Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system.
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.
Kedees
M H
MH
Zentrum für Hygiene und Medizinische Mikrobiologie, Philips-Universität Marburg, Germany.
Gerold
P
P
Azzouz
N
N
Blaschke
T
T
Shams-Eldin
H
H
Mühlberger
E
E
Holder
A A
AA
Klenk
H D
HD
Schwarz
R T
RT
Eckert
V
V
D000818:Animals; D000953:Antigens, Protozoan; D016367:Baculoviridae; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D008358:Mannose; D020066:Merozoite Surface Protein 1 / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum; D011134:Polysaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000953:Antigens, Protozoan; D017261:Glycosylphosphatidylinositols; D020066:Merozoite Surface Protein 1; D011134:Polysaccharides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C047168:SAG1 antigen, Toxoplasma; D008358:Mannose