Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis
Procarboxypeptidase U [proCPU, thrombin‐activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the...
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| Veröffentlicht in: | The FEBS journal 2006-02, Vol.273 (4), p.778-792 |
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| creator | Knecht, Wolfgang Willemse, Johan Stenhamre, Hanna Andersson, Mats Berntsson, Pia Furebring, Christina Harrysson, Anna Hager, Ann‐Christin Malmborg Wissing, Britt‐Marie Hendriks, Dirk Cronet, Philippe |
| description | Procarboxypeptidase U [proCPU, thrombin‐activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down‐regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2–4 mutations in human CPU can prolong the half‐life of CPU's activity at 37 °C from 0.2 h of wild‐type CPU to 0.5–5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis. |
| doi_str_mv | 10.1111/j.1742-4658.2006.05110.x |
| format | Article |
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ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down‐regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2–4 mutations in human CPU can prolong the half‐life of CPU's activity at 37 °C from 0.2 h of wild‐type CPU to 0.5–5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis.</description><identifier>ISSN: 1742-464X</identifier><identifier>EISSN: 1742-4658</identifier><identifier>DOI: 10.1111/j.1742-4658.2006.05110.x</identifier><identifier>PMID: 16441664</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Science Ltd</publisher><subject>Amino Acid Sequence ; Animals ; Biochemistry ; Blood Coagulation ; carboxypeptidase ; Carboxypeptidase B2 - chemistry ; Carboxypeptidase B2 - genetics ; Carboxypeptidase B2 - metabolism ; Cell Line ; coagulation ; directed evolution ; Down-Regulation ; Enzyme Activation ; Enzyme Stability ; Enzymes ; Fibrin - genetics ; Fibrin - metabolism ; Fibrinolysis ; Hot Temperature ; Humans ; Lysine - metabolism ; Molecular Sequence Data ; Mutagenesis ; Mutation ; Peptides ; Point Mutation ; protease ; Protein Denaturation ; Protein Precursors - chemistry ; Protein Precursors - genetics ; Protein Precursors - metabolism ; Sequence Alignment</subject><ispartof>The FEBS journal, 2006-02, Vol.273 (4), p.778-792</ispartof><rights>2006 The Authors Journal compilation 2006 FEBS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3950-dcbf50a40466ebd9e5eb56bcfcc3bee26b70c74cd1269eb7c1dfdf7486e6e7ec3</citedby><cites>FETCH-LOGICAL-c3950-dcbf50a40466ebd9e5eb56bcfcc3bee26b70c74cd1269eb7c1dfdf7486e6e7ec3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fj.1742-4658.2006.05110.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1742-4658.2006.05110.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>315,781,785,1418,1434,27929,27930,45579,45580,46414,46838</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16441664$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Knecht, Wolfgang</creatorcontrib><creatorcontrib>Willemse, Johan</creatorcontrib><creatorcontrib>Stenhamre, Hanna</creatorcontrib><creatorcontrib>Andersson, Mats</creatorcontrib><creatorcontrib>Berntsson, Pia</creatorcontrib><creatorcontrib>Furebring, Christina</creatorcontrib><creatorcontrib>Harrysson, Anna</creatorcontrib><creatorcontrib>Hager, Ann‐Christin Malmborg</creatorcontrib><creatorcontrib>Wissing, Britt‐Marie</creatorcontrib><creatorcontrib>Hendriks, Dirk</creatorcontrib><creatorcontrib>Cronet, Philippe</creatorcontrib><title>Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis</title><title>The FEBS journal</title><addtitle>FEBS J</addtitle><description>Procarboxypeptidase U [proCPU, thrombin‐activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. 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We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Biochemistry</subject><subject>Blood Coagulation</subject><subject>carboxypeptidase</subject><subject>Carboxypeptidase B2 - chemistry</subject><subject>Carboxypeptidase B2 - genetics</subject><subject>Carboxypeptidase B2 - metabolism</subject><subject>Cell Line</subject><subject>coagulation</subject><subject>directed evolution</subject><subject>Down-Regulation</subject><subject>Enzyme Activation</subject><subject>Enzyme Stability</subject><subject>Enzymes</subject><subject>Fibrin - genetics</subject><subject>Fibrin - metabolism</subject><subject>Fibrinolysis</subject><subject>Hot Temperature</subject><subject>Humans</subject><subject>Lysine - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis</subject><subject>Mutation</subject><subject>Peptides</subject><subject>Point Mutation</subject><subject>protease</subject><subject>Protein Denaturation</subject><subject>Protein Precursors - chemistry</subject><subject>Protein Precursors - genetics</subject><subject>Protein Precursors - metabolism</subject><subject>Sequence