjects. We argue that CRT, either on the membrane surface of cells or in soluble form, is a potent stimulatory molecule to B cells and macrophages via the TLR-4/CD14 pathway and plays important roles in the pathogenesis of autoimmune diseases.</description><subject>Adolescent</subject><subject>Adult</subject><subject>Aged</subject><subject>Aged, 80 and over</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Arthritis, Rheumatoid - immunology</subject><subject>Autoimmunity - immunology</subject><subject>B-Lymphocytes - immunology</subject><subject>B-Lymphocytes - metabolism</subject><subject>Blotting, Western</subject><subject>Calreticulin - chemistry</subject><subject>Calreticulin - immunology</subject><subject>Calreticulin - metabolism</subject><subject>Cell Separation</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>Escherichia coli</subject><subject>Female</subject><subject>Flow Cytometry</subject><subject>Humans</subject><subject>Lipopolysaccharide Receptors - immunology</subject><subject>Lipopolysaccharide Receptors - metabolism</subject><subject>Lupus Erythematosus, Systemic - immunology</subject><subject>Lymphocyte Activation - immunology</subject><subject>Macrophage Activation - immunology</subject><subject>Male</subject><subject>Mice</subject><subject>Mice, Inbred BALB C</subject><subject>Mice, Inbred C57BL</subject><subject>Mice, Nude</subject><subject>Middle Aged</subject><subject>Molecular Sequence Data</subject><subject>Polymerase Chain Reaction</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - immunology</subject><subject>Recombinant Proteins - metabolism</subject><subject>Signal Transduction - immunology</subject><subject>Toll-Like Receptor 4 - immunology</subject><subject>Toll-Like Receptor 4 - metabolism</subject><subject>Young Adult</subject><issn>0022-1767</issn><issn>1550-6606</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><recordid>eNqFkU9LJDEQxYO46Kx69yS5eWq3Ov_Hm4juLgh7Wc9NOl09E0l3xqRb8HP4hY3OKHgSihRUXv1ewiPktIYLAWL568EPwzzGcFEDgORqjyxqKaFSCtQ-WQAwVtVa6UPyM-eHolHAxAE5ZGCkNJovyMvtPLrJx9EGasvxnH2msacJXRxaP9pxos6GhJN3c_Aj7ZNdDVimfFkxzS6pHzbBO_vGyLSPiW7f1PoY4qpcFG4xePKTx3fyF1qpNdowrYt5Rzuf0WY8Jj96GzKe7PoRub-9-X_9p7r79_vv9dVd5QQ3UyVtC0vsnZBGcDCiBSWZsSiN0h1K6XpnZcuXzApUhmHfQle-30lXJB1yfkTOt9xNio8z5qkZfHYYgh0xzrkxUmnNhKq_VWpZDLjSqihhq3Qp5pywbzbJDzY9NzU0b5k1H5k1u8zKytkOPrcDdp8LHyHxV9K2l7M</recordid><startdate>20101015</startdate><enddate>20101015</enddate><creator>Hong, Chao</creator><creator>Qiu, Xiang</creator><creator>Li, Yue</creator><creator>Huang, Qianrong</creator><creator>Zhong, Zhaoyan</creator><creator>Zhang, Yan</creator><creator>Liu, Xiangyuan</creator><creator>Sun, Lin</creator><creator>Lv, Ping</creator><creator>Gao, Xiao-Ming</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>7T5</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>20101015</creationdate><title>Functional analysis of recombinant calreticulin fragment 39-272: implications for immunobiological activities of calreticulin in health and disease</title><author>Hong, Chao ; Qiu, Xiang ; Li, Yue ; Huang, Qianrong ; Zhong, Zhaoyan ; Zhang, Yan ; Liu, Xiangyuan ; Sun, Lin ; Lv, Ping ; Gao, Xiao-Ming</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c438t-5ab09efc45843084b06528ae5867de55cfca5b392a4e682efb0d024d5ce58de33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Adolescent</topic><topic>Adult</topic><topic>Aged</topic><topic>Aged, 80 and over</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Arthritis, Rheumatoid - immunology</topic><topic>Autoimmunity - immunology</topic><topic>B-Lymphocytes - immunology</topic><topic>B-Lymphocytes - metabolism</topic><topic>Blotting, Western</topic><topic>Calreticulin - chemistry</topic><topic>Calreticulin - immunology</topic><topic>Calreticulin - metabolism</topic><topic>Cell Separation</topic><topic>Enzyme-Linked Immunosorbent Assay</topic><topic>Escherichia coli</topic><topic>Female</topic><topic>Flow Cytometry</topic><topic>Humans</topic><topic>Lipopolysaccharide Receptors - immunology</topic><topic>Lipopolysaccharide Receptors - metabolism</topic><topic>Lupus Erythematosus, Systemic - immunology</topic><topic>Lymphocyte Activation - immunology</topic><topic>Macrophage Activation - immunology</topic><topic>Male</topic><topic>Mice</topic><topic>Mice, Inbred BALB C</topic><topic>Mice, Inbred C57BL</topic><topic>Mice, Nude</topic><topic>Middle Aged</topic><topic>Molecular Sequence Data</topic><topic>Polymerase