PHISTc protein family members localize to different subcellular organelles and bind Plasmodium falciparum major virulence factor PfEMP‐1

Abstract

Plasmodium falciparum encodes a novel repertoire of the Plasmodium helical interspersed subtelomeric (PHIST) family of exported proteins, which play diverse roles in infected red blood cells, contributing to malaria pathogenesis. PHIST proteins are central to parasite biology and modify human erythrocytes by interacting with parasite and host proteins. Here, we have attempted to understand the localization and function of two unexplored proteins of the PHISTc subfamily, PFD1140w and PF11_0503, and compared these with a well-characterized member, PFI1780w. We demonstrate that Phist domains assume different oligomeric states owing to a distinct array of subunit interface residues. Colocalization of a Maurer's cleft signature protein, P. falciparum skeleton-binding protein-1 (PfSBP-1), and P. falciparum erythrocyte membrane protein-1 (PfEMP-1) revealed different subcellular destinations for these PHIST members. We further show the binding of recombinant PHIST proteins to the cytoplasmic tail of PfEMP-1 and a novel interaction with PfSBP-1. Interestingly, PFD1140w interacts with PfEMP-1 and PfSBP-1 simultaneously in vitro leading to formation of a complex. These two distant PHISTc members also bind PfEMP-1 on distinct sites, despite sharing the Phist domain. Our data re-emphasize a supportive role for PHIST proteins in cytoadhesion, and identify a new binding partner, PfSBP-1, for members of this family. This information therefore adds another chapter to the understanding of P. falciparum biology and highlights the significance of the unexplored PHIST family.

Cholesterol boundPlasmodium falciparumco-chaperone ‘PFA0660w’ complexes with major virulence factor ‘PfEMP1’ via chaperone ‘PfHsp70-x’

“…1C) owing to protein degradation. This is evident from western blots of recombinant proteins probed with anti-hexahistidine monoclonal antibodies (ATS 36 , Fig. S1A for Hsp40 and Hsp70 constructs).…”

“…ATPase activity, which is connected by a linker region to a SBD present in the C-terminal region ( Recombinant Hsp40 and Hsp70 proteins were purified using Ni-NTA affinity and gel permeation chromatographies, while acidic terminal segment (ATS) domain of PfEMP1 was purified as reported earlier 36,37 . Purified PfHsp70-x-S, PfHsp70-x-C, PFA0660w-C, PFA0660w-S and ATS run as species of approximately 45 kDa, 70 kDa, 38 kDa, 28 kDa and 45 kDa respectively on SDS-PAGE ( Fig.…”

“…Since PfHsp70-x binds both PFA0660w and ATS of PfEMP1, we tested its potential to attach with both proteins simultaneously to form a complex using in-vitro aminoplus coupling resin based pull down assays 36 . Recombinant PfHsp70-x-C was coupled to the resin, and allowed to bind PFA0660w-C, followed by ATS.…”

CX3CL1 binding protein-2 (CBP2) of Plasmodium falciparum binds nucleic acids

“…Interaction of ATP and CBP2 was also analysed by 1 These proteins were purified according to protocols described in Kumar et al [28] and Kaur et al [49]. rcCBP2 incubated with DNA (plasmid/ ss-DNA oligomers) was also run on 15% native-PAGE and transferred on NC membrane.…”

“…These blots were washed twice each with PBST (PBS with 0.025% tween 20) and PBS, and then incubated for 1hr with anti-CBP2 antibodies (1:5000) followed by anti-rabbit HRP conjugated secondary antibodies (1:2000) before developing with DAB/H2O2 chromogenic substrate. BR-5 region of PfSBP1 and ATS domain of PfEMP1 were expressed and purified as described by Kumar et al [28].…”