Structure of the Plasmodium falciparum circumsporozoite protein, a leading malaria vaccine candidate.
The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R(h) 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21-25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.
Plassmeyer
Matthew L
ML
Malaria Vaccine Development Branch, NIAID, National Institutes of Health, Rockville, Maryland 20852, USA.
Reiter
Karine
K
Shimp
Richard L
RL
Kotova
Svetlana
S
Smith
Paul D
PD
Hurt
Darrell E
DE
House
Brent
B
Zou
Xiaoyan
X
Zhang
Yanling
Y
Hickman
Merrit
M
Uchime
Onyinyechukwu
O
Herrera
Raul
R
Nguyen
Vu
V
Glen
Jacqueline
J
Lebowitz
Jacob
J
Jin
Albert J
AJ
Miller
Louis H
LH
MacDonald
Nicholas J
NJ
Wu
Yimin
Y
Narum
David L
DL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000953:Antigens, Protozoan / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006493:Heparin / Q000031:analogs & derivatives / Q000378:metabolism; D022781:Hepatocytes / Q000276:immunology / Q000469:parasitology / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D017780:Malaria Vaccines / Q000276:immunology*; D008958:Models, Molecular; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D034101:Sporozoites / Q000276:immunology* / Q000378:metabolism; D013696:Temperature; D014461:Ultracentrifugation
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D017261:Glycosylphosphatidylinositols; D017780:Malaria Vaccines; D011509:Proteoglycans; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C041595:circumsporozoite protein, Protozoan; C036090:heparin proteoglycan; D006493:Heparin