Chain Reaction</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - immunology</topic><topic>Recombinant Proteins - metabolism</topic><topic>Signal Transduction - immunology</topic><topic>Toll-Like Receptor 4 - immunology</topic><topic>Toll-Like Receptor 4 - metabolism</topic><topic>Young Adult</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hong, Chao</creatorcontrib><creatorcontrib>Qiu, Xiang</creatorcontrib><creatorcontrib>Li, Yue</creatorcontrib><creatorcontrib>Huang, Qianrong</creatorcontrib><creatorcontrib>Zhong, Zhaoyan</creatorcontrib><creatorcontrib>Zhang, Yan</creatorcontrib><creatorcontrib>Liu, Xiangyuan</creatorcontrib><creatorcontrib>Sun, Lin</creatorcontrib><creatorcontrib>Lv, Ping</creatorcontrib><creatorcontrib>Gao, Xiao-Ming</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>Immunology Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>The Journal of immunology (1950)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hong, Chao</au><au>Qiu, Xiang</au><au>Li, Yue</au><au>Huang, Qianrong</au><au>Zhong, Zhaoyan</au><au>Zhang, Yan</au><au>Liu, Xiangyuan</au><au>Sun, Lin</au><au>Lv, Ping</au><au>Gao, Xiao-Ming</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Functional analysis of recombinant calreticulin fragment 39-272: implications for immunobiological activities of calreticulin in health and disease</atitle><jtitle>The Journal of immunology (1950)</jtitle><addtitle>J Immunol</addtitle><date>2010-10-15</date><risdate>2010</risdate><volume>185</volume><issue>8</issue><spage>4561</spage><epage>4569</epage><pages>4561-4569</pages><issn>0022-1767</issn><eissn>1550-6606</eissn><abstract>Although calreticulin (CRT) is a major Ca(2+)-binding luminal resident protein, it can also appear on the surface of various types of cells and it functions as an immunopotentiating molecule. However, molecular mechanisms underlying the potent immunobiological activity of cell surface CRT are still unclear. In the present study, a recombinant fragment (rCRT/39-272) covering the lectin-like N domain and partial P domain of murine CRT has been expressed in Escherichia coli. The affinity-purified rCRT/39-272 assembles into homodimers and oligomers in solution and exhibits high binding affinity to various glycans, including carrageenan, alginic acids, and hyaluronic acids. Functionally, rCRT/39-272 is capable of driving the activation and maturation of B cells and cytokine production by macrophages in a TLR-4-dependent manner in vitro. It specifically binds recombinant mouse CD14, but not BAFFR and CD40. It is also able to trigger Ig class switching by B cells in the absence of T cell help both in vitro and in vivo. Furthermore, this fragment of CRT exhibits strong adjuvanticity when conjugated to polysaccharides or expressed as part of a fusion protein. Soluble CRT can be detected in the sera of patients with rheumatoid arthritis or systemic lupus erythematosus, but not in healthy subjects. We argue that CRT, either on the membrane surface of cells or in soluble form, is a potent stimulatory molecule to B cells and macrophages via the TLR-4/CD14 pathway and plays important roles in the pathogenesis of autoimmune diseases.</abstract><cop>United States</cop><pmid>20855873</pmid><doi>10.4049/jimmunol.1000536</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Adolescent Adult Aged Aged, 80 and over Amino Acid Sequence Animals Arthritis, Rheumatoid - immunology Autoimmunity - immunology B-Lymphocytes - immunology B-Lymphocytes - metabolism Blotting, Western Calreticulin - chemistry Calreticulin - immunology Calreticulin - metabolism Cell Separation Enzyme-Linked Immunosorbent Assay Escherichia coli Female Flow Cytometry Humans Lipopolysaccharide Receptors - immunology Lipopolysaccharide Receptors - metabolism Lupus Erythematosus, Systemic - immunology Lymphocyte Activation - immunology Macrophage Activation - immunology Male Mice Mice, Inbred BALB C Mice, Inbred C57BL Mice, Nude Middle Aged Molecular Sequence Data Polymerase Chain Reaction Recombinant Proteins - chemistry Recombinant Proteins - immunology Recombinant Proteins - metabolism Signal Transduction - immunology Toll-Like Receptor 4 - immunology Toll-Like Receptor 4 - metabolism Young Adult |
title | Functional analysis of recombinant calreticulin fragment 39-272: implications for immunobiological activities of calreticulin in health and disease |
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