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 : 002230 ( Istex/Corpus ); précédent : 002229; suivant : 002231Double-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 ; Chris Burbage ; Edward Tagge ; John Chandler ; Mark Willingham ; Arthur FrankelSource :
- 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
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ISTEX:7C1819B940792562DFC0BE3CC4B7423241542012Le document en format XML
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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><mods:affiliation>Departments of Medicine, Surgery, and Pathology, Medical University of South Carolina, Charleston, SouthCarolina 29425</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Medicine.</mods:affiliation></affiliation></author><author><name sortKey="Burbage, Chris" sort="Burbage, Chris" uniqKey="Burbage C" first="Chris" last="Burbage">Chris Burbage</name><affiliation><mods:affiliation>Departments of Medicine, Surgery, and Pathology, Medical University of South Carolina, Charleston, SouthCarolina 29425</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Medicine.</mods:affiliation></affiliation></author><author><name sortKey="Tagge, Edward" sort="Tagge, Edward" uniqKey="Tagge E" first="Edward" last="Tagge">Edward Tagge</name><affiliation><mods:affiliation>Departments of Medicine, Surgery, and Pathology, Medical University of South Carolina, Charleston, SouthCarolina 29425</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Surgery.</mods:affiliation></affiliation></author><author><name sortKey="Chandler, John" sort="Chandler, John" uniqKey="Chandler J" first="John" last="Chandler">John Chandler</name><affiliation><mods:affiliation>Departments of Medicine, Surgery, and Pathology, Medical University of South Carolina, Charleston, SouthCarolina 29425</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Surgery.</mods:affiliation></affiliation></author><author><name sortKey="Willingham, Mark" sort="Willingham, Mark" uniqKey="Willingham M" first="Mark" last="Willingham">Mark Willingham</name><affiliation><mods:affiliation>Departments of Medicine, Surgery, and Pathology, Medical University of South Carolina, Charleston, SouthCarolina 29425</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Pathology.</mods:affiliation></affiliation></author><author><name sortKey="Frankel, Arthur" sort="Frankel, Arthur" uniqKey="Frankel A" first="Arthur" last="Frankel">Arthur Frankel</name><affiliation><mods:affiliation>Departments of Medicine, Surgery, and Pathology, Medical University of South Carolina, Charleston, SouthCarolina 29425</mods:affiliation></affiliation><affiliation><mods:affiliation> Department of Medicine.</mods:affiliation></affiliation><affiliation><mods:affiliation> Address correspondence to this author at thefollowingaddress: Hollings Cancer Center, Rm 306, 86 JonathanLucasSt., Charleston, SC 29425. Telephone: 803-792-1450.Fax: 803-792-3200.</mods:affiliation></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. 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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. 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Have Residual Galactose Binding: Evidence for More Than Two
Lectin Sites on the Ricin Toxin B Chain</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 1996 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">1996</date><date type="Copyright" when="1996">1996</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><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 xml:id="author-0000"><persName><surname>Fu</surname><forename type="first">Tao</forename></persName><affiliation><orgName type="division">Departments of Medicine</orgName><address><addrLine>Surgery</addrLine><addrLine>and Pathology</addrLine><addrLine>Medical University of South Carolina</addrLine><addrLine>Charleston</addrLine><addrLine>South Carolina 29425</addrLine></address></affiliation><note place="foot" n="bc960056bAF2"><ref>†</ref><p>
Department of Medicine.</p></note></author><author xml:id="author-0001"><persName><surname>Burbage</surname><forename type="first">Chris</forename></persName><affiliation><orgName type="division">Departments of Medicine</orgName><address><addrLine>Surgery</addrLine><addrLine>and Pathology</addrLine><addrLine>Medical University of South Carolina</addrLine><addrLine>Charleston</addrLine><addrLine>South Carolina 29425</addrLine></address></affiliation><note place="foot" n="bc960056bAF2"><ref>†</ref><p>
Department of Medicine.</p></note></author><author xml:id="author-0002"><persName><surname>Tagge</surname><forename type="first">Edward</forename></persName><affiliation><orgName type="division">Departments of Medicine</orgName><address><addrLine>Surgery</addrLine><addrLine>and Pathology</addrLine><addrLine>Medical University of South Carolina</addrLine><addrLine>Charleston</addrLine><addrLine>South Carolina 29425</addrLine></address></affiliation><note place="foot" n="bc960056bAF3"><ref>‡</ref><p>
Department of Surgery.</p></note></author><author xml:id="author-0003"><persName><surname>Chandler</surname><forename type="first">John</forename></persName><affiliation><orgName type="division">Departments of Medicine</orgName><address><addrLine>Surgery</addrLine><addrLine>and Pathology</addrLine><addrLine>Medical University of South Carolina</addrLine><addrLine>Charleston</addrLine><addrLine>South Carolina 29425</addrLine></address></affiliation><note place="foot" n="bc960056bAF3"><ref>‡</ref><p>
Department of Surgery.</p></note></author><author xml:id="author-0004"><persName><surname>Willingham</surname><forename type="first">Mark</forename></persName><affiliation><orgName type="division">Departments of Medicine</orgName><address><addrLine>Surgery</addrLine><addrLine>and Pathology</addrLine><addrLine>Medical University of South Carolina</addrLine><addrLine>Charleston</addrLine><addrLine>South Carolina 29425</addrLine></address></affiliation><note place="foot" n="bc960056bAF4"><ref>§</ref><p>
Department of Pathology.</p></note></author><author xml:id="author-0005" role="corresp"><persName><surname>Frankel</surname><forename type="first">Arthur</forename></persName><note place="foot" n="bc960056bAF2"><ref>†</ref><p>
Department of Medicine.</p></note><affiliation role="corresp"> Address correspondence to this author at the following address: Hollings Cancer Center, Rm 306, 86 Jonathan Lucas St., Charleston, SC 29425. Telephone: 803-792-1450. Fax: 803-792-3200.</affiliation></author><idno type="istex">7C1819B940792562DFC0BE3CC4B7423241542012</idno><idno type="ark">ark:/67375/TPS-3DMJ2R0G-C</idno><idno type="DOI">10.1021/bc960056b</idno></analytic><monogr><title level="j" type="main">Bioconjugate Chemistry</title><title level="j" type="abbrev">Bioconjugate Chem.</title><idno type="acspubs">bc</idno><idno type="coden">bcches</idno><idno type="pISSN">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></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p>Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor
bean seeds, contains a cell
binding lectin subunit (RTB) disulfide linked to an RNA
<hi rend="italic">N</hi>-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.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bc</journal-id><journal-id journal-id-type="coden">bcches</journal-id><journal-title-group><journal-title>Bioconjugate Chemistry</journal-title><abbrev-journal-title>Bioconjugate Chem.</abbrev-journal-title></journal-title-group><issn pub-type="ppub">1043-1802</issn><issn pub-type="epub">1520-4812</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/bc</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bc960056b</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-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</article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Fu</surname><given-names>Tao</given-names></name><xref rid="bc960056bAF2"><sup>†</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Burbage</surname><given-names>Chris</given-names></name><xref rid="bc960056bAF2"><sup>†</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Tagge</surname><given-names>Edward</given-names></name><xref rid="bc960056bAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Chandler</surname><given-names>John</given-names></name><xref rid="bc960056bAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Willingham</surname><given-names>Mark</given-names></name><xref rid="bc960056bAF4"><sup>§</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Frankel</surname><given-names>Arthur</given-names></name><xref rid="bc960056bAF1">*</xref><xref rid="bc960056bAF2"><sup>†</sup></xref></contrib><aff>Departments of Medicine, Surgery, and Pathology, Medical University of South Carolina, Charleston, South
Carolina 29425</aff></contrib-group><author-notes><fn id="bc960056bAF2"><label>†</label><p>
Department of Medicine.</p></fn><fn id="bc960056bAF3"><label>‡</label><p>
Department of Surgery.</p></fn><fn id="bc960056bAF4"><label>§</label><p>
Department of Pathology.</p></fn><corresp id="bc960056bAF1">
Address correspondence to this author at the
following
address: Hollings Cancer Center, Rm 306, 86 Jonathan
Lucas
St., Charleston, SC 29425. Telephone: 803-792-1450.
