Abstract
Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.
Highlights
Most studies on merozoite surface protein 1 (MSP1) have focused on its role in erythrocyte invasion and its potential as a vaccine candidate, based on the ability of MSP1specific antibodies to inhibit invasion
In the present study we have explored in detail the post-invasion fate of MSP119 using a combination of metabolic labeling, immunofluorescence assay (IFA) and IEM techniques applied to highly synchronized parasite cultures, together with antibodies directed against well-defined regions of MSP1
Antibodies A number of antibodies against different regions of MSP1 were used: monoclonal antibody 1E1 [8] specific for MSP119; rabbit polyclonal antibodies raised against either affinity purified MSP1 (Wellcome type) [45] or recombinant GST-MSP119 [46]; and a pool of rabbit polyclonal antibodies raised against recombinant protein epitopes spanning parts of MSP1 that are N-terminal of MSP119 [47]
Summary
Most studies on merozoite surface protein 1 (MSP1) have focused on its role in erythrocyte invasion and its potential as a vaccine candidate, based on the ability of MSP1specific antibodies to inhibit invasion. At the time of red blood cell (RBC) invasion a second proteolytic cleavage of the 42-kDa polypeptide, by the enzyme SUB2 [6], releases the protein complex from the parasite surface except for a 19-kDa C-terminal GPI-linked fragment (MSP119). The latter comprises two epidermal growth factor (EGF)-like domains and is carried into the interior of the infected-RBC on the merozoite surface [7], MSP119 has been detected on the surface of the early ring-stage parasite by both IFA [1,2], and IEM [1]. The fate of MSP119 after invasion has not been studied in any detail
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