Recombinant human thrombomodulincsa+: a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate
Identifieur interne : 001D18 ( Istex/Corpus ); précédent : 001D17; suivant : 001D19Recombinant human thrombomodulincsa+: a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate
Auteurs : Daniel Parzy ; Thierry Fusaï ; Bruno Pouvelle ; Marilyne Torrentino ; Frédéric Eustacchio ; Catherine Lépolard ; Artur Scherf ; Jürg GysinSource :
- Microbes and Infection [ 1286-4579 ] ; 2000.
English descriptors
Abstract
The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-Plasmodium falciparum-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies.
Url:
DOI: 10.1016/S1286-4579(00)90357-5
Links to Exploration step
ISTEX:D6C8872F4A2C891D308F233A4FDFCBEDC89DD785Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Recombinant human thrombomodulincsa+: a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate</title><author><name sortKey="Parzy, Daniel" sort="Parzy, Daniel" uniqKey="Parzy D" first="Daniel" last="Parzy">Daniel Parzy</name><affiliation><mods:affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</mods:affiliation></affiliation></author><author><name sortKey="Fusai, Thierry" sort="Fusai, Thierry" uniqKey="Fusai T" first="Thierry" last="Fusaï">Thierry Fusaï</name><affiliation><mods:affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</mods:affiliation></affiliation></author><author><name sortKey="Pouvelle, Bruno" sort="Pouvelle, Bruno" uniqKey="Pouvelle B" first="Bruno" last="Pouvelle">Bruno Pouvelle</name><affiliation><mods:affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</mods:affiliation></affiliation></author><author><name sortKey="Torrentino, Marilyne" sort="Torrentino, Marilyne" uniqKey="Torrentino M" first="Marilyne" last="Torrentino">Marilyne Torrentino</name><affiliation><mods:affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</mods:affiliation></affiliation></author><author><name sortKey="Eustacchio, Frederic" sort="Eustacchio, Frederic" uniqKey="Eustacchio F" first="Frédéric" last="Eustacchio">Frédéric Eustacchio</name><affiliation><mods:affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</mods:affiliation></affiliation></author><author><name sortKey="Lepolard, Catherine" sort="Lepolard, Catherine" uniqKey="Lepolard C" first="Catherine" last="Lépolard">Catherine Lépolard</name><affiliation><mods:affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</mods:affiliation></affiliation></author><author><name sortKey="Scherf, Artur" sort="Scherf, Artur" uniqKey="Scherf A" first="Artur" last="Scherf">Artur Scherf</name><affiliation><mods:affiliation>Unité de Biologie des Interactions Hôte-Parasite, CNRS URA 1960, Institut Pasteur, 75724 Paris, France</mods:affiliation></affiliation></author><author><name sortKey="Gysin, Jurg" sort="Gysin, Jurg" uniqKey="Gysin J" first="Jürg" last="Gysin">Jürg Gysin</name><affiliation><mods:affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:D6C8872F4A2C891D308F233A4FDFCBEDC89DD785</idno><date when="2000" year="2000">2000</date><idno type="doi">10.1016/S1286-4579(00)90357-5</idno><idno type="url">https://api.istex.fr/document/D6C8872F4A2C891D308F233A4FDFCBEDC89DD785/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">001D18</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001D18</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Recombinant human thrombomodulincsa+: a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate</title><author><name sortKey="Parzy, Daniel" sort="Parzy, Daniel" uniqKey="Parzy D" first="Daniel" last="Parzy">Daniel Parzy</name><affiliation><mods:affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</mods:affiliation></affiliation></author><author><name sortKey="Fusai, Thierry" sort="Fusai, Thierry" uniqKey="Fusai T" first="Thierry" last="Fusaï">Thierry Fusaï</name><affiliation><mods:affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</mods:affiliation></affiliation></author><author><name sortKey="Pouvelle, Bruno" sort="Pouvelle, Bruno" uniqKey="Pouvelle B" first="Bruno" last="Pouvelle">Bruno Pouvelle</name><affiliation><mods:affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</mods:affiliation></affiliation></author><author><name sortKey="Torrentino, Marilyne" sort="Torrentino, Marilyne" uniqKey="Torrentino M" first="Marilyne" last="Torrentino">Marilyne Torrentino</name><affiliation><mods:affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</mods:affiliation></affiliation></author><author><name sortKey="Eustacchio, Frederic" sort="Eustacchio, Frederic" uniqKey="Eustacchio F" first="Frédéric" last="Eustacchio">Frédéric Eustacchio</name><affiliation><mods:affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</mods:affiliation></affiliation></author><author><name sortKey="Lepolard, Catherine" sort="Lepolard, Catherine" uniqKey="Lepolard C" first="Catherine" last="Lépolard">Catherine Lépolard</name><affiliation><mods:affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</mods:affiliation></affiliation></author><author><name sortKey="Scherf, Artur" sort="Scherf, Artur" uniqKey="Scherf A" first="Artur" last="Scherf">Artur Scherf</name><affiliation><mods:affiliation>Unité de Biologie des Interactions Hôte-Parasite, CNRS URA 1960, Institut Pasteur, 75724 Paris, France</mods:affiliation></affiliation></author><author><name sortKey="Gysin, Jurg" sort="Gysin, Jurg" uniqKey="Gysin J" first="Jürg" last="Gysin">Jürg Gysin</name><affiliation><mods:affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Microbes