Falciparum malaria parasitized erythrocytes bind to a carboxy-terminal thrombospondin fragment and not the amino-terminal heparin-binding region.
We investigated Plasmodium falciparum parasitized erythrocyte binding to proteolytic fragments of thrombospondin and the effects of anti-thrombospondin monoclonal antibodies on this binding. Purified human platelet thrombospondin was cleaved by trypsin, chymotrypsin or thrombin. Fragments were separated by heparin-agarose affinity chromatography, removing the amino-terminal heparin-binding region. Trypsin at 5.0 micrograms ml-1 of thrombospondin cleaved thrombospondin to reduced 140 and 120 kDa fragments plus a reduced 25-kDa heparin-binding fragment. Infected erythrocytes bound to intact thrombospondin (3420 +/- 460 infected erythrocytes mm-2) and the carboxy-terminal fragment, yielding 120-140-kDa fragments on sulfhydryl reduction, but not to the 25-kDa fragment (144 +/- 104 infected erythrocytes mm-2 (mean +/- s.d., N = 4). Similar results were obtained with chymotrypsin and thrombin cleavage. When the anti-thrombospondin monoclonal antibody MA-I was added to immobilized thrombospondin prior to infected erythrocytes, adherence was inhibited by 99%. At the same concentration, MA-I inhibited adherence to C32 melanoma cells by only 35%. MA-I binds to a calcium-dependent structure at the C-terminal globular region of thrombospondin. Monoclonal antibody MA-II inhibited adherence to thrombospondin by 46%, while MA-III had no effect. These antibodies bind to the N-terminal globular region which includes the heparin-binding site and the segment connecting the two globular regions, respectively. The site(s) for infected erythrocyte binding on thrombospondin reside in the large, 140- or 120-kDa, proteolytic cleavage fragments, and not in the N-terminal heparin-binding region.
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases; National Institutes of Health, Bethesda, MD.
J A
JA
Sherwood
D D
DD
Roberts
S L
SL
Spitalnik
J W
JW
Lawler
L H
LH
Miller
R J
RJ
Howard
D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D002448:Cell Adhesion; D002846:Chromatography, Affinity; D004912:Erythrocytes / Q000469:parasitology*; D006493:Heparin; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000276:immunology; D019699:Thrombospondins; D014407:Tumor Cells, Cultured
D000913:Antibodies, Protozoan; D008562:Membrane Glycoproteins; D019699:Thrombospondins; D006493:Heparin