Recombinant human thrombomodulin(csa+): a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate.
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.
Parzy
D
D
Unité de parasitologie IMTSSA, Jardin du Pharo, Boulevard Charles Livon, Marseille, France.
Fusai
T
T
Pouvelle
B
B
Torrentino
M
M
Eustacchio
F
F
Lépolard
C
C
Scherf
A
A
Gysin
J
J
D000818:Animals; D000906:Antibodies / Q000097:blood; D016466:CHO Cells; D019556:COS Cells; D002448:Cell Adhesion; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004912:Erythrocytes / Q000469:parasitology / Q000502:physiology; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D066298:In Vitro Techniques; D010641:Phenotype; D010963:Plasmodium falciparum / Q000166:cytology / Q000502:physiology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012453:Saimiri; D018180:Thrombomodulin / Q000737:chemistry / Q000502:physiology*
D000906:Antibodies; D011994:Recombinant Proteins; D018180:Thrombomodulin; D002809:Chondroitin Sulfates