and Infection</title><title level="j" type="abbrev">MICINF</title><idno type="ISSN">1286-4579</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000">2000</date><biblScope unit="volume">2</biblScope><biblScope unit="issue">7</biblScope><biblScope unit="page" from="779">779</biblScope><biblScope unit="page" to="788">788</biblScope></imprint><idno type="ISSN">1286-4579</idno></series><idno type="istex">D6C8872F4A2C891D308F233A4FDFCBEDC89DD785</idno><idno type="DOI">10.1016/S1286-4579(00)90357-5</idno><idno type="PII">S1286-4579(00)90357-5</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1286-4579</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Plasmodium falciparum</term><term>chondroitin-4-sulfate</term><term>cytoadhesion</term><term>receptors</term><term>thrombomodulin</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-Plasmodium falciparum-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>Daniel Parzy</name><affiliations><json:string>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</json:string></affiliations></json:item><json:item><name>Thierry Fusaï</name><affiliations><json:string>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</json:string></affiliations></json:item><json:item><name>Bruno Pouvelle</name><affiliations><json:string>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</json:string></affiliations></json:item><json:item><name>Marilyne Torrentino</name><affiliations><json:string>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</json:string></affiliations></json:item><json:item><name>Frédéric Eustacchio</name><affiliations><json:string>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</json:string></affiliations></json:item><json:item><name>Catherine Lépolard</name><affiliations><json:string>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</json:string></affiliations></json:item><json:item><name>Artur Scherf</name><affiliations><json:string>Unité de Biologie des Interactions Hôte-Parasite, CNRS URA 1960, Institut Pasteur, 75724 Paris, France</json:string></affiliations></json:item><json:item><name>Jürg Gysin</name><affiliations><json:string>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>chondroitin-4-sulfate</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>thrombomodulin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>cytoadhesion</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Plasmodium falciparum</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>receptors</value></json:item></subject><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-Plasmodium falciparum-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies.</abstract><qualityIndicators><score>8</score><pdfVersion>1.2</pdfVersion><pdfPageSize>580 x 765 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>5</keywordCount><abstractCharCount>2042</abstractCharCount><pdfWordCount>5524</pdfWordCount><pdfCharCount>34455</pdfCharCount><pdfPageCount>10</pdfPageCount><abstractWordCount>267</abstractWordCount></qualityIndicators><title>Recombinant human thrombomodulincsa+: a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate</title><pii><json:string>S1286-4579(00)90357-5</json:string></pii><refBibs><json:item><author><json:item><name>M Aikawa</name></json:item></author><host><volume>20</volume><pages><last>10</last><first>3</first></pages><author></author><title>AMJ Trop. Med. Hyg.</title></host><title>Human cerebral malaria</title></json:item><json:item><author><json:item><name>H Fujioka</name></json:item><json:item><name>M Aikawa</name></json:item></author><host><volume>146</volume><pages><last>72</last><first>63</first></pages><author></author><title>Microb. Pathog.</title></host><title>The molecular basis of pathogenesis of cerebral malaria</title></json:item><json:item><author><json:item><name>C Robert</name></json:item><json:item><name>B Pouvelle</name></json:item><json:item><name>P Meyer</name></json:item><json:item><name>K Muanza</name></json:item><json:item><name>H Fujioka</name></json:item><json:item><name>M Aikawa</name></json:item><json:item><name>A Scherf</name></json:item><json:item><name>J Gysin</name></json:item></author><host><volume>147</volume><pages><last>393</last><first>383</first></pages><author></author><title>Res. Immunol.</title></host><title>Chondroitin-4-sulphate (proteoglycan), a receptor for Plasmodium falciparum-infected erythrocyte adherence on brain microvascular endothelial cells</title></json:item><json:item><author><json:item><name>K Muanza</name></json:item><json:item><name>F Gay</name></json:item><json:item><name>C Behr</name></json:item><json:item><name>A Scherf</name></json:item></author><host><volume>182</volume><pages><last>163</last><first>149</first></pages><author></author><title>Res. Immunol.</title></host><title>Primary culture of human lung microvessel endothelial cells: a useful in vitro model for studying Plasmodium falciparum-infected erythrocyte cytoadherence</title></json:item><json:item><author><json:item><name>S.J Rogerson</name></json:item><json:item><name>S.C Chaiyaroj</name></json:item><json:item><name>K Ng</name></json:item><json:item><name>J.C Reeder</name></json:item><json:item><name>G.V Brown</name></json:item></author><host><volume>272</volume><pages><last>20</last><first>15</first></pages><author></author><title>J. Exp. M</title></host><title>Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes</title></json:item><json:item><author><json:item><name>M Fried</name></json:item><json:item><name>P.E Duffy</name></json:item></author><host><volume>86</volume><pages><last>1504</last><first>1502</first></pages><author></author><title>Science</title></host><title>Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta</title></json:item><json:item><author><json:item><name>S.J Rogerson</name></json:item><json:item><name>S Novakovic</name></json:item><json:item><name>B.M Cooke</name></json:item><json:item><name>G.V Brown</name></json:item></author><host><volume>88</volume><pages><last>18</last><first>8</first></pages><author></author><title>Exp. Parasitol.