Double-Lectin Site Ricin B Chain Mutants Expressed in Insect Cells Have Residual Galactose Binding: Evidence for More Than Two Lectin Sites on the Ricin Toxin B Chain
Identifieur interne : 003E92 ( Main/Exploration ); précédent : 003E91; suivant : 003E93Double-Lectin Site Ricin B Chain Mutants Expressed in Insect Cells Have Residual Galactose Binding: Evidence for More Than Two Lectin Sites on the Ricin Toxin B Chain
Auteurs : Tao Fu [États-Unis] ; Chris Burbage [États-Unis] ; Edward Tagge [États-Unis] ; John Chandler [États-Unis] ; Mark Willingham [États-Unis] ; Arthur Frankel [États-Unis]Source :
- Bioconjugate Chemistry [ 1043-1802 ] ; 1996.
Abstract
Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, contains a cell binding lectin subunit (RTB) disulfide linked to an RNA N-glycosidase protein synthesis-inactivating subunit (RTA). Investigations of the molecular nature of the lectin sites in RTB by X-ray crystallography, equilibrium dialysis, chemical modification, and mutational analysis have yielded conflicting results as to the number, location, and affinity of sugar-combining sites. An accurate assessment of the amino acid residues of RTB involved in galactose binding is needed both for correlating structure−function of a number of plant lectins and for the design and synthesis of targeted toxins for cancer and autoimmune disease therapy. We have performed oligonucleotide-directed mutagenesis on cDNA encoding RTB and expressed the mutant RTBs in insect cells. Partially purified recombinant proteins obtained from infected cell supernatants and cell extracts were characterized as to yields, immunoreactivities, asialofetuin binding, cell binding, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Two single-site mutants (subdomain 1α or 2γ) and two double-site mutants (subdomains 1α and 2γ) were produced and studied. Yields varied by two logs with lower recoveries of double-site mutants. All the mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. Single-lectin site mutants displayed up to a 1 log decrease in asialofetuin binding avidity, while the double-site mutants showed close to a 2 log decrease in sugar binding. However, for each of the double-site mutants, residual sugar binding was demonstrated to both immobilized asialofetuin and cells, and this binding was specifically inhibitable with α-lactose. All mutants reassociated with RTA, and the mutant heterodimers were cytotoxic to mammalian cells with potencies 1000-fold or more times that of unreassociated wild-type RTA or RTB. These data support a model for three or more lectin binding subdomains in RTB.
Url:
DOI: 10.1021/bc960056b
Affiliations:
Links toward previous steps (curation, corpus...)
- to stream Istex, to step Corpus: 002230
- to stream Istex, to step Curation: 002230
- to stream Istex, to step Checkpoint: 001628
- to stream Main, to step Merge: 003F47
- to stream Main, to step Curation: 003E92
Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Double-Lectin Site Ricin B Chain Mutants Expressed in Insect Cells Have Residual Galactose Binding: Evidence for More Than Two Lectin Sites on the Ricin Toxin B Chain</title><author><name sortKey="Fu, Tao" sort="Fu, Tao" uniqKey="Fu T" first="Tao" last="Fu">Tao Fu</name></author><author><name sortKey="Burbage, Chris" sort="Burbage, Chris" uniqKey="Burbage C" first="Chris" last="Burbage">Chris Burbage</name></author><author><name sortKey="Tagge, Edward" sort="Tagge, Edward" uniqKey="Tagge E" first="Edward" last="Tagge">Edward Tagge</name></author><author><name sortKey="Chandler, John" sort="Chandler, John" uniqKey="Chandler J" first="John" last="Chandler">John Chandler</name></author><author><name sortKey="Willingham, Mark" sort="Willingham, Mark" uniqKey="Willingham M" first="Mark" last="Willingham">Mark Willingham</name></author><author><name sortKey="Frankel, Arthur" sort="Frankel, Arthur" uniqKey="Frankel A" first="Arthur" last="Frankel">Arthur Frankel</name></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:7C1819B940792562DFC0BE3CC4B7423241542012</idno><date