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 (KD=40 nM) compared with the A domain (KD=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.
- A domain
- heparin binding
- hepatocyte invasion
- malaria sporozoite
- monomeric domain
- surface-plasmon resonance
- thrombospondin-related anonymous protein
Abbreviations used: CS, circumsporozoite; ECD, extracellular domain; GdnHCl, guanidine hydrochloride; MIDAS, metal-ion-dependent adhesion site; Ni-NTA, Ni2+-nitrilotriacetate; SPR, surface plasmon resonance; TRAP, thrombospondin-related anonymous protein; TRM, thrombospondin repeat motif; vWF, von Willebrand factor.
- The Biochemical Society, London ©2004