Fax:
803-792-3200.</corresp></author-notes><pub-date pub-type="epub"><day>27</day><month>11</month><year>1996</year></pub-date><pub-date pub-type="ppub"><day>27</day><month>11</month><year>1996</year></pub-date><volume>7</volume><issue>6</issue><fpage>651</fpage><lpage>658</lpage><history><date date-type="received"><day>31</day><month>05</month><year>1996</year></date><date date-type="issue-pub"><day>27</day><month>11</month><year>1996</year></date></history><permissions><copyright-statement>Copyright © 1996 American Chemical Society</copyright-statement><copyright-year>1996</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor
bean seeds, contains a cell
binding lectin subunit (RTB) disulfide linked to an RNA
<italic toggle="yes">N</italic>-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.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bc960056b</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bc960056bAF7"><label>✗</label><p>
Abstract published in <italic toggle="yes">Advance ACS
Abstracts,</italic> November
1, 1996.</p></notes></front><body><sec id="d7e170"><title>Introduction</title><p>Lectins such as ricin toxin from the <italic toggle="yes">Ricinus
communis</italic>
plant mediate a wide range of biological effects due to
binding cell surface carbohydrates. The B chain
subunit
of ricin (RTB)<xref rid="bc960056bb00001" ref-type="bibr"></xref> binds to mammalian cell
membranes by
recognizing galactose-containing receptors, and this reaction is the first necessary step for intoxication of cells
(<italic toggle="yes"><xref rid="bc960056bb00001" ref-type="bibr"></xref></italic>). RTB binds β-galactosides (<italic toggle="yes"><xref rid="bc960056bb00002" ref-type="bibr"></xref></italic>) with
association constants from 10<sup>3</sup> to 3 × 10<sup>4</sup> M<sup>-1</sup>
for simple sugars (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00003" ref-type="bibr"></xref>, <xref rid="bc960056bb00004" ref-type="bibr"></xref></named-content></italic>)
and 10<sup>7</sup>−10<sup>8</sup> M<sup>-1</sup> for free and
cell surface-bound glycoproteins (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00005" ref-type="bibr"></xref>, <xref rid="bc960056bb00006" ref-type="bibr"></xref></named-content></italic>). These results lead to the
hypothesis that
multiple low-affinity sugar binding on ricin interact with
complex oligosaccharides and cells to yield high-affinity
binding. Clustering of target sugars in the proper
geometry to enhance lectin binding via interactions at
multiple sites has been observed for the hepatic galactose
<italic toggle="yes">N</italic>-acetylgalactosamine-binding receptor (<italic toggle="yes"><xref rid="bc960056bb00007" ref-type="bibr"></xref></italic>) and
the macrophage mannose receptor (<italic toggle="yes"><xref rid="bc960056bb00008" ref-type="bibr"></xref></italic>).</p><p>The X-ray crystallographic structure of ricin provides
a structural basis for this hypothesis. RTB has two
domains each with three subdomains (<italic toggle="yes"><xref rid="bc960056bb00009" ref-type="bibr"></xref></italic>). The six
subdomains have similar folding and primary amino acid
sequences and resemble the primitive galactose binding
fold in discoidin I from the slime mold
<italic toggle="yes">Dictyostelium
discoideum</italic>. Tripeptide kinks in the loops from
subdomains 1α, 1β, 2α, and 2γ may interact with
galactosides.
Each of these subdomains has aromatic residues which
can interact with the nonpolar face of galactose, and
three
of the four subdomain folds (1α, 1β, and 2γ) have
polar
residues for hydrogen bond formation to the sugar
hydroxyls. Cocrystallization of α-lactose at low
concentrations (5 mM) with ricin permitted identification of
bonds between lactose and amino acid residues of subdomains 1α and 2γ.</p><p>Biochemical modification studies have identified one
to three sugar binding sites per RTB molecule.
<italic toggle="yes">N</italic>-Bromosuccinimide modification of Trp-37 reduced sugar
binding, demonstrating a sugar-binding site in the subdomain 1α fold (<italic toggle="yes"><xref rid="bc960056bb00010" ref-type="bibr"></xref></italic>). <italic toggle="yes">N</italic>-Acetylimidazole
O-acetylation of
two tyrosines reduced sugar binding, implicating sites
in subdomains 1β pocket and 2γ pocket (<italic toggle="yes"><xref rid="bc960056bb00011" ref-type="bibr"></xref></italic>).
Further,
three distinct sites on ricin were cross-linked by radiolabeled fetuin glycopeptide containing a dichlorotriazine-activated 6-(<italic toggle="yes">N</italic>-methylamino)-6-deoxy-<sc>d</sc>-galactose
moiety,
supporting the concept of three sugar-binding sites
(<italic toggle="yes"><xref rid="bc960056bb00012" ref-type="bibr"></xref></italic>).</p><p>Mutational analysis of recombinant RTBs produced in
Cos cells, bacteriophage, and <italic toggle="yes">Xenopus laevis</italic> ooctyes
has
suggested either one active lectin site in subdomain 2γ
or two lectin sites in subdomains 1α and 2γ
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00013" ref-type="bibr"></xref>−<xref rid="bc960056bb00014" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc960056bb00015" ref-type="bibr"></xref></named-content></italic>). RTB
mutant N255A produced in Cos cells and RTB mutants
K40M/N46G/N255G and D22Q/V23A/R24N/D234A/V235A/R236T synthesized in <italic toggle="yes">Xenopus</italic> oocytes lacked
sugar
binding (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00013" ref-type="bibr"></xref>, <xref rid="bc960056bb00014" ref-type="bibr"></xref></named-content></italic>). However, very small
amounts of
proteins were made in each case, and purification and
immunologic characterization of products were not done.
In all three mutational studies, decreased sugar
binding
due to misfolding or aggregation of recombinant RTBs
could lead to an overestimation of the effect of
individual
modifications.</p><p>To obtain more accurate quantitative information on
the RTB lectin sites, our laboratory has expressed,
partially purified, and characterized wild-type, single-site, and double-site RTB mutants in insect cells
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00016" ref-type="bibr"></xref>−<xref rid="bc960056bb00017" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc960056bb00018" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc960056bb00019" ref-type="bibr"></xref></named-content></italic>). We obtained microgram to milligram yields of
recombinant proteins and were able to purify the lectins
to 10−50% purity by Coomassie-stained SDS−PAGE.
Wild-type recombinant RTB bound asialofetuin and cell
surface oligosaccharides in a manner similar to that of
plant RTB with half-maximal binding concentrations of
about 5 × 10<sup>-9</sup> M (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00016" ref-type="bibr"></xref>, <xref rid="bc960056bb00017" ref-type="bibr"></xref></named-content></italic>).