when="1996" year="1996">1996</date><idno type="doi">10.1021/bc960056b</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-3DMJ2R0G-C/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002230</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002230</idno><idno type="wicri:Area/Istex/Curation">002230</idno><idno type="wicri:Area/Istex/Checkpoint">001628</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Checkpoint">001628</idno><idno type="wicri:doubleKey">1043-1802:1996:Fu T:double:lectin:site</idno><idno type="wicri:Area/Main/Merge">003F47</idno><idno type="wicri:Area/Main/Curation">003E92</idno><idno type="wicri:Area/Main/Exploration">003E92</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Double-Lectin Site Ricin B Chain Mutants Expressed in Insect Cells
Have Residual Galactose Binding: Evidence for More Than Two
Lectin Sites on the Ricin Toxin B Chain</title><author><name sortKey="Fu, Tao" sort="Fu, Tao" uniqKey="Fu T" first="Tao" last="Fu">Tao Fu</name><affiliation wicri:level="4"><orgName type="university">Université de Caroline du Sud</orgName><country>États-Unis</country><placeName><settlement type="city">Columbia (Caroline du Sud)</settlement><region type="state">Caroline du Sud</region></placeName></affiliation><affiliation></affiliation></author><author><name sortKey="Burbage, Chris" sort="Burbage, Chris" uniqKey="Burbage C" first="Chris" last="Burbage">Chris Burbage</name><affiliation wicri:level="4"><orgName type="university">Université de Caroline du Sud</orgName><country>États-Unis</country><placeName><settlement type="city">Columbia (Caroline du Sud)</settlement><region type="state">Caroline du Sud</region></placeName></affiliation><affiliation></affiliation></author><author><name sortKey="Tagge, Edward" sort="Tagge, Edward" uniqKey="Tagge E" first="Edward" last="Tagge">Edward Tagge</name><affiliation wicri:level="4"><orgName type="university">Université de Caroline du Sud</orgName><country>États-Unis</country><placeName><settlement type="city">Columbia (Caroline du Sud)</settlement><region type="state">Caroline du Sud</region></placeName></affiliation><affiliation></affiliation></author><author><name sortKey="Chandler, John" sort="Chandler, John" uniqKey="Chandler J" first="John" last="Chandler">John Chandler</name><affiliation wicri:level="4"><orgName type="university">Université de Caroline du Sud</orgName><country>États-Unis</country><placeName><settlement type="city">Columbia (Caroline du Sud)</settlement><region type="state">Caroline du Sud</region></placeName></affiliation><affiliation></affiliation></author><author><name sortKey="Willingham, Mark" sort="Willingham, Mark" uniqKey="Willingham M" first="Mark" last="Willingham">Mark Willingham</name><affiliation wicri:level="4"><orgName type="university">Université de Caroline du Sud</orgName><country>États-Unis</country><placeName><settlement type="city">Columbia (Caroline du Sud)</settlement><region type="state">Caroline du Sud</region></placeName></affiliation><affiliation></affiliation></author><author><name sortKey="Frankel, Arthur" sort="Frankel, Arthur" uniqKey="Frankel A" first="Arthur" last="Frankel">Arthur Frankel</name><affiliation wicri:level="4"><orgName type="university">Université de Caroline du Sud</orgName><country>États-Unis</country><placeName><settlement type="city">Columbia (Caroline du Sud)</settlement><region type="state">Caroline du Sud</region></placeName></affiliation><affiliation></affiliation><affiliation wicri:level="2"><country xml:lang="fr">États-Unis</country><placeName><region type="state">Caroline du Sud</region></placeName><wicri:cityArea> Address correspondence to this author at thefollowingaddress: Hollings Cancer Center, Rm 306, 86 JonathanLucasSt., Charleston</wicri:cityArea></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Bioconjugate Chemistry</title><title level="j" type="abbrev">Bioconjugate Chem.