Membrane translocation of diphtheria toxin A-fragment: role of carboxy-terminal region
The C-terminal end of diphtheria toxin A-fragment was altered and the consequences for toxicity and translocation of the A-fragment to the cytosol were studied. Mutations and deletions in the protease-sensitive, disulfide-bridged region linking the two functional parts of the toxin, the A- and B-fra...
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| Veröffentlicht in: | Biochemistry (Easton) 1993-01, Vol.32 (1), p.83-90 |
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| container_title | Biochemistry (Easton) |
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| creator | Ariansen, Sarah Afanasiev, Boris N Moskaug, Jan Oeivind Stenmark, Harald Madshus, Inger Helene Olsnes, Sjur |
| description | The C-terminal end of diphtheria toxin A-fragment was altered and the consequences for toxicity and translocation of the A-fragment to the cytosol were studied. Mutations and deletions in the protease-sensitive, disulfide-bridged region linking the two functional parts of the toxin, the A- and B-fragments, reduced the toxicity of the protein as such, but when the mutant toxins were cleaved ("nicked") by trypsin before being added to cells, the toxicity was restored. Prevention of disulfide formation by removal of Cys186 resulted in complete loss of toxicity. To circumvent the nicking step, toxin was formed by reconstitution from separate A- and B-fragments where the A-fragments varied in the C-terminal sequences. The amino acids C-terminal to Cys186 were found not to be required for translocation. Furthermore, both charged and uncharged residues near the C-terminal end were compatible with translocation. The data indicate that the C-terminal amino acid sequence is not decisive for translocation of diphtheria toxin A-fragment to the cytosol. |
| doi_str_mv | 10.1021/bi00052a012 |
| format | Article |
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Mutations and deletions in the protease-sensitive, disulfide-bridged region linking the two functional parts of the toxin, the A- and B-fragments, reduced the toxicity of the protein as such, but when the mutant toxins were cleaved ("nicked") by trypsin before being added to cells, the toxicity was restored. Prevention of disulfide formation by removal of Cys186 resulted in complete loss of toxicity. To circumvent the nicking step, toxin was formed by reconstitution from separate A- and B-fragments where the A-fragments varied in the C-terminal sequences. The amino acids C-terminal to Cys186 were found not to be required for translocation. Furthermore, both charged and uncharged residues near the C-terminal end were compatible with translocation. The data indicate that the C-terminal amino acid sequence is not decisive for translocation of diphtheria toxin A-fragment to the cytosol.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi00052a012</identifier><identifier>PMID: 8418864</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>Amino Acid Sequence ; Animals ; Base Sequence ; Biological and medical sciences ; Biological Transport ; Cell Membrane - metabolism ; Cell physiology ; Corynebacterium diphtheriae ; Cytosol - metabolism ; Diphtheria Toxin - chemistry ; Diphtheria Toxin - genetics ; Diphtheria Toxin - metabolism ; Electrochemistry ; Escherichia coli - genetics ; Fundamental and applied biological sciences. Psychology ; Hydrogen-Ion Concentration ; Membrane and intracellular transports ; Molecular and cellular biology ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Peptide Fragments - chemistry ; Peptide Fragments - genetics ; Peptide Fragments - metabolism ; Plasmids ; Structure-Activity Relationship ; Trypsin - metabolism ; Vero Cells</subject><ispartof>Biochemistry (Easton), 1993-01, Vol.32 (1), p.83-90</ispartof><rights>1993 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a414t-c5752409417e95eb60806108c3c14f6e0118b555b91c9739569c10c32b8e58ee3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi00052a012$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi00052a012$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,776,780,2752,27053,27901,27902,56713,56763</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4837138$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8418864$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ariansen, Sarah</creatorcontrib><creatorcontrib>Afanasiev, Boris