Plasmodium falciparum merozoite surface protein 2 is unstructured and forms amyloid-like fibrils

Abstract

Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies have confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying β-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular β-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites.

Sequential Processing of Merozoite Surface Proteins during and after Erythrocyte Invasion by Plasmodium falciparum

“…Importantly, both the 3D7 and FC27 allelic forms of MSP2 showed the same phenotype of being carried into RBCs followed by rapid degradation. The 3D7 and FC27 alleles of MSP2 share N-and C-terminal regions but vary significantly in the central strain-specific region of the protein (46,(76)(77)(78). The observation that both MSP2 variants show the same localization and very precise timing of degradation postinvasion suggests that the functions of the 3D7 and FC27 forms of MSP2 are the same, despite any structural differences.…”

“…MSP2 is the second most abundant merozoite surface protein in terms of copy number (42), and while a function for MSP2 has not been identified, this protein is refractory to genetic deletion and is thought to be essential (43). MSP2 is highly polymorphic; however, all alleles can be grouped as either 3D7-like or FC27-like based on tandem sequence repeats and flanking nonrepetitive dimorphic sequences, with both alleles sharing N-and C-terminal regions (44)(45)(46). Currently, the fate of MSP2 during invasion is unclear, and there is no evidence of MSP2 in culture supernatants (20,47).…”

Plasmodium falciparum Merozoite Surface Protein 3

“…However, it was also observed that regions of MSP2 outside the conserved N-terminal domain were also important for fibril formation by MSP2. Interestingly, failure of mAb 6D8 in situ that reacted with the N-terminal conserved region of MSP2 was taken as indirect evidence of the presence of MSP2 on the merozoite surface as a homooligomer; no direct evidence for MSP2 fibril has been reported (53). A major difference between these two highly abundant blood stage proteins with regard to their intrinsic propensity to form fibrils is that although MSP2 is anchored to the merozoite surface via the GPI anchor, MSP3 is supposed to be present on the merozoite surface through association with other surface proteins (47,48).…”

“…Oligomerization of MSP3, as a soluble protein, is facile and likely inherent to its structure that includes the presence of leucine zipper-like sequences at the C terminus (40). In both the proteins, although small peptides based on the domains involved in fibril formation also form fibrils like the full-length proteins themselves, the sequences outside the fibril forming domains are also involved and/or assist in fibril formation by the two proteins (52,53). Results described in this study, however, clearly show that MSP3-based fibrils are present on the merozoite surface and seem to interact with erythrocytes.…”

Malaria vaccine development based on merozoite surface proteins ofPlasmodium falciparum

“…The recombinant forms of MSP-2 have been reported to form amyloid fibrils in solution 38 and it is believed that native MSP-2 based higher order aggregates like those seen in the amyloid fibrils may be present in the fibrillar surface coat of the merozoites. 38 The unstructured properties of recombinant forms of MSP-2 would need to be strongly considered in pre-clinical vaccine development programs with this antigen. Monomeric recombinant MSP-2 may not represent the dominant conformation adopted by native MSP-2 on the merozoite surface.…”