Single-site mutants of both
the 1α subdomain and the 2γ subdomain had reduced
binding affinity for asialofetuin and KB mammalian cells
(<italic toggle="yes"><xref rid="bc960056bb00018" ref-type="bibr"></xref></italic>). However, the reduction in binding affinity was
1
log or less in each case. After reassociation with
plant
RTA, each single-site mutant retained HUT102 human
leukemia cell cytotoxicity with ID<sub>50</sub>'s within 1−1.5
logs
of that of the wild-type heterodimer. The minor
effect
of genetic modification of lectin sites in subdomains 1α
and 2γ suggested incomplete inactivation of lectin sites
or additional sugar-combining sites on RTB. We then
expressed, partially purified, and characterized three
double-site mutant RTBs and one additional single-site
mutant RTB (<italic toggle="yes"><xref rid="bc960056bb00019" ref-type="bibr"></xref></italic>). Two of the double-site mutants
had
unique properties suggesting either persistence of two
main galactose binding sites operating at reduced levels
or the theory of three ricin lectin sites. To resolve
the
issue of incomplete inactivation of the lectin sites in
the
single- and double-site mutants, we now report the
biological properties of two additional single-site
mutants
and two additional double-site mutants. Modifications
at each site were chosen to maximally alter each lectin
pocket. Our findings support the hypothesis of at
least
three independent sugar-combining sites on ricin.
</p></sec><sec id="d7e306"><title>Experimental Procedures</title><p><bold>Mutagenesis.</bold> pUC119-RTB plasmid containing
a
<italic toggle="yes">Bam</italic>HI-<italic toggle="yes">Eco</italic>RI DNA fragment coding for ADP−RTB
was
propagated in INVαF‘ <italic toggle="yes">Escherichia coli</italic> cells
(InVitrogen,
San Diego, CA) as previously described (<italic toggle="yes"><xref rid="bc960056bb00016" ref-type="bibr"></xref></italic>).
Single-stranded DNA was produced by infection of transformants with M13K07 phage (Stratagene, La Jolla, CA)
as previously described (<italic toggle="yes"><xref rid="bc960056bb00020" ref-type="bibr"></xref></italic>). Oligonucleotides
were
synthesized on an Applied Biosystems 380B DNA synthesizer and desalted with butan-1-ol. 39-mers were
prepared with the modified codon flanked by 18 bases
on each side matching RTB sequence and lacking an
<italic toggle="yes">Nci</italic>I
site. Site-specific mutagenesis was performed by the
Eckstein method using the Sculptor <italic toggle="yes">in vitro</italic>
mutagenesis
system (Amersham, Arlington Heights, IL) and the
manufacturer's instructions (<italic toggle="yes"><xref rid="bc960056bb00021" ref-type="bibr"></xref></italic>). Modifications
were
made at both the 1α and 2γ subdomains on the basis of
the X-ray crystallographic model of the lectin binding
sites (Figure <xref rid="bc960056bf00001"></xref>) to either alter key polar
residues which
provide hydrogen bonds to sugar hydroxyls (D234E,
N46G/K40M/N255G, and D22Q/V23A/R24N/D234A/
V235A/R236T) or change aromatic ring residues which
provide van der Waals interactions between the protein
and sugar (Y248H). Sequences of mutant RTB DNAs
were confirmed by double-stranded dideoxy sequencing
by the Sanger method using the Sequenase kit (USB,
Cleveland, OH) (<italic toggle="yes"><xref rid="bc960056bb00022" ref-type="bibr"></xref></italic>).<fig id="bc960056bf00001" position="float" orientation="portrait"><label>1</label><caption><p>Model of subdomains 1α and 2γ of ricin B chain showing amino acid residues altered in this study. Coordinates derived from Rutenber and Robertus (<italic toggle="yes"><xref rid="bc960056bb00009" ref-type="bibr"></xref></italic>). Ball and stick diagram
display of Brookhaven coordinates on SYBYL software on Silicon
Graphics
Iris Indigo workstation with α carbon backbone displayed in gray and
modified amino acid residues in black. Lactose
molecules
displayed in black also.</p></caption><graphic xlink:href="bc960056bf00001.gif" position="float" orientation="portrait"></graphic></fig></p><p><bold>Construction of Transfer Vectors Encoding Mutant RTBs.</bold> Mutant RTB-encoding pUC119 DNAs were
then restricted with <italic toggle="yes">Bam</italic>HI and <italic toggle="yes">Eco</italic>RI, and the
RTB-encoding fragments were subcloned into pAcGP67A plasmid (PharMingen, San Diego, CA) and used to transform
INVαF‘ <italic toggle="yes">E</italic>. <italic toggle="yes">coli</italic> cells. Transfer vectors
with mutant RTBs
were then purified by cesium chloride gradient centrifugation.</p><p><bold>Isolation of Recombinant Baculoviruses.</bold> The
Sf9
<italic toggle="yes">Spodoptera</italic> <italic toggle="yes">frugiperda</italic> ovarian cell line was
maintained
on TMNFH medium supplemented with 10% fetal calf
serum and gentamicin sulfate (10 μg/mL). pAcGP67A−mutant RTB DNAs (4 μg) were cotransfected with 0.5
μg of BaculoGold AcNPV DNA (PharMingen) into 2 ×
10<sup>6</sup> Sf9 insect cells following the recommendations of
the
supplier. On day 7 post-transfection, media were
centrifuged and the supernatants tested in
limiting dilution
assays with Sf9 cells. Sf9 cells (2 × 10<sup>4</sup>) were
incubated
with 10-fold dilutions of supernatants in 96-well plates.
Seven days postinfection, supernatants were saved and
cells in each assay well were lysed with NaOH and the
lysates transferred to nitrocellulose. The
nitrocellulose
was then blocked with Blotto and reacted with random
primer [<sup>32</sup>P]-dCTP-labelled RTB DNA. After
hybridization for 16 h at 67 °C, the dot blot membranes were
washed with 0.1 × (150 mM NaCl/15 mM sodium citrate)/1% SDS, dried, and exposed to X-ray film. Positive
wells
were identified and supernatants reassayed by limiting
dilution until all wells up to 10<sup>-8</sup> dilution were
positive.
Two rounds of selection were required for each
mutant.
Recombinant viruses in the supernatants were then
amplified by infecting Sf9 cells at a multiplicity of
infection (moi) of 0.1, followed by collection of day 7
supernatants.</p><p><bold>Expression of Mutant B Chains in Sf9 Cells.</bold>
Recombinant baculoviruses were used to infect 2 ×
10<sup>8</sup>
Sf9 cells at an moi of 5 in EX-CELL 400 media (JRH
Scientific, Lexena, KS) with 50 mM α-lactose in spinner
flasks. Media supernatants containing mutant RTBs
were collected on day 6 postinfection.</p><p><bold>Purification of Mutant RTBS.</bold> Media
supernatants
were adjusted to 0.01% sodium azide and maintained
through all purification steps at 4 °C. The
supernatants
were concentrated 15-fold by vacuum dialysis, centrifuged at 3000<italic toggle="yes">g</italic> for 10 min to remove precipitate,
dialyzed
against 50 mM NaCl, 25 mM Tris (pH 8), 1 mM EDTA,
0.01% sodium azide, and 25 mM α-lactose (NTEAL),
ultracentrifuged at 100000<italic toggle="yes">g</italic> for 1 h, and loaded onto
a
P2 monoclonal antibody−acrylamide column previously
described (<italic toggle="yes"><xref rid="bc960056bb00016" ref-type="bibr"></xref></italic>). The affinity column was then
washed
sequentially with NTEAL and 500 mM NaCl, 25 mM Tris
(pH 9), 1 mM EDTA, 0.01% sodium azide, 25 mM
α-lactose, and 0.1% Tween 20 (NTEALT), and mutant
RTBs were eluted with 0.1 M triethylamine (pH 11).
