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Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus
M H Kedees1, P Gerold, N Azzouz
1Zentrum für Hygiene und Medizinische Mikrobiologie, Philips-Universität Marburg, Germany.
Abstract:
We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.
Insights
Recombinant Plasmodium falciparum merozoite surface protein 1 (MSP-1) C-terminal fragments were expressed in insect cells but remained intracellular. This intracellular retention, unlike Toxoplasma gondii SAG-1, suggests protein-specific transport differences impacting malaria vaccine development.
Area of Science:
- Molecular Biology
- Parasitology
- Cell Biology
Background:
- The merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a key malaria parasite antigen.
- Understanding the post-translational modification and cellular transport of MSP-1 is crucial for malaria vaccine development.
Purpose of the Study:
- To investigate the expression, glycosylation, and intracellular localization of C-terminal fragments of Plasmodium falciparum MSP-1 in insect cells.
- To compare the transport of MSP-1 fragments with a control protein and the surface antigen SAG-1 of Toxoplasma gondii.
Main Methods:
- Expression of MSP-1 C-terminal fragments and a control protein in insect cells using the baculovirus system.
- Analysis of protein localization via immunofluorescence and N-glycan characterization.
- Comparison with the transport of Toxoplasma gondii SAG-1.
Main Results:
- Both MSP-1 fragments were retained intracellularly, primarily in the endoplasmic reticulum.
- N-glycans on MSP-1 fragments were not processed in post-endoplasmic reticulum compartments.
- Toxoplasma gondii SAG-1 was efficiently transported to the cell surface, unlike MSP-1.
- Absence of detected GPI-anchors for both MSP-1 constructs and SAG-1.
Conclusions:
- The intracellular retention of MSP-1 fragments in insect cells is a protein-specific characteristic, not due to the absence of a GPI-anchor.
- Differences in intracellular processing and transport between recombinant proteins in insect cells and native proteins in parasites have implications for malaria vaccine strategies.