Activity after Site-Directed Mutagenesis of CD59 on Complement-Mediated Cytolysis
CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex （MAC）. However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is sti...
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| Veröffentlicht in: | Cellular & molecular immunology 2008-04, Vol.5 (2), p.141-146 |
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| creator | Zhu, Xinhong Gao, Meihua Ren, Shurong Wang, Qiubo Lin, Cunzhi |
| description | CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex （MAC）. However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site （Mutant 1, M1） or to change C39W40K41 to W39W40W41 （Mutant 2, M2）. Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CI-IO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly. Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors. Cellular ＆ Molecular Immunology. |
| doi_str_mv | 10.1038/cmi.2008.17 |
| format | Article |
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However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site （Mutant 1, M1） or to change C39W40K41 to W39W40W41 （Mutant 2, M2）. Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CI-IO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly. Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors. Cellular ＆ Molecular Immunology.</description><identifier>ISSN: 1672-7681</identifier><identifier>EISSN: 2042-0226</identifier><identifier>DOI: 10.1038/cmi.2008.17</identifier><identifier>PMID: 18445344</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>Animals ; Antibodies ; Biomedical and Life Sciences ; Biomedicine ; CD59 ; CD59 antigen ; CD59 Antigens - genetics ; CD59 Antigens - metabolism ; Cell membranes ; CHO Cells ; Complement system ; Complement System Proteins - immunology ; Cricetinae ; Cricetulus ; Cytolysis ; Cytolytic activity ; Cytotoxicity, Immunologic - genetics ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation ; Genetic Vectors ; Humans ; Hydrophobicity ; Immunology ; In Situ Hybridization ; Medical Microbiology ; Membrane attack complex ; Microbiology ; Mutagenesis ; Mutagenesis, Site-Directed ; Mutants ; RNA, Messenger - metabolism ; Site-directed mutagenesis ; Transfection ; Tumor cells ; Tumorigenesis ; Vaccine ; 基因突变 ; 细胞活性</subject><ispartof>Cellular & molecular immunology, 2008-04, Vol.5 (2), p.141-146</ispartof><rights>The Chinese Society of Immunology 2008</rights><rights>Copyright Nature Publishing Group Apr 2008</rights><rights>The Chinese Society of Immunology 2008.</rights><rights>Copyright © Wanfang Data Co. 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All Rights Reserved.</rights><rights>Copyright © 2008 The Chinese Society of Immunology 2008 The Chinese Society of Immunology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c591t-842f67a1973ae3dc1fe93745bb8bf78a34314c68be6663a0eff599bce8e6f6d83</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://image.cqvip.com/vip1000/qk/87787X/87787X.jpg</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651242/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651242/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,315,728,781,785,886,27928,27929,53795,53797</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18445344$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zhu, Xinhong</creatorcontrib><creatorcontrib>Gao, Meihua</creatorcontrib><creatorcontrib>Ren, Shurong</creatorcontrib><creatorcontrib>Wang, Qiubo</creatorcontrib><creatorcontrib>Lin, Cunzhi</creatorcontrib><title>Activity after Site-Directed Mutagenesis of CD59 on Complement-Mediated Cytolysis</title><title>Cellular & molecular immunology</title><addtitle>Cell Mol Immunol</addtitle><addtitle>Cellular ＆ Molecular Immunology</addtitle><description>CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex （MAC）. However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site （Mutant 1, M1） or to change C39W40K41 to W39W40W41 （Mutant 2, M2）. Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CI-IO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly. Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors. Cellular ＆ Molecular Immunology.