The
alkaline eluants were immediately neutralized with 1 M
sodium phosphate (pH 4.8) and stored at −20 °C until
they were assayed. Optical densities at 280 nm were
determined and aliquots mixed with reducing 2 × SDS
sample buffer, boiled for 4 min, submitted to a 15% SDS−PAGE, stained with Coomassie Blue R-250, and destained
with acetic acid/methanol. Gels were scanned on an
IBAS automatic image analysis system (Kontron, Germany) to estimate the fraction of the protein with a
molecular mass of 32 kDa.</p><p>Several preparations of mutant RTBs were made from
cell pellets by dissolving pellets in 10 volumes of 20 mM
Tris (pH 8), 50 mM NaCl, 1% NP40, 1 mM PMSF, 2 μg/mL aprotinin, 1.5 μg/mL pepstatin, and 1.5 μg/mL
leupeptin. The extracts were frozen at −70 °C,
thawed,
and centrifuged at 22000<italic toggle="yes">g</italic> for 15 min at 4 °C.
Extracts
were then dialyzed against NTEAL and treated in the
same manner as dialyzed concentrated cell supernatants.</p><p><bold>Immunological Properties of Mutant RTBs.</bold>
Aliquots of mutant RTBs, plant RTB (Inland Laboratories,
Austin, TX), wild-type recombinant RTB, and prestained
low-molecular mass standards (BioRad, Hercules, CA)
were mixed with reducing 2 × SDS sample buffer, boiled
for 4 min, submitted to a 15% SDS−PAGE, and electrophoresed for 90 min. Gels, Whatman 3M #1 paper, and
nitrocellulose were equilibrated for 15 min in Towbin
buffer (20 mM Tris/0.1 M glycine/20% methanol) and
placed in a Semi-dry Trans-blot cell (BioRad). After
electrophoresis at 15 V for 20 min, the nitrocellulose was
blocked with 10% Carnation's nonfat dry milk/0.1% BSA/0.1% Tween 20/0.02% sodium azide. The blots were
then
washed with PBS plus 0.05% Tween 20 and PBS,
incubated with rabbit anti-ricin antibody (Sigma, St.
Louis, MO) at 1:400 in PBS plus 0.5% BSA plus 0.01%
sodium azide for 1 h, washed again, incubated with
alkaline phosphatase-conjugated goat anti-(rabbit IgG)
(Sigma) at 1:1000 in PBS plus 0.5% BSA plus 0.01%
sodium azide for 1 h, washed again, and developed with
the Vectastain alkaline phosphatase kit, following the
manufacturer's recommendations (Vector Laboratories,
Burlingame, CA). Blots were scanned as above to
compare 32 kDa <italic toggle="yes">M</italic><sub>r</sub> band intensities.</p><p>Monoclonal antibody P2, P8, or P10 (gifts of Dr. Walter
Blattler, ImmunoGen, Cambridge, MA) (100 μL) at 5 μg/mL in PBS was incubated in Costar EIA microtiter wells
overnight at 4 °C. Samples of plant RTB, wild-type
recombinant RTB, and mutant RTBs were treated for 20
min at room temperature with 5% β-mercaptoethanol and
then dilutions made in EX-CELL 400. The antibody-coated microtiter wells were then washed with PBS plus
0.1% Tween 20, blocked with 3% BSA/PBS/0.01% sodium
azide, rewashed and incubated with dilutions of the
reduced RTB samples, rewashed and incubated with
rabbit anti-ricin antibody at 1:400 in PBS plus 0.5% BSA
plus 0.01% sodium azide, washed again, incubated with
alkaline phosphatase-conjugated goat anti-(rabbit IgG)
at 1:1000 in PBS plus 0.05% BSA plus 0.01% sodium
azide, washed and developed with
(<italic toggle="yes">p</italic>-nitrophenyl)phosphate at 1 mg/mL in 50 mM diethanolamine buffer (pH
9.8), and read on a BioRad 450 microplate reader at 405
nm. For each experiment, 12 different concentrations
of
plant RTB and recombinant RTBs were tested. A plot
of absorbance versus dilution was made for plant RTB
and recombinant proteins. Dilutions yielding half-maximal binding were used to calculate concentrations.</p><p><bold>Lectin Activity of Mutant RTBs.</bold> Volumes (100
μL)
of 1 μg/mL asialofetuin in PBS were added to wells of a
Costar EIA plate and incubated overnight at 4 °C.
Samples of plant RTB, wild-type recombinant RTB, or
mutant RTBs in EX-CELL 400 were exposed to 5%
β-mercaptoethanol for 20 min at room temperature to
remove homodimers and dilutions made in EX-CELL 400
medium with or without 100 μg/mL asialofetuin or 100
mM α-lactose. The asialofetuin-coated microtiter
wells
were then washed with PBS/0.1% Tween 20, blocked with
3% BSA/PBS/0.01% sodium azide, and rewashed. The
dilutions of various reduced RTBs were added to wells
for 1 h and then removed and the wells washed again.
Rabbit anti-ricin antibody was added (1:400 dilution
in
0.5% BSA/PBS/0.01% sodium azide) for 1 h, and the wells
were washed again; alkaline phosphatase-conjugated
goat anti-(rabbit IgG) (1:5000 in 0.5% BSA/PBS/0.01%
sodium azide) was incubated in the wells, and finally,
the wells were washed and reacted with 1 mg/mL
(<italic toggle="yes">p</italic>-nitrophenyl)phosphate in 50 mM diethanolamine
buffer
(pH 9.6) and measured in a microtiter plate reader at
405 nm. In each experiment, 12 different
concentrations
of plant RTB and recombinant protein were tested. As
in the antibody ELISA, relative reactivity to plant RTB
was calculated from concentrations giving half-maximal
binding. The effects of 100 μg/mL asialofetuin or 100
mM
α-lactose on half-maximal binding were calculated for
plant RTB, wild-type recombinant RTB, and mutant
RTBs.</p><p>KB cells were washed with PBS and attached to
polylysine-coated tissue culture dishes and centrifuged
at 2000<italic toggle="yes">g</italic> for 10 min. The cells were then incubated
live
at 4 °C. The cells were washed with 2 mg/mL BSA in
PBS and incubated in PBS plus BSA with or without 100
μg/mL asialofetuin and with 1 μg/mL freshly reduced
plant RTB, recombinant wild-type RTB, or mutant RTB.
The incubation was done at 4 °C. The cells were
then
washed with PBS and incubated with rabbit
anti-ricin
antibody at 1:400 in PBS plus BSA for 30 min at 4 °C.