N</creatorcontrib><creatorcontrib>Moskaug, Jan Oeivind</creatorcontrib><creatorcontrib>Stenmark, Harald</creatorcontrib><creatorcontrib>Madshus, Inger Helene</creatorcontrib><creatorcontrib>Olsnes, Sjur</creatorcontrib><title>Membrane translocation of diphtheria toxin A-fragment: role of carboxy-terminal region</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>The C-terminal end of diphtheria toxin A-fragment was altered and the consequences for toxicity and translocation of the A-fragment to the cytosol were studied. Mutations and deletions in the protease-sensitive, disulfide-bridged region linking the two functional parts of the toxin, the A- and B-fragments, reduced the toxicity of the protein as such, but when the mutant toxins were cleaved ("nicked") by trypsin before being added to cells, the toxicity was restored. Prevention of disulfide formation by removal of Cys186 resulted in complete loss of toxicity. To circumvent the nicking step, toxin was formed by reconstitution from separate A- and B-fragments where the A-fragments varied in the C-terminal sequences. The amino acids C-terminal to Cys186 were found not to be required for translocation. Furthermore, both charged and uncharged residues near the C-terminal end were compatible with translocation. The data indicate that the C-terminal amino acid sequence is not decisive for translocation of diphtheria toxin A-fragment to the cytosol.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Biological Transport</subject><subject>Cell Membrane - metabolism</subject><subject>Cell physiology</subject><subject>Corynebacterium diphtheriae</subject><subject>Cytosol - metabolism</subject><subject>Diphtheria Toxin - chemistry</subject><subject>Diphtheria Toxin - genetics</subject><subject>Diphtheria Toxin - metabolism</subject><subject>Electrochemistry</subject><subject>Escherichia coli - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hydrogen-Ion Concentration</subject><subject>Membrane and intracellular transports</subject><subject>Molecular and cellular biology</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Peptide Fragments - chemistry</subject><subject>Peptide Fragments - genetics</subject><subject>Peptide Fragments - metabolism</subject><subject>Plasmids</subject><subject>Structure-Activity Relationship</subject><subject>Trypsin - metabolism</subject><subject>Vero Cells</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkMFvFCEUh4mxqWv15NlkDkYPZvS9GWDAW9Nqq6la09orYeibljozbIFNtv-9NLvZePACgd_HL4-PsVcIHxAa_Nh7ABCNBWyesAWKBmqutXjKFuVe1o2W8Iw9T-muHDl0fJ_tK45KSb5gV99p6qOdqcplTWNwNvswV2Gorv3yNt9S9LbKYe3n6rAeor2ZaM6fqhhGeoScjX1YP9SZ4uRnO1aRbsr7F2xvsGOil9v9gP3-8vny6LQ--3ny9ejwrLYcea6d6ETDQXPsSAvqJSiQCMq1DvkgCRBVL4ToNTrdtVpI7RBc2_SKhCJqD9jbTe8yhvsVpWwmnxyNY_lRWCWDUiCU0gK-34AuhpQiDWYZ_WTjg0EwjxbNPxYL_Xpbu-onut6xW20lf7PNbXJ2LFpm59MO46rtsFUFqzeYT5nWu9jGP0Z2bSfM5fmFgV8XV99Ojn-Y88K_2_DWJXMXVrEITf8d8C8XlZP2</recordid><startdate>19930112</startdate><enddate>19930112</enddate><creator>Ariansen, Sarah</creator><creator>Afanasiev, Boris N</creator><creator>Moskaug, Jan Oeivind</creator><creator>Stenmark, Harald</creator><creator>Madshus, Inger Helene</creator><creator>Olsnes, Sjur</creator><general>American Chemical Society</general><scope>BSCLL</scope><scope>IQODW</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>7QL</scope><scope>7T7</scope><scope>7U7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>19930112</creationdate><title>Membrane translocation of diphtheria toxin A-fragment: role of carboxy-terminal region</title><author>Ariansen, Sarah ; Afanasiev, Boris N ; Moskaug, Jan Oeivind ; Stenmark, Harald ; Madshus, Inger Helene ; Olsnes, Sjur</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a414t-c5752409417e95eb60806108c3c14f6e0118b555b91c9739569c10c32b8e58ee3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Biological Transport</topic><topic>Cell Membrane - metabolism</topic><topic>Cell