Alignment</subject><issn>1742-464X</issn><issn>1742-4658</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc9u1DAQxiNERUvhFZDFCQ67tRPH2VyQ2lUXKq1UJLoSN8t_JluvEjvYDt3ceAQeiKfhSXC6q8IRXzya-X3fjPRlGSJ4TtK72M1JRfMZZeVinmPM5rgkabZ_lp09DZ4_1fTrafYyhB3GRUnr-kV2ShilhDF6lv1am85E0KgbotiChWACMlZ5EAECiveAQhTStCaOyDXofuiERUp46fZjD300OoFog97dXa5uxHskrEYaOmdD9CIeLUzXOx-FVTB5LD9v_jX9Dh713k1d5RJiJ6FxNp2BtHuwv3_89LAd2tS0W9QY6Y117ZgOfZWdNKIN8Pr4n2eb1fXd8tNsffvxZnm5nqmiLvFMK9mUWFBMGQOpayhBlkyqRqlCAuRMVlhVVGmSsxpkpYhudFPRBQMGFajiPHt78E1nfhsgRL5zg7dpJc8xJbRMugQtDpDyLgQPDe-96YQfOcF8So3v-BQIn8LhU2r8MTW-T9I3R_9BdqD_Co8xJeDDAXgwLYz_bcxX11dfprL4Ax8QrXE</recordid><startdate>200602</startdate><enddate>200602</enddate><creator>Knecht, Wolfgang</creator><creator>Willemse, Johan</creator><creator>Stenhamre, Hanna</creator><creator>Andersson, Mats</creator><creator>Berntsson, Pia</creator><creator>Furebring, Christina</creator><creator>Harrysson, Anna</creator><creator>Hager, Ann‐Christin Malmborg</creator><creator>Wissing, Britt‐Marie</creator><creator>Hendriks, Dirk</creator><creator>Cronet, Philippe</creator><general>Blackwell Science Ltd</general><general>Blackwell Publishing Ltd</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>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7TM</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></search><sort><creationdate>200602</creationdate><title>Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis</title><author>Knecht, Wolfgang ; Willemse, Johan ; Stenhamre, Hanna ; Andersson, Mats ; Berntsson, Pia ; Furebring, Christina ; Harrysson, Anna ; Hager, Ann‐Christin Malmborg ; Wissing, Britt‐Marie ; Hendriks, Dirk ; Cronet, Philippe</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3950-dcbf50a40466ebd9e5eb56bcfcc3bee26b70c74cd1269eb7c1dfdf7486e6e7ec3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Biochemistry</topic><topic>Blood Coagulation</topic><topic>carboxypeptidase</topic><topic>Carboxypeptidase B2 - chemistry</topic><topic>Carboxypeptidase B2 - genetics</topic><topic>Carboxypeptidase B2 - metabolism</topic><topic>Cell Line</topic><topic>coagulation</topic><topic>directed evolution</topic><topic>Down-Regulation</topic><topic>Enzyme Activation</topic><topic>Enzyme Stability</topic><topic>Enzymes</topic><topic>Fibrin - genetics</topic><topic>Fibrin - metabolism</topic><topic>Fibrinolysis</topic><topic>Hot Temperature</topic><topic>Humans</topic><topic>Lysine - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis</topic><topic>Mutation</topic><topic>Peptides</topic><topic>Point Mutation</topic><topic>protease</topic><topic>Protein Denaturation</topic><topic>Protein Precursors - chemistry</topic><topic>Protein Precursors - genetics</topic><topic>Protein Precursors - metabolism</topic><topic>Sequence Alignment</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Knecht, Wolfgang</creatorcontrib><creatorcontrib>Willemse, Johan</creatorcontrib><creatorcontrib>Stenhamre, Hanna</creatorcontrib><creatorcontrib>Andersson, Mats</creatorcontrib><creatorcontrib>Berntsson, Pia</creatorcontrib><creatorcontrib>Furebring, Christina</creatorcontrib><creatorcontrib>Harrysson, Anna</creatorcontrib><creatorcontrib>Hager, Ann‐Christin Malmborg</creatorcontrib><creatorcontrib>Wissing, Britt‐Marie</creatorcontrib><creatorcontrib>Hendriks, Dirk</creatorcontrib><creatorcontrib>Cronet, Philippe</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids 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><jtitle>The FEBS journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Knecht, Wolfgang</au><au>Willemse, Johan</au><au>Stenhamre, Hanna</au><au>Andersson, Mats</au><au>Berntsson, Pia</au><au>Furebring, Christina</au><au>Harrysson, Anna</au><au>Hager, Ann‐Christin Malmborg</au><au>Wissing, Britt‐Marie</au><au>Hendriks, Dirk</au><au>Cronet, Philippe</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis</atitle><jtitle>The FEBS journal</jtitle><addtitle>FEBS J</addtitle><date>2006-02</date><risdate>2006</risdate><volume>273</volume><issue>4</issue><spage>778</spage><epage>792</epage><pages>778-792</pages><issn>1742-464X</issn><eissn>1742-4658</eissn><abstract>Procarboxypeptidase U [proCPU, thrombin‐activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down‐regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2–4 mutations in human CPU can prolong the half‐life of CPU's activity at 37 °C from 0.2 h of wild‐type CPU to 0.5–5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis.</abstract><cop>Oxford, UK</cop><pub>Blackwell Science Ltd</pub><pmid>16441664</pmid><doi>10.1111/j.1742-4658.2006.05110.x</doi><tpages>15</tpages></addata></record> |
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| subjects | Amino Acid Sequence Animals Biochemistry Blood Coagulation carboxypeptidase Carboxypeptidase B2 - chemistry Carboxypeptidase B2 - genetics Carboxypeptidase B2 - metabolism Cell Line coagulation directed evolution Down-Regulation Enzyme Activation Enzyme Stability Enzymes Fibrin - genetics Fibrin - metabolism Fibrinolysis Hot Temperature Humans Lysine - metabolism Molecular Sequence Data Mutagenesis Mutation Peptides Point Mutation protease Protein Denaturation Protein Precursors - chemistry Protein Precursors - genetics Protein Precursors - metabolism Sequence Alignment |
| title | Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down‐regulating fibrinolysis |
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