The cells were then washed with PBS and reacted with
goat anti-(rabbit Ig) conjugated to rhodamine at 25 μg/mL for 30 min at 4 °C. The cells were washed again
in
PBS, fixed in 3.7% formaldehyde in PBS, mounted under
a #1 coverslip in glycerol−PBS (90:10), and examined
under a Zeiss Axioplan epifluorescence microscope.</p><p><bold>Reassociation of Mutant RTBs with Plant RTA
To Form Heterodimers.</bold> Portions (30 μg) of plant
RTB
and wild-type recombinant RTB and 1−5 μg of mutant
RTBs were mixed with a 4-fold molar excess of plant RTA
(Inland Laboratories) in a total volume of 0.5 mL of 0.1
M triethylamine/sodium phosphate (pH 7) shaking overnight at room temperature. The reaction mixture was
then analyzed by a modified ricin ELISA. Wells of an
EIA plate were coated with 10 μg/mL P2 monoclonal
antibody to RTB diluted in PBS in a volume of 100 μL
overnight at 4 °C. The wells were washed with PBS
plus
0.1% Tween 20, blocked with 3% BSA in PBS plus 0.02%
sodium azide, rewashed, and incubated with dilutions of
ricin or reassociated heterodimers. The wells were
again
washed and incubated with biotin-conjugated αBR12
monoclonal antibody (αBR12 mouse monoclonal antibody
reactive with RTA was a gift of Dr. Walter Blattler,
ImmunoGen) at 5 μg/mL in PBS/0.5% BSA/0.01% sodium
azide. Biotinylation was performed using
(<italic toggle="yes">N</italic>-hydroxysuccinimido)biotin (Sigma) following the
manufacturer's
instructions. Wells were washed and incubated with
alkaline phosphatase-conjugated strepavidin (Sigma) at
1:1000 in PBS/0.5% BSA/0.01% sodium azide, washed
again, and developed with (<italic toggle="yes">p</italic>-nitrophenyl)phosphate
at
1 mg/mL in 50 mM diethanolamine (pH 9.8). Absorbance
at 405 nm was read on a microtiter plate reader.
Reassociated mixtures were also analyzed by nonreducing SDS−PAGE followed by immunoblots with αBR12
anti-RTA monoclonal antibody and P10 anti-RTB monoclonal antibody. Densitometric scanning using the IBAS
2000 automatic image analysis system was done to
quantify shift of immunoreactive material from 30 to 60
kDa.</p><p><bold>Cytotoxicity of Recombinant Mutant Heterodimers.</bold> HUT102 human T leukemia cells (1.5 ×
10<sup>4</sup>)
in 100 μL were placed in 96-well flat-bottomed plates in
leucine-poor RPMI1640 containing 10% dialyzed fetal
bovine serum. Fifty microliters of ricin (Inland
Laboratories), recombinant wild-type RTB−plant RTA heterodimer, and mutant RTB−plant RTA heterodimers at
varying concentrations were added in the same medium
and the cells incubated at 37 °C in 5% CO<sub>2</sub> for 24 h.
[<sup>3</sup>H]-Leucine, 0.5 μCi per well (120 mCi/mmol), in 50 μL of
the same medium was added and incubated for 4 h.
Cells
were then harvested with a PhD cell harvester onto glass-fiber filter mats. The filters were dried, mixed with
3
mL of liquid scintillation fluid, and counted in an LKB-Wallac liquid scintillation counter gated for <sup>3</sup>H.
Cells
cultured with medium alone served as controls. All
assays were performed in triplicate. The ID<sub>50</sub> was
the
concentration of protein which inhibited protein synthesis
by 50% compared with control.
</p></sec><sec id="d7e479"><title>Results</title><p><bold>Yields and Immunoreactivity of Mutant RTBs.</bold>
Two new single-site mutants were prepared (D22E and
Y248H). Y248H was previously described as part of a
single-domain RTB−gene 3 protein fusion on fd phage
(<italic toggle="yes"><xref rid="bc960056bb00023" ref-type="bibr"></xref></italic>), but the properties of a full length RTB with
the
single mutation have not been reported. <italic toggle="yes">R</italic>.
<italic toggle="yes">communis</italic>
agglutinin B chain and ricin E B chain have
histidinesat position 248 which may contribute to their lower
galactose avidities (<italic toggle="yes"><xref rid="bc960056bb00024" ref-type="bibr"></xref></italic>). The two double-site
mutants,
N46G/K40M/N255G and D22Q/V23A/R24N/D234A/V235A/R236T, which were prepared had been previously expressed in <italic toggle="yes">Xenopus</italic> oocytes but have not been
expressed
and purified from insect cells (<italic toggle="yes"><xref rid="bc960056bb00014" ref-type="bibr"></xref></italic>).</p><p>The yields were estimated from the optical density at
280 nm of neutralized alkaline eluants with postaffinity
chromatography (plant RTB OD = 1.44 for 1 mg/mL) and
densitometry of Coomassie-stained reducing SDS−PAGE
(10−30% of the protein migrated at 33 kDa, Figure
<xref rid="bc960056bf00002"></xref>A).
These results were confirmed by densitometry of immunoblots with polyclonal rabbit anti-ricin antibody. As
shown in Figure <xref rid="bc960056bf00002"></xref>B, the single-site and double-site
mutants were reactive with the polyclonal antibody.
Finally, a monoclonal antibody anti-RTB ELISA was
used
to verify concentrations of each mutant. All three
assays
gave similar values. The yields of each of the four
new
mutants and 12 previously prepared mutants
are shown
in Table <xref rid="bc960056bt00001"></xref>. The lower yields of 3/11
single-site mutants
(N255A, K40M/N46G, and K40M) and 4/5 double-site
mutants (D22E/D234E, W37S/Y248S, N46G/K40M/
N255G, and D22Q/V23A/R24N/D234A/V235A/R236T) may
be due to degradation of improperly folded proteins.
Other investigators reported bioactivity of mutants
without purification or characterization (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00013" ref-type="bibr"></xref>−<xref rid="bc960056bb00014" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc960056bb00015" ref-type="bibr"></xref></named-content></italic>).
Their assumption that modification of lectin site residues would
not affect secondary structure may be inaccurate and
overemphasize the role of individual residues and subdomains in sugar binding and cytotoxicity. Yields from
cells extracts were similar to yields from supernatants
in the four mutants tested (K40M, Q35N, N46G/K40M/N255G, and D22Q/V23A/R24N/D234A/V235A/R236T).<fig id="bc960056bf00002" position="float" orientation="portrait"><label>2</label><caption><p>Insect-derived wild-type and mutant RTBs. (A) Coomassie-stained 15% reducing SDS−PAGE of mutant RTBs: lane 1, low-molecular mass prestained BioRad protein standards; lane 2, wild type; lane 3, D234E; lane 4, Y248H; lane 5, N46G/K40M/N255G; and lane 6, D22Q/V23A/R24N/D234A/V235A/R236T. (B) Immunoblot using rabbit anti-ricin antibody of 15% reducing SDS−PAGE of wild-type and mutant RTBs. Lanes are the same as in panel A.</p></caption><graphic xlink:href="bc960056bf00002.gif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bc960056bt00001" position="float" orientation="portrait"><label>1</label><caption><p>Yields of Mutant RTBs Produced in Insect Cells<italic toggle="yes"><sup>a</sup></italic></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="4"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1">protein</oasis:entry><oasis:entry colname="2">type of mutant
(subdomain)</oasis:entry><oasis:entry colname="3">amount of partially
purified mutant per
liter of culture (μg)</oasis:entry><oasis:entry colname="4">reference
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">1500</oasis:entry><oasis:entry colname="4">present study
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248S</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">640</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248H</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">420</oasis:entry><oasis:entry colname="4">present study
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">wild-type</oasis:entry><oasis:entry colname="2">−</oasis:entry><oasis:entry colname="3">400</oasis:entry><oasis:entry colname="4">16
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">264</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E/A237R</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">240</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248H</oasis:entry><oasis:entry colname="2">1α 2γ</oasis:entry><oasis:entry colname="3">205</oasis:entry><oasis:entry colname="4">19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255G</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">160</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">150</oasis:entry><oasis:entry colname="4">19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Q35N</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">90</oasis:entry><oasis:entry colname="4">18, 19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255A</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">40</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M/N46G</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">32</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">24</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E/D234E</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">23</oasis:entry><oasis:entry colname="4">19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248S</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">22</oasis:entry><oasis:entry colname="4">19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N46G/K40M/N255G</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">10</oasis:entry><oasis:entry colname="4">present study
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22Q/V234A/R24N/D234A/V235A/R236T</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">2</oasis:entry><oasis:entry colname="4">present study</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic> Quantity of RTB based on P2 ELISA and product
of densitometry of Coomassie-stained gels and absorbance at 280 nm of
patially
purified protein. At least four preparations of each mutant made.