</title><idno type="ISSN">1043-1802</idno><idno type="eISSN">1520-4812</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">1996</date><date type="published">1996</date><biblScope unit="vol">7</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="651">651</biblScope><biblScope unit="page" to="658">658</biblScope></imprint><idno type="ISSN">1043-1802</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1043-1802</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, contains a cell binding lectin subunit (RTB) disulfide linked to an RNA N-glycosidase protein synthesis-inactivating subunit (RTA). Investigations of the molecular nature of the lectin sites in RTB by X-ray crystallography, equilibrium dialysis, chemical modification, and mutational analysis have yielded conflicting results as to the number, location, and affinity of sugar-combining sites. An accurate assessment of the amino acid residues of RTB involved in galactose binding is needed both for correlating structure−function of a number of plant lectins and for the design and synthesis of targeted toxins for cancer and autoimmune disease therapy. We have performed oligonucleotide-directed mutagenesis on cDNA encoding RTB and expressed the mutant RTBs in insect cells. Partially purified recombinant proteins obtained from infected cell supernatants and cell extracts were characterized as to yields, immunoreactivities, asialofetuin binding, cell binding, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Two single-site mutants (subdomain 1α or 2γ) and two double-site mutants (subdomains 1α and 2γ) were produced and studied. Yields varied by two logs with lower recoveries of double-site mutants. All the mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. Single-lectin site mutants displayed up to a 1 log decrease in asialofetuin binding avidity, while the double-site mutants showed close to a 2 log decrease in sugar binding. However, for each of the double-site mutants, residual sugar binding was demonstrated to both immobilized asialofetuin and cells, and this binding was specifically inhibitable with α-lactose. All mutants reassociated with RTA, and the mutant heterodimers were cytotoxic to mammalian cells with potencies 1000-fold or more times that of unreassociated wild-type RTA or RTB. These data support a model for three or more lectin binding subdomains in RTB.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Caroline du Sud</li></region><settlement><li>Columbia (Caroline du Sud)</li></settlement><orgName><li>Université de Caroline du Sud</li></orgName></list><tree><country name="États-Unis"><region name="Caroline du Sud"><name sortKey="Fu, Tao" sort="Fu, Tao" uniqKey="Fu T" first="Tao" last="Fu">Tao Fu</name></region><name sortKey="Burbage, Chris" sort="Burbage, Chris" uniqKey="Burbage C" first="Chris" last="Burbage">Chris Burbage</name><name sortKey="Chandler, John" sort="Chandler, John" uniqKey="Chandler J" first="John" last="Chandler">John Chandler</name><name sortKey="Frankel, Arthur" sort="Frankel, Arthur" uniqKey="Frankel A" first="Arthur" last="Frankel">Arthur Frankel</name><name sortKey="Frankel, Arthur" sort="Frankel, Arthur" uniqKey="Frankel A" first="Arthur" last="Frankel">Arthur Frankel</name><name sortKey="Tagge, Edward" sort="Tagge, Edward" uniqKey="Tagge E" first="Edward" last="Tagge">Edward Tagge</name><name sortKey="Willingham, Mark" sort="Willingham, Mark" uniqKey="Willingham M" first="Mark" last="Willingham">Mark Willingham</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Main/Exploration
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 003E92 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Main/Exploration/biblio.hfd -nk 003E92 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= MersV1
|flux= Main
|étape= Exploration
|type= RBID
|clé= ISTEX:7C1819B940792562DFC0BE3CC4B7423241542012
|texte= Double-Lectin Site Ricin B Chain Mutants Expressed in Insect Cells Have Residual Galactose Binding: Evidence for More Than Two Lectin Sites on the Ricin Toxin B Chain
}}
| This area was generated with Dilib version V0.6.33. |  |