</title></host><title>Plasmodium falciparum-infected erythrocytes adhere to the proteoglycan thrombomodulin in static and flow-based systems</title></json:item><json:item><author><json:item><name>J Gysin</name></json:item><json:item><name>B Pouvelle</name></json:item><json:item><name>M Le Tonqueze</name></json:item><json:item><name>L Edelman</name></json:item><json:item><name>M.C Boffa</name></json:item></author><host><volume>58</volume><pages><last>271</last><first>267</first></pages><author></author><title>Mol. Biochem. Parasitol.</title></host><title>Chondroitin sulfate of thrombomodulin is an adhesion receptor for Plasmodium falciparum-infected erythrocytes</title></json:item><json:item><author><json:item><name>B Pouvelle</name></json:item><json:item><name>T Fusai</name></json:item><json:item><name>J Gysin</name></json:item></author><host><volume>33</volume><pages><last>198</last><first>187</first></pages><author></author><title>Med. Trop.</title></host><title>Plasmodium falciparum and chondroitin-4-sulfate: the new key couple in sequestration</title></json:item><json:item><author><json:item><name>M.C Boffa</name></json:item><json:item><name>R.W Jackman</name></json:item><json:item><name>N Peyri</name></json:item><json:item><name>J.F Boffa</name></json:item><json:item><name>B George</name></json:item></author><host><volume>556</volume><pages><last>429</last><first>423</first></pages><author></author><title>Nouv. Rev. Fr. Hematol.</title></host><title>Thrombomodulin in the central nervous system</title></json:item><json:item><author><json:item><name>V.L.Y Wong</name></json:item><json:item><name>F.M Hofman</name></json:item><json:item><name>H Ishii</name></json:item><json:item><name>M Fisher</name></json:item></author><host><volume>55</volume><pages><last>5</last><first>1</first></pages><author></author><title>Brain Res.</title></host><title>Regional distribution of thrombomodulin in human brain</title></json:item><json:item><author><json:item><name>S.C Chaiyaroj</name></json:item><json:item><name>P Angkasekwinai</name></json:item><json:item><name>A Buranakiti</name></json:item><json:item><name>S Looareesuwan</name></json:item><json:item><name>S.J Rogerson</name></json:item><json:item><name>G.V Brown</name></json:item></author><host><volume>66</volume><pages><last>80</last><first>76</first></pages><author></author><title>Am. J. Trop. Med. Hyg.</title></host><title>Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate a as a cytoadherence receptor</title></json:item><json:item><author><json:item><name>B Pouvelle</name></json:item><json:item><name>T Fusai</name></json:item><json:item><name>C Lepolard</name></json:item><json:item><name>J Gysin</name></json:item></author><host><volume>66</volume><pages><last>4956</last><first>4950</first></pages><author></author><title>Infect. Immun.</title></host><title>Biological and biochemical characteristics of cytoadhesion of Plasmodium falciparum-infected erythrocytes to chondroitin-4-sulfate</title></json:item><json:item><author><json:item><name>J Gysin</name></json:item><json:item><name>M Hommel</name></json:item><json:item><name>L.P Da Silva</name></json:item></author><host><volume>32</volume><pages><last>1009</last><first>1003</first></pages><author></author><title>J. Parasitol.</title></host><title>Experimental infection of the squirrel monkey (Saimiri sciureus) with Plasmodium falciparum</title></json:item><json:item><author><json:item><name>J Gysin</name></json:item><json:item><name>T Fandeur</name></json:item></author><host><volume>17</volume><pages><last>467</last><first>461</first></pages><author></author><title>Am. J. Trop. Med. Hyg.</title></host><title>Saimiri sciureus (karyotype 14-7): an alternative experimental model of Plasmodium falciparum infection</title></json:item><json:item><author><json:item><name>A Scherf</name></json:item><json:item><name>R Hernandez-Rivas</name></json:item><json:item><name>P Buffet</name></json:item><json:item><name>E Bottius</name></json:item><json:item><name>C Benatar</name></json:item><json:item><name>B Pouvelle</name></json:item><json:item><name>J Gysin</name></json:item><json:item><name>M Lanzer</name></json:item></author><host><volume>184</volume><pages><last>5426</last><first>5418</first></pages><author></author><title>Embo J.</title></host><title>Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum</title></json:item><json:item><author><json:item><name>F Gay</name></json:item><json:item><name>C Robert</name></json:item><json:item><name>B Pouvelle</name></json:item><json:item><name>S Peyrol</name></json:item><json:item><name>A Scherf</name></json:item><json:item><name>J Gysin</name></json:item></author><host><volume>103</volume><pages><last>28</last><first>15</first></pages><author></author><title>J. Immunol. Meth.