Values given are mean. Agreement between densitometry and
ELISA
within 30% in each case.</p></table-wrap-foot></table-wrap><table-wrap id="bc960056bt00002" position="float" orientation="portrait"><label>2</label><caption><p>Binding of Monoclonal Anti-RTB Antibodies to Mutants<italic toggle="yes"><sup>a</sup></italic></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="4"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry namest="3" nameend="4">% relative to
monoclonal P2</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">protein</oasis:entry><oasis:entry colname="2">type
(subdomain)</oasis:entry><oasis:entry colname="3">P8</oasis:entry><oasis:entry colname="4">P10
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E/D234E</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">60</oasis:entry><oasis:entry colname="4">100
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">wild-type</oasis:entry><oasis:entry colname="2">−</oasis:entry><oasis:entry colname="3">88</oasis:entry><oasis:entry colname="4">20
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248H</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">90</oasis:entry><oasis:entry colname="4">710
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248S</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">94</oasis:entry><oasis:entry colname="4">160
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Q35N</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">96</oasis:entry><oasis:entry colname="4">250
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">100</oasis:entry><oasis:entry colname="4">100
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22Q/V23A/R24N/D234A/
V235A/R236T</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">100</oasis:entry><oasis:entry colname="4">250
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">100</oasis:entry><oasis:entry colname="4">100
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E/A237R</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">113</oasis:entry><oasis:entry colname="4">53
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N46G/K40M</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">114</oasis:entry><oasis:entry colname="4">80
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255G</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">117</oasis:entry><oasis:entry colname="4">80
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">119</oasis:entry><oasis:entry colname="4">55
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255A</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">130</oasis:entry><oasis:entry colname="4">350
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248H</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">143</oasis:entry><oasis:entry colname="4">202
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">145</oasis:entry><oasis:entry colname="4">90
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N46G/K40M/N255G</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">200</oasis:entry><oasis:entry colname="4">600
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248S</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">250</oasis:entry><oasis:entry colname="4">80</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic> Measured by P2, P8, and P10 ELISA calibrated
with known
amounts of plant RTB. Each assay employed 5%
β-mercaptoethanol to reduce homodimers. Average of two to four assays
on
each mutant.</p></table-wrap-foot></table-wrap></p><p>Reactivites of mutant RTBs with different monoclonal
antibodies to RTB (P2, P8, and P10) were tested by
substituting different monoclonal antibodies as capture
reagents in the antibody ELISA. Equivalent results
were
observed for each antibody in most cases, suggesting
similar folding of the mutants (Table <xref rid="bc960056bt00002"></xref>).</p><p><bold>Sugar Binding of Mutant RTBs.</bold> Binding of
partially purified mutants to immobilized asialofetuin was
quantitated by ELISA, and the results are shown in
Table <xref rid="bc960056bt00003"></xref>. The two new single-site mutants
showed a less
than 1 log drop in binding relative to recombinant or
plant RTB. The two new double-site mutants showed a
close to 2 log drop in sugar binding. The limit of
detection
of the assay was a 2.5−3 log decrease in sugar binding
avidity.<table-wrap id="bc960056bt00003" position="float" orientation="portrait"><label>3</label><caption><p>Binding of Mutant RTBs to Asialofetuin<italic toggle="yes"><sup>a</sup></italic></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="4"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1">protein</oasis:entry><oasis:entry colname="2">type of
mutant</oasis:entry><oasis:entry colname="3">relative binding
to asialofetuin (%)</oasis:entry><oasis:entry colname="4">reference
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">wild-type</oasis:entry><oasis:entry colname="2">−</oasis:entry><oasis:entry colname="3">83</oasis:entry><oasis:entry colname="4">16
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255A</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">70</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Q35N</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">50</oasis:entry><oasis:entry colname="4">18, 19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">33</oasis:entry><oasis:entry colname="4">this report
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255G</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">33</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248S</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">32</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E/A237R</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">30</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">25</oasis:entry><oasis:entry colname="4">19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">24</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248S</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">20</oasis:entry><oasis:entry colname="4">19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248H</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">17</oasis:entry><oasis:entry colname="4">this report
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">15</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M/N46G</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">12</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22Q/V23A/R24N/
D234A/V235A/R236T</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">10</oasis:entry><oasis:entry colname="4">this report
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M/N46G/N255G</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">4</oasis:entry><oasis:entry colname="4">this report
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248H</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">2</oasis:entry><oasis:entry colname="4">19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E/D234E</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">2</oasis:entry><oasis:entry colname="4">19</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic> Quantity of RTB based on P2 ELISA and
asialofetuin binding
measured by asialofetuin ELISA. Both assays employed
5%
β-mercaptoethanol to reduce homodimers. Average of three to
four
assays run on each mutant. Δ32 nonsense mutant, DRA,
and
AcNPV antibody matrix eluants treated identically gave
values
of 0.01 μg/mL for both the P2 and asialofetuin ELISA.
Experimentals had P2 values of 0.2−150 μg/mL and asialofetuin
values
of 0.02−58 μg/mL. Q35N assays within 1 week of
preparation.</p></table-wrap-foot></table-wrap></p><p>An independent measure of mutant RTB binding to
glycoproteins was made by detecting mutant RTB bound
to cell surfaces. All mutants bound KB cells at 4
°C
(Figure <xref rid="bc960056bf00003"></xref>).<fig id="bc960056bf00003" position="float" orientation="portrait"><label>3</label><caption><p>Binding of mutant RTBs to KB cells. Cells were attached to polylysine-coated tissue culture dishes, and all incubations were done at 4 °C. The cells were washed with 2 mg/mL BSA in PBS and the PBS plus BSA plus 1 μg/mL purified mutant RTB or wild-type RTB, rewashed, incubated with 1:200 rabbit anti-ricin antibody (Sigma) in PBS plus BSA, rewashed, incubated with affinity-urified goat anti-(rabbit Ig) coupled to rhodamine (Jackson ImmunoResearch) at 25 μg/mL, washed again, and fixed in 3.7% formaldehyde in PBS (magnification = 250×; bar = 20 μm): (A, C, E, G, and I) without 100 μg/mL asialofetuin and (B, D, F, H, and J) with asialofetuin. (A and B) wild-type RTB, (C and D) D234E, (E and F) Y248H, (G and H) N46G/K40M/N255G, and (I and J) D22Q/V23A/R24N/D234A/V235A/R236T.</p></caption><graphic xlink:href="bc960056bf00003.gif" position="float" orientation="portrait"></graphic></fig></p><p><bold>Competition Experiments.</bold> Binding of
recombinant
proteins to immobilized asialofetuin was performed in the
presence of 100 mM α-lactose or 100 μg/mL
asialofetuin.
