The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages

Abstract

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strains so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich in glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that the Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton

“…Pf332 was detected as a band of >584 kDa (indicated by the arrow) in the 3D7 TX100 insoluble fraction and in the pIOVs sample, but not in the IOVs prepared from normal human RBCs. Although originally described as a megadalton protein [25, 27], Pf332 is currently predicted to be a 700kDa protein, and was observed in our hands as having a molecular mass in excess of 584kDa. It is not uncommon for highly charged repetitive malaria proteins, such as Pf332, to resolve at anomalous higher-than-predicted molecular masses in SDS-PAGE [47].…”

“…700 kDa) that is exported into the IRBC cytosol [25, 26]. Pf332 is highly charged and possesses an extensive region of highly degenerate glutamic acid-rich repeats that span over 4300 residues (Fig 1A; [27]). The pf332 gene shares features common to other genes that encode exported parasite proteins, including a two exon gene structure and a P. falciparum protein trafficking motif (PEXEL or Vacuolar Transport Signal (VTS); [28, 29]) encoded within exon 1 (Fig.…”

A Novel DBL-Domain of the P. falciparum 332 Molecule Possibly Involved in Erythrocyte Adhesion

“…This conclusion was confirmed by Western blot analysis using three different α-Pf332 antibodies to various regions of the polypeptide. Exon II of Pf332 was initially identified from a gDNA expression library containing only the region of repetitive sequence without additional 5′-RACE sequencing to identify the 5′-end of the transcript [15], [27]. The existence of the additional upstream exon in the gene of Pf332 has therefore previously been overlooked and exon I (PF11_0506) was as a consequence annotated in the P. falciparum genome as a separate gene encoding a hypothetical protein.…”

Characterization of the Duffy-Binding-Like Domain ofPlasmodium falciparumBlood-Stage Antigen 332

“…One explanation might be that although living close to each other geographically and experiencing the same malaria transmission rate, the different tribes had encountered parasites presenting different PfEMP1 molecules on the iRBC surface. Whilst the Pf332 gene is present as a single copy gene in most parasite isolates studied so far [9], the var genes encoding the PfEMP1 molecule are present in approximately 60 copies per genome [49, 50]. In Mali, the Fulani who are less susceptible to malaria were found to have significantly higher antibody levels to PfEMP1-DBL1 α than their sympatric tribe Dogon.…”

“…Previous studies on Pf332 have primarily focused on EB200, a 157 amino-acid-long Glu-rich fragment, located in the central repeat region of the protein (Figure 1(a)) [9]. Individuals living in malaria endemic areas have been shown to have antibodies recognizing this region [12, 13] and these antibodies were associated with fewer clinical malaria attacks [14].…”