</description><subject>Animals</subject><subject>Antibodies</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>CD59</subject><subject>CD59 antigen</subject><subject>CD59 Antigens - genetics</subject><subject>CD59 Antigens - metabolism</subject><subject>Cell membranes</subject><subject>CHO Cells</subject><subject>Complement system</subject><subject>Complement System Proteins - immunology</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>Cytolysis</subject><subject>Cytolytic activity</subject><subject>Cytotoxicity, Immunologic - genetics</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>Gene Expression Regulation</subject><subject>Genetic Vectors</subject><subject>Humans</subject><subject>Hydrophobicity</subject><subject>Immunology</subject><subject>In Situ Hybridization</subject><subject>Medical Microbiology</subject><subject>Membrane attack complex</subject><subject>Microbiology</subject><subject>Mutagenesis</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutants</subject><subject>RNA, Messenger - metabolism</subject><subject>Site-directed mutagenesis</subject><subject>Transfection</subject><subject>Tumor cells</subject><subject>Tumorigenesis</subject><subject>Vaccine</subject><subject>基因突变</subject><subject>细胞活性</subject><issn>1672-7681</issn><issn>2042-0226</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9ks1v1DAQxSMEokvhxB0iuCAgi-34KxekasuX1Aoh4Gw53nHqkjhbOylk_3ocdrUUhLjYh_n5zZt5zrKHGC0xKuUr07klQUgusbiVLQiipECE8NvZAnNBCsElPsruxXiJEJNU0LvZEZaUspLSRfbpxAzu2g1Tru0AIf_sBihOXQAzwDo_HwfdgIfoYt7bfHXKqrz3-arvNi104IfiHNZOz-hqGvp2SuD97I7VbYQH-_s4-_r2zZfV--Ls47sPq5OzwrAKD4WkxHKhcSVKDeXaYAtVKSira1lbIXVJS0wNlzVwzkuNwFpWVbUBCdzytSyPs9c73c1Yd7A2yU3QrdoE1-kwqV479WfFuwvV9NeKcoYJJUng-U7gu_ZW-0Zd9mPwybLaNt30Y7tVMK8VzUeCn-27hf5qhDiozkUDbas99GNUWFSoKhkTIqFP_0IPwkTwlBitGPofhYVkgnBKq0S92FEm9DEGsIf5MFJz-iqlr2aH6VWiH91cyW92H3cCXu6AmEq-gXCj6T_1Hu9wr4cxwEEvMTPyi3iy93fR--Yqaapam2_WtZCGJenDMVz-BKQ3zrc</recordid><startdate>20080401</startdate><enddate>20080401</enddate><creator>Zhu, Xinhong</creator><creator>Gao, Meihua</creator><creator>Ren, Shurong</creator><creator>Wang, Qiubo</creator><creator>Lin, Cunzhi</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><general>Department of Immunology, Medical College of Qingdao University,Qingdao, Shandong, China%Clinical Immune Institution, Qingdao Chest Hospital, 896 Chongqing Road,Qingdao 266043, China</general><scope>2RA</scope><scope>92L</scope><scope>CQIGP</scope><scope>W91</scope><scope>~WA</scope><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>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7T5</scope><scope>H94</scope><scope>2B.</scope><scope>4A8</scope><scope>92I</scope><scope>93N</scope><scope>PSX</scope><scope>TCJ</scope><scope>5PM</scope></search><sort><creationdate>20080401</creationdate><title>Activity after Site-Directed Mutagenesis of CD59 on Complement-Mediated Cytolysis</title><author>Zhu, Xinhong ; Gao, Meihua ; Ren, Shurong ; Wang, Qiubo ; Lin, Cunzhi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c591t-842f67a1973ae3dc1fe93745bb8bf78a34314c68be6663a0eff599bce8e6f6d83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Animals</topic><topic>Antibodies</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedicine</topic><topic>CD59</topic><topic>CD59 antigen</topic><topic>CD59 Antigens - 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However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site （Mutant 1, M1） or to change C39W40K41 to W39W40W41 （Mutant 2, M2）. Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CI-IO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly. Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors. Cellular ＆ Molecular Immunology.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>18445344</pmid><doi>10.1038/cmi.2008.17</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Antibodies Biomedical and Life Sciences Biomedicine CD59 CD59 antigen CD59 Antigens - genetics CD59 Antigens - metabolism Cell membranes CHO Cells Complement system Complement System Proteins - immunology Cricetinae Cricetulus Cytolysis Cytolytic activity Cytotoxicity, Immunologic - genetics Enzyme-Linked Immunosorbent Assay Gene Expression Regulation Genetic Vectors Humans Hydrophobicity Immunology In Situ Hybridization Medical Microbiology Membrane attack complex Microbiology Mutagenesis Mutagenesis, Site-Directed Mutants RNA, Messenger - metabolism Site-directed mutagenesis Transfection Tumor cells Tumorigenesis Vaccine 基因突变 细胞活性 |
| title | Activity after Site-Directed Mutagenesis of CD59 on Complement-Mediated Cytolysis |
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