In each case, the sugar binding of the RTB protein
was
inhibited by a competitor (Table <xref rid="bc960056bt00004"></xref>).
Lactose blocked wild-type and mutant RTB sugar binding between 2- and 81-
fold. Asialofetuin blocked sugar
binding 3−400-fold. The
inhibition of soluble carbohydrates of double-site mutant
binding was a lower estimate as the assay sensitivity was
only 5−10-fold less than the observed binding to immobilized asialofetuin in the absence of added sugars for
four of the double-site mutants. Each experiment was
repeated three times, and 12 different concentrations of
recombinant protein and plant RTB were used to compare half-maximal binding concentrations. Binding of
mutant RTBs to KB cells was blocked by 100 μg/mL
asialofetuin (Figure <xref rid="bc960056bf00003"></xref>).<table-wrap id="bc960056bt00004" position="float" orientation="portrait"><label>4</label><caption><p>Competition of Mutant RTB Lectin Binding by Sugars<italic toggle="yes"><sup>a</sup></italic></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="4"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry namest="3" nameend="4">fold inhibition</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">protein</oasis:entry><oasis:entry colname="2">type
(subdomain)</oasis:entry><oasis:entry colname="3">lactose</oasis:entry><oasis:entry colname="4">asialofetuin
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Q35N</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">81</oasis:entry><oasis:entry colname="4">150
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248H</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">27</oasis:entry><oasis:entry colname="4">9
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M/N46G</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">27</oasis:entry><oasis:entry colname="4">50
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">27</oasis:entry><oasis:entry colname="4">243
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">27</oasis:entry><oasis:entry colname="4">100
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255A</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">15</oasis:entry><oasis:entry colname="4">150
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">15</oasis:entry><oasis:entry colname="4">81
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">9</oasis:entry><oasis:entry colname="4">15
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">wild-type</oasis:entry><oasis:entry colname="2">−</oasis:entry><oasis:entry colname="3">9</oasis:entry><oasis:entry colname="4">400
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248S</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">9</oasis:entry><oasis:entry colname="4">243
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255G</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">9</oasis:entry><oasis:entry colname="4">243
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22Q/V23A/R24N/D234A/
V235A/R236T</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">9</oasis:entry><oasis:entry colname="4">9
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M/N46G/N255G</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">3</oasis:entry><oasis:entry colname="4">3
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248S</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">3</oasis:entry><oasis:entry colname="4">200
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248H</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">3</oasis:entry><oasis:entry colname="4">5
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E/A237R</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">3</oasis:entry><oasis:entry colname="4">150
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E/D234E</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">2</oasis:entry><oasis:entry colname="4">3</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic> Asialofetuin ELISA performed in the presence
or absence of
100 mM α-lactose or 100 μg/mL asialofetuin. Twelve
different
concentrations of each protein tested and half-maximal
binding
concentrations compared to assess fold inhibition of
binding.</p></table-wrap-foot></table-wrap></p><p><bold>Reassociation of Mutant RTBs with Plant RTA.</bold>
Incubation of mutant RTBs at 3 × 10<sup>-8</sup> to 3 ×
10<sup>-7</sup> M
with plant RTA at a 4-fold molar excess overnight at
room temperature led to 11−100% reassociation (Table
<xref rid="bc960056bt00005"></xref> and Figure <xref rid="bc960056bf00004"></xref>).
Similar levels of reassociation were seen
using plant RTB or recombinant wild-type RTB with
plant RTA at the same concentrations. The heterodimer
concentrations were quantitated by a modified ELISA
which identified molecules with both RTA and RTB
epitopes and by densitometry of 65 kDa bands of immunoblots with anti-RTB or anti-RTA antibodies. Both
ELISA and immunoblots gave similar values and showed
all mutants reassociated well with plant RTA and had
minimal homodimer formation.<fig id="bc960056bf00004" position="float" orientation="portrait"><label>4</label><caption><p>Reassociation of mutant RTBs with plant RTA. (A) Immunoblots of 15% nonreducing SDS−PAGE of reassociated mutant RTB−plant RTA. Low-molecular mass BioRad protein standards are 106, 80, 49.5, 32.5, 27.5, and 18.5 kDa. The heterodimer appears at 60 kDa, and subunits appear at 30 kDa. Immunoblot uses monoclonal antibody P2 and P10 anti-RTB. (B) Same as panel A except immunoblot uses monoclonal antibody αBR12 anti-RTA: lane 1, low-molecular mass prestained BioRad protein standards; lane 2, D234E−RTA; lane 3, Y248H−RTA; lane 4, N46G/K40M/N255G−RTA; lane 5, D22Q/V23A/R24N/D234A/V235A/R236T−RTA; and lane 6, ricin.</p></caption><graphic xlink:href="bc960056bf00004.gif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bc960056bt00005" position="float" orientation="portrait"><label>5</label><caption><p>Reassociation of Mutant RTBs with RTA<italic toggle="yes"><sup>a</sup></italic></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="3"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1">protein</oasis:entry><oasis:entry colname="2">type
(subdomain)</oasis:entry><oasis:entry colname="3">heterodimer
formed (%)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">11
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N46G/K40M</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">22
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248S</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">25
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">25
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">27
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255A</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">34
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248S</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">35
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248H</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">43
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">wild-type</oasis:entry><oasis:entry colname="2">−</oasis:entry><oasis:entry colname="3">45
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E/D234E</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">50
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E/A237R</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">50
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Q35N</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">65
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248H</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">70
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">70
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M/N46G/N255G</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">81
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22Q/V23A/R24N/D234A/
V235A/R236T</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">90
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255G</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">100</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic> Assayed by modified ricin ELISA using P2
anti-RTB antibody
capture and biotinylated αBR12 anti-RTA antibody
detection
reagent and confirmed by densitometry of immunoblots of
nonreducing SDS−PAGE.</p></table-wrap-foot></table-wrap></p><p><bold>Cytotoxicity of Mutant Heterodimers.</bold> The
ID<sub>50</sub> of
ricin and recombinant mutant RTB−RTA heterodimers
on HUT102 human leukemia cells is shown in Figure
<xref rid="bc960056bf00005"></xref>
and Table <xref rid="bc960056bt00006"></xref>. The reduction in potency for
single-site and
double-site mutant RTB−RTA heterodimers compared to
that for wild-type RTB−RTA and plant ricin was 1−2
log. Nevertheless, the potency for all the mutants
was
at least 3 log more than that for wild-type RTA or RTB
alone.
<fig id="bc960056bf00005" position="float" orientation="portrait"><label>5</label><caption><p>HUT102 cell cytotoxicity. Assay as described in text: (·) ricin, IC<sub>50</sub> = 4 × 10<sup>-12</sup> M;
(○) D234E−RTA, IC<sub>50</sub> = 1
× 10<sup>-10</sup> M; (▪) Y248H−RTA, IC<sub>50</sub> = 8
× 10<sup>-11</sup> M; (□) N46G/K40M/N255G−RTA, IC<sub>50</sub> = 2 × 10<sup>-10</sup> M; and
(▴) D22Q/V23A/R24N/D234A/V235A/R236T−RTA, IC<sub>50</sub> = 5 ×
10<sup>-11</sup> M. Yield
of reassociated heterodimer tested by modified ELISA.
