Figure 1.
Figure 1. PfEMP1 structure and chimeric proteins expressed in P falciparum. (A) PfEMP1 has a putative Tm region (Tm; red segment) followed by a conserved acidic terminal segment (ATS; dark blue). The extracellular domain is variable in sequence and size but assembled from 3 main building blocks: NTS (N-terminal segment; dark yellow), DBL (Duffy-binding–like) domain (yellow and purple segments), and CIDR (cysteine-rich interdomain region; pink and black segments). Sizes of each domain are not to scale. (B) Structures of the proteins expressed from plasmid constructs. The color of each domain is the same as in panel A. All are chimeric proteins with GFP at the C-terminus. (i) NTS and DBL1α. (ii) Twenty amino acids (AAs) of CIDR2β, PfEMP1 Tm, and 113 amino acids of ATS (ATSs). (iii) The CIDR2β fragment, PfEMP1 Tm, and 447 amino acids of ATS. (iv) The signal peptide of ACP (amino acids 1-20; gray segment), the CIDR2β fragment, PfEMP1 Tm, and ATSs. (v) The first 60 amino acids of KAHRP (light blue), the CIDR2β fragment, PfEMP1 Tm, and ATSs. (vi) KAHRP1-60, CIDR2β fragment, PfEMP1 Tm, and full-length ATS. (vii) KAHRP1-119 including the His-rich domain, CIDR2β domain, PfEMP1 Tm, and full-length ATS. (viii) KAHRP1-60, 20 amino acids of the AMA1 exodomain (green), AMA1 Tm, and ATSs. The arrow shows the position of the PEXEL in each construct. (C) Transgene expression of GFP chimeric proteins in P falciparum transfected with the PfEMP1 constructs. The 3D7-NTS/DBL1α, 3D7-TmATSs, 3D7-ACPTmATSs, 3D7-K60TmATSs, 3D7, and 3D7-K60Tm(ama1)ATSs were subjected to Western analysis and probed with αGFP antibody. The top band in 3D7-K60TmATSs may represent full-length chimeric protein before cleavage of the KAHRP signal peptide. Shown below is the same blot probed with αhsp70 antibodies. (D) The 3D7, 3D7-TmATS, 3D7-K60TmATS, and 3D7-K119TmATS were probed with αGFP antibody. Shown below is the same blot probed with αhsp70 antibodies. (E) The 3D7-K119TmATS parasites were synchronized and samples were taken at various intervals after invasion. Shown below is the same blot probed with αhsp70 antibodies. (F) The 3D7-K60TmATS, 3D7-K119TmATS, D10-K119TmATS, and D10 parasitized RBCs were probed with αKAHRP-His, αGFP, αKAHRP-C (3′ repeat region of C-terminus), and αhsp70 antibodies. The arrow indicates the 120-kDa K119-TmATS-GFP protein.

PfEMP1 structure and chimeric proteins expressed in P falciparum. (A) PfEMP1 has a putative Tm region (Tm; red segment) followed by a conserved acidic terminal segment (ATS; dark blue). The extracellular domain is variable in sequence and size but assembled from 3 main building blocks: NTS (N-terminal segment; dark yellow), DBL (Duffy-binding–like) domain (yellow and purple segments), and CIDR (cysteine-rich interdomain region; pink and black segments). Sizes of each domain are not to scale. (B) Structures of the proteins expressed from plasmid constructs. The color of each domain is the same as in panel A. All are chimeric proteins with GFP at the C-terminus. (i) NTS and DBL1α. (ii) Twenty amino acids (AAs) of CIDR2β, PfEMP1 Tm, and 113 amino acids of ATS (ATSs). (iii) The CIDR2β fragment, PfEMP1 Tm, and 447 amino acids of ATS. (iv) The signal peptide of ACP (amino acids 1-20; gray segment), the CIDR2β fragment, PfEMP1 Tm, and ATSs. (v) The first 60 amino acids of KAHRP (light blue), the CIDR2β fragment, PfEMP1 Tm, and ATSs. (vi) KAHRP1-60, CIDR2β fragment, PfEMP1 Tm, and full-length ATS. (vii) KAHRP1-119 including the His-rich domain, CIDR2β domain, PfEMP1 Tm, and full-length ATS. (viii) KAHRP1-60, 20 amino acids of the AMA1 exodomain (green), AMA1 Tm, and ATSs. The arrow shows the position of the PEXEL in each construct. (C) Transgene expression of GFP chimeric proteins in P falciparum transfected with the PfEMP1 constructs. The 3D7-NTS/DBL1α, 3D7-TmATSs, 3D7-ACPTmATSs, 3D7-K60TmATSs, 3D7, and 3D7-K60Tm(ama1)ATSs were subjected to Western analysis and probed with αGFP antibody. The top band in 3D7-K60TmATSs may represent full-length chimeric protein before cleavage of the KAHRP signal peptide. Shown below is the same blot probed with αhsp70 antibodies. (D) The 3D7, 3D7-TmATS, 3D7-K60TmATS, and 3D7-K119TmATS were probed with αGFP antibody. Shown below is the same blot probed with αhsp70 antibodies. (E) The 3D7-K119TmATS parasites were synchronized and samples were taken at various intervals after invasion. Shown below is the same blot probed with αhsp70 antibodies. (F) The 3D7-K60TmATS, 3D7-K119TmATS, D10-K119TmATS, and D10 parasitized RBCs were probed with αKAHRP-His, αGFP, αKAHRP-C (3′ repeat region of C-terminus), and αhsp70 antibodies. The arrow indicates the 120-kDa K119-TmATS-GFP protein.

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