</title></host><title>Isolation and characterization of brain microvascular endothelial cells from Saimiri monkeys. An in vitro model for sequestration of Plasmodium falciparum-infected erythrocytes</title></json:item><json:item><author><json:item><name>T Shirai</name></json:item><json:item><name>S Shiojiri</name></json:item><json:item><name>H Ito</name></json:item><json:item><name>S Yamamoto</name></json:item><json:item><name>H Kusomoto</name></json:item><json:item><name>Y Deyashiki</name></json:item><json:item><name>I Maruyama</name></json:item><json:item><name>K Suzuki</name></json:item></author><host><volume>74</volume><pages><last>285</last><first>281</first></pages><author></author><title>J. Biochem.</title></host><title>Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C</title></json:item><json:item><author><json:item><name>F Sanger</name></json:item><json:item><name>S Nicken</name></json:item><json:item><name>A.R Coulson</name></json:item></author><host><volume>90</volume><pages><last>5467</last><first>5463</first></pages><author></author><title>Proc. Natl. Acad. Sci. USA</title></host><title>DNA sequencing with chain-terminating inhibitors</title></json:item><json:item><author><json:item><name>C.B Glaser</name></json:item><json:item><name>J Morser</name></json:item><json:item><name>J.H Clarke</name></json:item><json:item><name>E Blasko</name></json:item><json:item><name>K Mc Lean</name></json:item><json:item><name>I Khun</name></json:item><json:item><name>R.J Chang</name></json:item><json:item><name>J.H Lin</name></json:item><json:item><name>L Vilander</name></json:item><json:item><name>W.H Andrews</name></json:item><json:item><name>D.R Light</name></json:item></author><host><volume>15</volume><pages><last>2573</last><first>2565</first></pages><author></author><title>J. Clin. Invest.</title></host><title>Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation</title></json:item><json:item><author><json:item><name>J.E Shively</name></json:item><json:item><name>H Conrad</name></json:item></author><host><volume>3</volume><pages><last>3942</last><first>3932</first></pages><author></author><title>Biochemistry</title></host><title>Formation of anhydrosugars in the chemical depolymerization of heparin</title></json:item><json:item><author><json:item><name>M Maccarana</name></json:item><json:item><name>U Lindhal</name></json:item></author><host><volume>27</volume><pages><last>277</last><first>271</first></pages><author></author><title>Glycobiology</title></host><title>Mode of interaction between platelet factor 4 and heparin</title></json:item><json:item><author><json:item><name>J.B Jensen</name></json:item><json:item><name>W Trager</name></json:item></author><host><volume>268</volume><pages><last>746</last><first>743</first></pages><author></author><title>Am. J. Trop. Med. Hyg.</title></host><title>Plasmodium falciparum in culture: establishment of additional strains</title></json:item><json:item><author><json:item><name>N Sugiura</name></json:item><json:item><name>K Sakurai</name></json:item><json:item><name>Y Hori</name></json:item><json:item><name>K Karasawa</name></json:item><json:item><name>S Suzuki</name></json:item><json:item><name>K Kimata</name></json:item></author><host><volume>259</volume><pages><last>15787</last><first>15779</first></pages><author></author><title>J. Biol. Chem.</title></host><title>Preparation of lipid-derivatized glycosaminoglycans to probe a regulatory function of the carbohydrate moieties of proteoglycans in cell-matrix interaction</title></json:item><json:item><author><json:item><name>K.C Carter</name></json:item><json:item><name>D Bowman</name></json:item><json:item><name>W Carrington</name></json:item><json:item><name>K Fogarty</name></json:item><json:item><name>J.A Mc Neil</name></json:item><json:item><name>F.S Fay</name></json:item><json:item><name>J.B Lawrence</name></json:item></author><host><volume>264</volume><pages><last>1335</last><first>1330</first></pages><author></author><title>Science</title></host><title>A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus [see comments]</title></json:item><json:item><author><json:item><name>D.J Stearns</name></json:item><json:item><name>S Kurosawa</name></json:item><json:item><name>C.T Esmon</name></json:item></author><host><volume>78</volume><pages><last>3356</last><first>3352</first></pages><author></author><title>J. Biol. Chem.</title></host><title>Microthrombomodulin. Residues 310-486 from the epidermal growth factor precursor homology domain of thrombomodulin will accelerate protein C activation</title></json:item><json:item><author><json:item><name>C.T Esmon</name></json:item><json:item><name>W.G Owen</name></json:item></author><host><volume>257</volume><pages><last>2252</last><first>2249</first></pages><author></author><title>Proc. Natl. Acad. Sci. USA</title></host><title>Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C</title></json:item><json:item><author><json:item><name>N.L Esmon</name></json:item><json:item><name>W.G Owen</name></json:item><json:item><name>C.T Esmon</name></json:item></author><host><volume>270</volume><pages><last>864</last><first>859</first></pages><author></author><title>J. Biol. Chem.</title></host><title>Isolation of a membrane-bound cofactor for thrombin catalyzed activation of protein C</title></json:item><json:item><author><json:item><name>M.C Bourin</name></json:item><json:item><name>U Lindahl</name></json:item></author><host><volume>265</volume><pages><last>425</last><first>419</first></pages><author></author><title>Biochem. J.