Each
experiment performed in triplicate.</p></caption><graphic xlink:href="bc960056bf00005.gif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bc960056bt00006" position="float" orientation="portrait"><label>6</label><caption><p>HUT102 Cell Sensitivity to Recombinant Heterodimers<italic toggle="yes"><sup>a</sup></italic></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="3"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1">protein</oasis:entry><oasis:entry colname="2">type
(subdomain)</oasis:entry><oasis:entry colname="3">IC<sub>50</sub> (M)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">wild-type</oasis:entry><oasis:entry colname="2">−</oasis:entry><oasis:entry colname="3">5 × 10<sup>-12</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248S−RTA</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">1 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S−RTA</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">2 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Q35N−RTA</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">2 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248S−RTA</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">5 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M/N46G−RTA</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">5 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22Q/V23A/R24N/D234A/
V235A/R236T−RTA</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">5 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Y248H−RTA</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">8 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E/A237R−RTA</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">8 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255G−RTA</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">8 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N255A−RTA</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">8 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E/D234E−RTA</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">9 × 10<sup>-11</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M−RTA</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">1 × 10<sup>-10</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D234E−RTA</oasis:entry><oasis:entry colname="2">2γ</oasis:entry><oasis:entry colname="3">1 × 10<sup>-10</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D22E−RTA</oasis:entry><oasis:entry colname="2">1α</oasis:entry><oasis:entry colname="3">2 × 10<sup>-10</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">K40M/N46G/N255G−RTA</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">2 × 10<sup>-10</sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">W37S/Y248H−RTA</oasis:entry><oasis:entry colname="2">1α, 2γ</oasis:entry><oasis:entry colname="3">2 × 10<sup>-10</sup></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic> HUT102 cell cytotoxicity as described in text.
Ricin IC<sub>50</sub> was
4 × 10<sup>-12</sup> M.</p></table-wrap-foot></table-wrap></p></sec><sec id="d7e1721"><title>Discussion</title><p>The molecular mechanism of interaction between ricin
and its saccharide receptors on the cell surface is the
first
necessary step in intoxication of cells.
Determination of
the number of lectin sites on RTB and their affinities and
participating amino acid residues are important both to
define the structure−function relationships of this and
other plant lectins, to interpret intracellular routing
signals, and to design and synthesize genetically engineered cell-selective toxins.</p><p>While initial studies with ricin by equilibrium dialysis
(<italic toggle="yes"><xref rid="bc960056bb00025" ref-type="bibr"></xref></italic>) and mutational analysis (<italic toggle="yes"><xref rid="bc960056bb00013" ref-type="bibr"></xref></italic>) suggested a
single
lectin site per molecule, subsequent equilibrium dialysis
measurements (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00004" ref-type="bibr"></xref>, <xref rid="bc960056bb00026" ref-type="bibr"></xref></named-content></italic>), X-ray diffraction analysis
(<italic toggle="yes"><xref rid="bc960056bb00009" ref-type="bibr"></xref></italic>),
chemical modification studies (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00010" ref-type="bibr"></xref>, <xref rid="bc960056bb00011" ref-type="bibr"></xref></named-content></italic>), and
mutational
analyses (<italic toggle="yes">14</italic>, <italic toggle="yes">15</italic>, <italic toggle="yes">23</italic>) suggested two
lectin sites in subdomains 1α and 2γ. However, for the mutational
analyses, no quantitation of soluble immunoreactive proteins
was reported, and hence, the true specific activities of
the recombinant proteins were unknown. If misfolding
with aggregation or proteolytic degradation occurred, no
residual lectin activity would be seen even if three or
more lectin sites were present.</p><p>To more accurately measure sugar binding avidities
of recombinant RTB proteins, we produced and purified
wild-type and mutant RTBs from insect cells using the
baculovirus system and monoclonal antibody affinity
chromatography (<italic toggle="yes">16</italic>−<italic toggle="yes">19</italic>, present study). One
log decreases in asialofetuin binding were observed with single-site mutants, and in 4/5 cases, 2 log decreases in
asialofetuin binding were seen with double-site mutants.
The persistent lactose and asialofetuin-inhibitable
sugar
and cell binding of a number of double-site (subdomain
1α and 2γ) mutants suggests either incomplete
inactivation of the two sites in the mutants or the existence of a
third lectin site. Since seven different residues in
one
subdomain (1α) and seven different residues in the other
domain (2γ) were tested (Figure <xref rid="bc960056bf00001"></xref>), we believe the first
hypothesis is unlikely and that ricin has three or more
lectin binding sites. Possible third lectin sites
include
subdomain 1β with aromatic ring residue
Y78 and a
lectin pocket kink and subdomain 2α with aromatic
residue W160, kink residues, and polar residues D153,
E170, and Q171. Ongoing mutational analysis using the
insect expression system should help address this question.</p><p>Other observations support the hypothesis of three
lectin sites on ricin. Three distinct sites on ricin
were
cross-linked by radiolabeled fetuin glycopeptides containing a dichlorotriazine-activated
6-(<italic toggle="yes">N</italic>-methylamino)-6-deoxy-<sc>d</sc>-galactose moiety (<italic toggle="yes"><xref rid="bc960056bb00012" ref-type="bibr"></xref></italic>). When ricin
molecules
containing either two or three affinity cross-linkers were
conjugated to monoclonal antibodies and tested for toxicity <italic toggle="yes">in vitro</italic> and <italic toggle="yes">in vivo</italic>, marked differences were
observed
(<italic toggle="yes"><xref rid="bc960056bb00027" ref-type="bibr"></xref></italic>). The residual nonspecific toxicity <italic toggle="yes">in
vitro</italic> of the
doubly blocked ricin conjugate could be blocked with
excess lactose.</p><p>The finding of three or more independent sugar-combining sites on a lectin is not unique to ricin and has
been reported for hepatic galactose
<italic toggle="yes">N</italic>-acetylgalactosamine
receptor and macrophage mannose receptor (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc960056bb00007" ref-type="bibr"></xref>, <xref rid="bc960056bb00008" ref-type="bibr"></xref></named-content></italic>).
The
results also have implications in the role of the residual
sugar-combining site of blocked ricin conjugates in intracellular trafficking (<italic toggle="yes"><xref rid="bc960056bb00028" ref-type="bibr"></xref></italic>) and in the genetic
engineering
of tumor cell selective ricin fusion toxins
(<italic toggle="yes"><xref rid="bc960056bb00029" ref-type="bibr"></xref></italic>).
</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank Joseph Vesely and Billie Harris for excellent
technical assistance, Starr Hazard for molecular graphics
analysis, James Nicholson for imaging analysis, Dr.
Walter Blattler for the anti-ricin monoclonal antibodies,
and Dr. Jerry Fulton for the plant ricin and ricin
subunits.
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Telephone: 803-792-1450.Fax: 803-792-3200.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">1996-11-27</dateCreated><dateIssued encoding="w3cdtf">1996-11-27</dateIssued><copyrightDate encoding="w3cdtf">1996</copyrightDate></originInfo><note type="footnote" ID="bc960056bAF7"> Abstract published in Advance ACS Abstracts, November 1, 1996.</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><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. 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