physiology</topic><topic>Corynebacterium diphtheriae</topic><topic>Cytosol - metabolism</topic><topic>Diphtheria Toxin - chemistry</topic><topic>Diphtheria Toxin - genetics</topic><topic>Diphtheria Toxin - metabolism</topic><topic>Electrochemistry</topic><topic>Escherichia coli - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hydrogen-Ion Concentration</topic><topic>Membrane and intracellular transports</topic><topic>Molecular and cellular biology</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Peptide Fragments - chemistry</topic><topic>Peptide Fragments - genetics</topic><topic>Peptide Fragments - metabolism</topic><topic>Plasmids</topic><topic>Structure-Activity Relationship</topic><topic>Trypsin - metabolism</topic><topic>Vero Cells</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ariansen, Sarah</creatorcontrib><creatorcontrib>Afanasiev, Boris N</creatorcontrib><creatorcontrib>Moskaug, Jan Oeivind</creatorcontrib><creatorcontrib>Stenmark, Harald</creatorcontrib><creatorcontrib>Madshus, Inger Helene</creatorcontrib><creatorcontrib>Olsnes, Sjur</creatorcontrib><collection>Istex</collection><collection>Pascal-Francis</collection><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>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Toxicology Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ariansen, Sarah</au><au>Afanasiev, Boris N</au><au>Moskaug, Jan Oeivind</au><au>Stenmark, Harald</au><au>Madshus, Inger Helene</au><au>Olsnes, Sjur</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Membrane translocation of diphtheria toxin A-fragment: role of carboxy-terminal region</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1993-01-12</date><risdate>1993</risdate><volume>32</volume><issue>1</issue><spage>83</spage><epage>90</epage><pages>83-90</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>The C-terminal end of diphtheria toxin A-fragment was altered and the consequences for toxicity and translocation of the A-fragment to the cytosol were studied. Mutations and deletions in the protease-sensitive, disulfide-bridged region linking the two functional parts of the toxin, the A- and B-fragments, reduced the toxicity of the protein as such, but when the mutant toxins were cleaved ("nicked") by trypsin before being added to cells, the toxicity was restored. Prevention of disulfide formation by removal of Cys186 resulted in complete loss of toxicity. To circumvent the nicking step, toxin was formed by reconstitution from separate A- and B-fragments where the A-fragments varied in the C-terminal sequences. The amino acids C-terminal to Cys186 were found not to be required for translocation. Furthermore, both charged and uncharged residues near the C-terminal end were compatible with translocation. The data indicate that the C-terminal amino acid sequence is not decisive for translocation of diphtheria toxin A-fragment to the cytosol.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>8418864</pmid><doi>10.1021/bi00052a012</doi><tpages>8</tpages></addata></record> |
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| ispartof | Biochemistry (Easton), 1993-01, Vol.32 (1), p.83-90 |
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| language | eng |
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| source | ACS Publications; MEDLINE |
| subjects | Amino Acid Sequence Animals Base Sequence Biological and medical sciences Biological Transport Cell Membrane - metabolism Cell physiology Corynebacterium diphtheriae Cytosol - metabolism Diphtheria Toxin - chemistry Diphtheria Toxin - genetics Diphtheria Toxin - metabolism Electrochemistry Escherichia coli - genetics Fundamental and applied biological sciences. Psychology Hydrogen-Ion Concentration Membrane and intracellular transports Molecular and cellular biology Molecular Sequence Data Mutagenesis, Site-Directed Peptide Fragments - chemistry Peptide Fragments - genetics Peptide Fragments - metabolism Plasmids Structure-Activity Relationship Trypsin - metabolism Vero Cells |
| title | Membrane translocation of diphtheria toxin A-fragment: role of carboxy-terminal region |
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