</title></host><title>Functional-role of the polysaccharide component of rabbit thrombomodulin proteoglycan. Effects on inactivation of thrombin by antithrombin, cleavage of fibrinogen by thrombin and thrombin-catalyzed activation of factor-V</title></json:item><json:item><author><json:item><name>M.C Bourin</name></json:item><json:item><name>E Lundgren-Akerlund</name></json:item><json:item><name>U Lindahl</name></json:item></author><host><volume>3</volume><pages><last>15431</last><first>15424</first></pages><author></author><title>J. Biol. Chem.</title></host><title>Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan</title></json:item><json:item><author><json:item><name>B Pouvelle</name></json:item><json:item><name>P Meyer</name></json:item><json:item><name>C Robert</name></json:item><json:item><name>L Bardel</name></json:item><json:item><name>J Gysin</name></json:item></author><host><volume>66</volume><pages><last>518</last><first>508</first></pages><author></author><title>Mol. M</title></host><title>Chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of Plasmodium falciparum infected erythrocytes</title></json:item><json:item><author><json:item><name>J.G Beeson</name></json:item><json:item><name>W Chai</name></json:item><json:item><name>S.J Rogerson</name></json:item><json:item><name>A.M Lawson</name></json:item><json:item><name>G.V Brown</name></json:item></author><host><volume>283</volume><pages><last>3402</last><first>3397</first></pages><author></author><title>Infect. Immun.</title></host><title>Inhibition of binding of malaria-infected erythrocytes by a tetradecasaccharide fraction from chondroitin sulfate A</title></json:item><json:item><author><json:item><name>J.F Parkinson</name></json:item><json:item><name>C.J Vlahos</name></json:item><json:item><name>S.C Yan</name></json:item><json:item><name>N.U Bang</name></json:item></author><host><volume>295</volume><pages><last>157</last><first>151</first></pages><author></author><title>Biochem. J.</title></host><title>Recombinant human thrombomodulin. Regulation of cofactor activity and anticoagulant function by a glycosaminoglycan side chain</title></json:item><json:item><author><json:item><name>B Gerlitz</name></json:item><json:item><name>T Hassell</name></json:item><json:item><name>C.J Vlahos</name></json:item><json:item><name>J.F Parkinson</name></json:item><json:item><name>N.U Bang</name></json:item><json:item><name>B.W Grinnell</name></json:item></author><host><volume>96</volume><pages><last>140</last><first>131</first></pages><author></author><title>Biochem. J.</title></host><title>Identification of the predominant glycosaminoglycan-attachment site in soluble recombinant human thrombomodulin: potential regulation of functionality by glycosyltransferase competition for serine474</title></json:item><json:item><author><json:item><name>P.A Buffet</name></json:item><json:item><name>B Gamain</name></json:item><json:item><name>C Scheidig</name></json:item><json:item><name>D Baruch</name></json:item><json:item><name>J.D Smith</name></json:item><json:item><name>R Hernandez-Rivas</name></json:item><json:item><name>B Pouvelle</name></json:item><json:item><name>S Oishi</name></json:item><json:item><name>N Fujii</name></json:item><json:item><name>T Fusai</name></json:item><json:item><name>D Parzy</name></json:item><json:item><name>L.H Miller</name></json:item><json:item><name>J Gysin</name></json:item><json:item><name>A Scherf</name></json:item></author><host><pages><last>12748</last><first>12743</first></pages><author></author><title>Proc. Natl. Acad. Sci. USA</title></host><title>Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: A receptor for human placental infection</title></json:item></refBibs><genre><json:string>research-article</json:string></genre><host><volume>2</volume><pii><json:string>S1286-4579(00)X0013-5</json:string></pii><pages><last>788</last><first>779</first></pages><issn><json:string>1286-4579</json:string></issn><issue>7</issue><genre><json:string>journal</json:string></genre><language><json:string>unknown</json:string></language><title>Microbes and Infection</title><publicationDate>2000</publicationDate></host><publicationDate>2000</publicationDate><copyrightDate>2000</copyrightDate><doi><json:string>10.1016/S1286-4579(00)90357-5</json:string></doi><id>D6C8872F4A2C891D308F233A4FDFCBEDC89DD785</id><score>0.14229298</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/document/D6C8872F4A2C891D308F233A4FDFCBEDC89DD785/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/document/D6C8872F4A2C891D308F233A4FDFCBEDC89DD785/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/D6C8872F4A2C891D308F233A4FDFCBEDC89DD785/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Recombinant human thrombomodulincsa+: a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>©2000 Éditions scientifiques et médicales Elsevier SAS</p></availability><date>2000</date></publicationStmt><notesStmt><note type="content">Section title: Original article</note><note type="content">Figure 1: Upper figure. Separation by anion exchange chromatography on Q Sepharose Fast Flow (Pharmacia) column of shrTMCSA+ and shrTMCSA– glycoforms from the supernatant of 4T2 B6/CHO confluent cell cultures metabolically labeled with 10 μCi/ml [3H]-glucosamine hydrochloride (–□–; left y axis) or 20 μCi/mL [14C]-leucine (–•–; right y axis). Differential elution was obtained using a 50-mM Tris-HCl pH 6.8 10 mM EDTA, 0.05% CHAPS buffer containing either 0.5 M NaCl (shrTMCSA–) or 1 M NaCl (shrTMCSA+). Radioactivity was counted by scintillation (Ultima Gold XR, Packard) with a Packard Minaxiβ counter and is expressed as dpm. Lower figure. CSTM structure was analyzed by exclusion chromatography on a Superose 6 (Pharmacia) column. Elution profiles are presented for a sample of 20000 dpm of [3H]-CSTM (♦) and fragments obtained by the hydrolysis of 20 000 dpm [3H]-CSTM by chondroitinase ABC (■). Vo = 8.5 ml and Vt = 24 ml. Radioactivity was counted by scintillation (Ultima Gold XR, Packard) with a Packard Minaxiβ counter and is expressed as dpm.