The Pf332 gene of Plasmodium falciparum codes for a giant protein that is translocated from the parasite to the membrane of infected erythrocytes

Abstract

We studied the gene structure of the Plasmodium falciparum antigen 332 (Ag332). The gene size was estimated to be approx. 20 kb based on the large size of both the transcript found in mature asexual blood stage parasites and mung bean nuclease fragment generated from genomic DNA. Sequence analysis of genomic and cDNA clones representing different regions of the Pf332 locus showed that the gene product contains a large number of highly degenerated glutamic acid (Glu)-rich repeats (32% Glu). The gene shows dramatic restriction fragment length polymorphism in various P. falciparum isolates and was mapped to the subtelomeric region of chromosome 11. The recombinant 332 fusion protein reacts strongly with the human monoclonal antibody (mAb) 33G2, which is able to inhibit the cytoadherence of parasitized red blood cells on the melanoma cell line C32 and merozoite invasion in in vitro assays. The epitope recognized by this mAb is found frequently in the reported sequence. Ag332 monospecific antibodies were obtained by immunization of mice with a recombinant fusion protein. These antibodies react with a large parasite molecule with an apparent molecular size of 2500 kDa of trophozoite and schizont-infected erythrocytes on Western blot and by immunoprecipitation analysis. Immunofluorescence studies using a confocal microscope showed that Ag332 is exported from the parasite to the infected red blood cell membrane within large vesicle-like structures of about 1 micron diameter.