Structural and functional dissection of the adhesive domains of Plasmodium falciparum thrombospondin-related anonymous protein (TRAP).
TRAP (thrombospondin-related anonymous protein) is a sporozoite surface protein that plays a central role in hepatocyte invasion. We have developed procedures for recombinant production of the entire ECD (extracellular domain) and A domain of TRAP using bacterial- and baculovirus-expression systems respectively. The ECD and A domain were purified to homogeneity and migrated on gel-filtration columns as non-aggregated, monomeric proteins. These adhesive modules bound to HepG2 cells in a dose-dependent and bivalent cation-independent manner. The binding of ECD and the A domain to HepG2 cells was inhibited poorly by an excess of sulphatide analogues, suggesting the presence of as yet unidentified receptors for the A domain on hepatocytes. Using surface-plasmon-resonance-based sensor technology (Biacore), we demonstrate that TRAP ECD has higher affinity for heparin (K(D)=40 nM) compared with the A domain (K(D)=79 nM). We also present a three-dimensional structure of the A domain based on the crystal structure of the homologous von Willebrand factor A1 domain. The TRAP A domain shows two spatially distinct ligand-binding surfaces. One surface on the A domain contains the MIDAS (metal-ion-dependent adhesion site) motif, where point mutations of Thr131 and Asp162 correlate with impairment of cell infectivity by sporozoites. The other surface contains a putative heparin-binding site and consists of a basic residue cluster. Our studies suggest that TRAP interacts with multiple receptors during the hepatocyte invasion process. Our results also pave the way for inclusion of these high-quality recombinant TRAP domains in subunit-based vaccines against malaria.
Akhouri
Reetesh Raj
RR
Malaria Research Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Bhattacharyya
Arindam
A
Pattnaik
Priyabrata
P
Malhotra
Pawan
P
Sharma
Amit
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D045744:Cell Line, Tumor; D006031:Glycosylation; D006493:Heparin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D000267:Tissue Adhesions; D014841:von Willebrand Factor / Q000737:chemistry
D010446:Peptide Fragments; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C069428:thrombospondin-related adhesive protein, protozoan; D006493:Heparin