</note><note type="content">Figure 2: Determination of the molecular weights of CHO-CSTM and of the CHO-shrTM protein core. A. The molecular weight of CSTM was determined by exclusion chromatography on a Superose 6 column calibrated with various radiolabeled standard GAGs: heparin (1.3, 3.3, 6.6 and 8.6 kDa) and hyaluronan (18.9, 30 and 43 kDa). The molecular weight of CSTM was estimated by calculating Kav as follows: Kav = Ve - Vo/Vt - Vo. B. Autoradiograph of a 7.5% polyacrylamide SDS-PAGE gel of three 20,000 dpm [14C]-shrTMCSA samples obtained by di-isopropyl fluorophosphate inactivated thrombin Affi-Gel 10 chromatography, undigested (lane 1) or after digestion with 0.5 U of chondroitinase ABC, at 37 °C, overnight (lane 2) or for 15 min (lane 3).</note><note type="content">Figure 3: Inhibition of cytoadhesion by shrTMCSA. Cytoadhesion of PACSA IEs to SBEC 1D was inhibited by various concentrations of CHO-shrTMCSA. The extent of inhibition was determined by comparing the inhibition obtained with each concentration of shrTMCSA with the maximal inhibition obtained with the commercial 50-kDa CSA.</note><note type="content">Figure 4: Detection of IECSA by modified shrTMCSA. A. ShrTMCSA-coated Dynabeads interact specifically with IECSA. This photograph was taken using the interference contrast technique. The smooth surface of the beads (diameter 4.5 μm) is clearly distinguishable from the rough appearance of IEs. IE/shrTMCSA-Dynabead agglutinates were observed exclusively with the CSA-binding phenotype. B. Fluorescent labeling of IECSA by FITC-shrTMCSA. Fluorescence was observed by exhaustive photon reassignment microscopy. Uninfected erythrocytes and CD36- or ICAM-1-binding IEs were not labeled by FITC-shrTMCSA.</note><note type="content">Figure 5: Adhesion of IECSA to shrTMCSA can be used to detect anti-CSA antibodies in immune sera. The capacity of 10 sera from multigravida women living in areas of endemic malaria in Cameroon and Senegal to inhibiton PACSA adhesion to immobilized shrTMCSA (■) and to SBEC 1D (□) was investigated. Inhibitory capacity was determined by comparing the adhesion obtained in the presence of a 1/10 dilution of each serum with that obtained with a 1/10 dilution of a nonimmune serum from a French volunteer.</note><note type="content">Table I: Specificity of the IE interaction with CSA-PE and CHO-shrTMCSA.</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Recombinant human thrombomodulincsa+: a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate</title><author xml:id="author-1"><persName><forename type="first">Daniel</forename><surname>Parzy</surname></persName><affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</affiliation></author><author xml:id="author-2"><persName><forename type="first">Thierry</forename><surname>Fusaï</surname></persName><affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</affiliation></author><author xml:id="author-3"><persName><forename type="first">Bruno</forename><surname>Pouvelle</surname></persName><affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</affiliation></author><author xml:id="author-4"><persName><forename type="first">Marilyne</forename><surname>Torrentino</surname></persName><affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</affiliation></author><author xml:id="author-5"><persName><forename type="first">Frédéric</forename><surname>Eustacchio</surname></persName><affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</affiliation></author><author xml:id="author-6"><persName><forename type="first">Catherine</forename><surname>Lépolard</surname></persName><affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</affiliation></author><author xml:id="author-7"><persName><forename type="first">Artur</forename><surname>Scherf</surname></persName><affiliation>Unité de Biologie des Interactions Hôte-Parasite, CNRS URA 1960, Institut Pasteur, 75724 Paris, France</affiliation></author><author xml:id="author-8"><persName><forename type="first">Jürg</forename><surname>Gysin</surname></persName><note type="correspondence"><p>Correspondence and reprints</p></note><affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</affiliation></author></analytic><monogr><title level="j">Microbes and Infection</title><title level="j" type="abbrev">MICINF</title><idno type="pISSN">1286-4579</idno><idno type="PII">S1286-4579(00)X0013-5</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="2000"></date><biblScope unit="volume">2</biblScope><biblScope unit="issue">7</biblScope><biblScope unit="page" from="779">779</biblScope><biblScope unit="page" to="788">788</biblScope></imprint></monogr><idno type="istex">D6C8872F4A2C891D308F233A4FDFCBEDC89DD785</idno><idno type="DOI">10.1016/S1286-4579(00)90357-5</idno><idno type="PII">S1286-4579(00)90357-5</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2000</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-Plasmodium falciparum-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>chondroitin-4-sulfate</term></item><item><term>thrombomodulin</term></item><item><term>cytoadhesion</term></item><item><term>Plasmodium falciparum</term></item><item><term>receptors</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2000">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/document/D6C8872F4A2C891D308F233A4FDFCBEDC89DD785/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="fx1" NDATA="IMAGE" name="fx1"></istex:entity><istex:entity SYSTEM="fx2" NDATA="IMAGE" name="fx2"></istex:entity><istex:entity SYSTEM="fx3" NDATA="IMAGE" name="fx3"></istex:entity><istex:entity SYSTEM="fx4" NDATA="IMAGE" name="fx4"></istex:entity><istex:entity SYSTEM="fx5" NDATA="IMAGE" name="fx5"></istex:entity><istex:entity SYSTEM="fx6" NDATA="IMAGE" name="fx6"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>MICINF</jid><aid>7</aid><ce:pii>S1286-4579(00)90357-5</ce:pii><ce:doi>10.1016/S1286-4579(00)90357-5</ce:doi><ce:copyright type="joint" year="2000">Éditions scientifiques et médicales Elsevier SAS</ce:copyright></item-info><head><ce:dochead><ce:textfn>Original article</ce:textfn></ce:dochead><ce:title>Recombinant human thrombomodulin<ce:sup>csa+</ce:sup>: a tool for analyzing <ce:bold><ce:italic>Plasmodium falciparum</ce:italic></ce:bold> adhesion to chondroitin-4-sulfate</ce:title><ce:author-group><ce:author><ce:given-name>Daniel</ce:given-name><ce:surname>Parzy</ce:surname><ce:cross-ref refid="ADR1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Thierry</ce:given-name><ce:surname>Fusaï</ce:surname><ce:cross-ref refid="ADR1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Bruno</ce:given-name><ce:surname>Pouvelle</ce:surname><ce:cross-ref refid="ADR2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Marilyne</ce:given-name><ce:surname>Torrentino</ce:surname><ce:cross-ref refid="ADR1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Frédéric</ce:given-name><ce:surname>Eustacchio</ce:surname><ce:cross-ref refid="ADR1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Catherine</ce:given-name><ce:surname>Lépolard</ce:surname><ce:cross-ref refid="ADR2"><ce:sup>b</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Artur</ce:given-name><ce:surname>Scherf</ce:surname><ce:cross-ref refid="ADR3"><ce:sup>c</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Jürg</ce:given-name><ce:surname>Gysin</ce:surname><ce:cross-ref refid="ADR2"><ce:sup>b</ce:sup></ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref></ce:author><ce:affiliation id="ADR1"><ce:label>a</ce:label><ce:textfn>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</ce:textfn></ce:affiliation><ce:affiliation id="ADR2"><ce:label>b</ce:label><ce:textfn>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</ce:textfn></ce:affiliation><ce:affiliation id="ADR3"><ce:label>c</ce:label><ce:textfn>Unité de Biologie des Interactions Hôte-Parasite, CNRS URA 1960, Institut Pasteur, 75724 Paris, France</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Correspondence and reprints</ce:text></ce:correspondence></ce:author-group><ce:date-received day="12" month="1" year="2000"></ce:date-received><ce:date-accepted day="3" month="4" year="2000"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-<ce:italic>Plasmodium falciparum</ce:italic>-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to <ce:italic>Saimiri</ce:italic> brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>chondroitin-4-sulfate</ce:text></ce:keyword><ce:keyword><ce:text>thrombomodulin</ce:text></ce:keyword><ce:keyword><ce:text>cytoadhesion</ce:text></ce:keyword><ce:keyword><ce:text><ce:bold><ce:italic>Plasmodium falciparum</ce:italic></ce:bold></ce:text></ce:keyword><ce:keyword><ce:text>receptors</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Recombinant human thrombomodulincsa+: a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Recombinant human thrombomodulin</title></titleInfo><name type="personal"><namePart type="given">Daniel</namePart><namePart type="family">Parzy</namePart><affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Thierry</namePart><namePart type="family">Fusaï</namePart><affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Bruno</namePart><namePart type="family">Pouvelle</namePart><affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Marilyne</namePart><namePart type="family">Torrentino</namePart><affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Frédéric</namePart><namePart type="family">Eustacchio</namePart><affiliation>Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, 13007 Marseille, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Catherine</namePart><namePart type="family">Lépolard</namePart><affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Artur</namePart><namePart type="family">Scherf</namePart><affiliation>Unité de Biologie des Interactions Hôte-Parasite, CNRS URA 1960, Institut Pasteur, 75724 Paris, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Jürg</namePart><namePart type="family">Gysin</namePart><affiliation>Unité de parasitologie expérimentale, faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France</affiliation><description>Correspondence and reprints</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article"></genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">2000</dateIssued><copyrightDate encoding="w3cdtf">2000</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract lang="en">The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-Plasmodium falciparum-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies.</abstract><note type="content">Section title: Original article</note><note type="content">Figure 1: Upper figure. Separation by anion exchange chromatography on Q Sepharose Fast Flow (Pharmacia) column of shrTMCSA+ and shrTMCSA– glycoforms from the supernatant of 4T2 B6/CHO confluent cell cultures metabolically labeled with 10 μCi/ml [3H]-glucosamine hydrochloride (–□–; left y axis) or 20 μCi/mL [14C]-leucine (–•–; right y axis). Differential elution was obtained using a 50-mM Tris-HCl pH 6.8 10 mM EDTA, 0.05% CHAPS buffer containing either 0.5 M NaCl (shrTMCSA–) or 1 M NaCl (shrTMCSA+). Radioactivity was counted by scintillation (Ultima Gold XR, Packard) with a Packard Minaxiβ counter and is expressed as dpm. Lower figure. CSTM structure was analyzed by exclusion chromatography on a Superose 6 (Pharmacia) column. Elution profiles are presented for a sample of 20000 dpm of [3H]-CSTM (♦) and fragments obtained by the hydrolysis of 20 000 dpm [3H]-CSTM by chondroitinase ABC (■). Vo = 8.5 ml and Vt = 24 ml. Radioactivity was counted by scintillation (Ultima Gold XR, Packard) with a Packard Minaxiβ counter and is expressed as dpm.</note><note type="content">Figure 2: Determination of the molecular weights of CHO-CSTM and of the CHO-shrTM protein core. A. The molecular weight of CSTM was determined by exclusion chromatography on a Superose 6 column calibrated with various radiolabeled standard GAGs: heparin (1.3, 3.3, 6.6 and 8.6 kDa) and hyaluronan (18.9, 30 and 43 kDa). The molecular weight of CSTM was estimated by calculating Kav as follows: Kav = Ve - Vo/Vt - Vo. B. Autoradiograph of a 7.5% polyacrylamide SDS-PAGE gel of three 20,000 dpm [14C]-shrTMCSA samples obtained by di-isopropyl fluorophosphate inactivated thrombin Affi-Gel 10 chromatography, undigested (lane 1) or after digestion with 0.5 U of chondroitinase ABC, at 37 °C, overnight (lane 2) or for 15 min (lane 3).</note><note type="content">Figure 3: Inhibition of cytoadhesion by shrTMCSA. Cytoadhesion of PACSA IEs to SBEC 1D was inhibited by various concentrations of CHO-shrTMCSA. The extent of inhibition was determined by comparing the inhibition obtained with each concentration of shrTMCSA with the maximal inhibition obtained with the commercial 50-kDa CSA.</note><note type="content">Figure 4: Detection of IECSA by modified shrTMCSA. A. ShrTMCSA-coated Dynabeads interact specifically with IECSA. This photograph was taken using the interference contrast technique. The smooth surface of the beads (diameter 4.5 μm) is clearly distinguishable from the rough appearance of IEs. IE/shrTMCSA-Dynabead agglutinates were observed exclusively with the CSA-binding phenotype. B. Fluorescent labeling of IECSA by FITC-shrTMCSA. Fluorescence was observed by exhaustive photon reassignment microscopy. Uninfected erythrocytes and CD36- or ICAM-1-binding IEs were not labeled by FITC-shrTMCSA.</note><note type="content">Figure 5: Adhesion of IECSA to shrTMCSA can be used to detect anti-CSA antibodies in immune sera. The capacity of 10 sera from multigravida women living in areas of endemic malaria in Cameroon and Senegal to inhibiton PACSA adhesion to immobilized shrTMCSA (■) and to SBEC 1D (□) was investigated. Inhibitory capacity was determined by comparing the adhesion obtained in the presence of a 1/10 dilution of each serum with that obtained with a 1/10 dilution of a nonimmune serum from a French volunteer.</note><note type="content">Table I: Specificity of the IE interaction with CSA-PE and CHO-shrTMCSA.</note><subject lang="en"><genre>Keywords</genre><topic>chondroitin-4-sulfate</topic><topic>thrombomodulin</topic><topic>cytoadhesion</topic><topic>Plasmodium falciparum</topic><topic>receptors</topic></subject><relatedItem type="host"><titleInfo><title>Microbes and Infection</title></titleInfo><titleInfo type="abbreviated"><title>MICINF</title></titleInfo><genre type="journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">200006</dateIssued></originInfo><identifier type="ISSN">1286-4579</identifier><identifier type="PII">S1286-4579(00)X0013-5</identifier><part><date>200006</date><detail type="volume"><number>2</number><caption>vol.</caption></detail><detail type="issue"><number>7</number><caption>no.</caption></detail><extent unit="issue pages"><start>725</start><end>848</end></extent><extent unit="pages"><start>779</start><end>788</end></extent></part></relatedItem><identifier type="istex">D6C8872F4A2C891D308F233A4FDFCBEDC89DD785</identifier><identifier type="DOI">10.1016/S1286-4579(00)90357-5</identifier><identifier type="PII">S1286-4579(00)90357-5</identifier><accessCondition type="use and reproduction" contentType="copyright">©2000 Éditions scientifiques et médicales Elsevier SAS</accessCondition><recordInfo><recordContentSource>ELSEVIER</recordContentSource><recordOrigin>Éditions scientifiques et médicales Elsevier SAS, ©2000</recordOrigin></recordInfo></mods></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Sante/explor/ParkinsonFranceV1/Data/Istex/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 001D18 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Istex/Corpus/biblio.hfd -nk 001D18 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Sante
|area= ParkinsonFranceV1
|flux= Istex
|étape= Corpus
|type= RBID
|clé= ISTEX:D6C8872F4A2C891D308F233A4FDFCBEDC89DD785
|texte= Recombinant human thrombomodulincsa+: a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate
}}
| This area was generated with Dilib version V0.6.29. |  |