Figure 1.
P. falciparum Transmembrane Adhesins Involved in Host-Cell Invasion
(A) A generalized schematic of the 14 Plasmodium adhesins analyzed in this study. Sizes of the molecules are meant to represent their relative sizes but are not to scale (N-termini are leftmost). Indicated domains are SP, signal peptide; TM, transmembrane domain; vW Adom, vonWillebrand A domain; T, thrombospondin; and Pro, prodomain. The Toxoplasma MIC2 adhesin is shown for comparison.
(B) Sequence of Plasmodium adhesin transmembrane domains (in capital letters) compared to those of Drosophila Spitz and Toxoplasma MIC2, with the putative membrane boundaries depicted as two vertical lines. Residues conducive for rhomboid cleavage are shown in green, while those that interfere with cleavage are in red.
(C) Cleavage of GFP-tagged Plasmodium adhesins by DmRho-1 (Dm) was examined in transiently transfected COS cells by anti-GFP Western analysis of media fractions. Cleaved adhesins are rapidly secreted into the cell culture media, while the cell fractions are shown as controls. Molecular weight standards in kDa are denoted to the right of each panel.
Figure 2.
Expression Levels and Proteolytic Activity of Plasmodium Rhomboid Proteins
(A) Expression levels of 3xHA-tagged rhomboid proteins in transiently transfected COS cells were detected by anti-HA Western analysis. The approximate positions of prestained molecular weight standards (in kDa) are indicated on the right. Tg denotes T. gondii, Pf is P. falciparum, Py is P. yoellii, and Pb is P. berghei.
(B) Cleavage of Drosophila Spitz protein in transiently transfected COS cells was analyzed by anti-GFP Western analysis of media. Labeling above each panel denotes which rhomboid enzyme was co-transfected with Spitz to assess cleavage. Cleaved Spitz is rapidly secreted into the cell culture media, while cell lysates indicate transfection levels. Note that transfection of high concentrations of PfROM DNA (hi) resulted in some cytotoxicity relative to lower amounts (lo), which is common for many rhomboid proteins
(C) Cleavage of Spitz versus Spitz with the top seven amino acids of its transmembrane domain mutated to VALVIGV. Diagram denotes Spitz (in black), and mutant region (in white), with its cytoplasmic region being downward and the membrane bilayer denoted by two horizontal lines. Spitz shedding by endogenous cellular proteases was reduced by including a metalloprotease inhibitor in the cell culture media (in all lanes except those labeled MP). Note that both forms of Spitz were cleaved efficiently by cellular metalloproteases (MP), indicating that both forms were expressed well and trafficked to the cell surface, but the mutant Spitz could not be cleaved by rhomboid enzymes.
(D) PfROM1 depends on its putative active site serine for activity against Spitz. Both wild-type and SA mutant PfROM1 proteins were expressed well in transfected COS cells, as revealed by anti-HA Western (lower panel).
Figure 3.
Cleavage of PfAMA1 by Parasite Rhomboid Enzymes
(A) GFP-tagged AMA1 was transiently co-transfected with various rhomboid genes into COS cells. Both TgROM5 and PfROM1 cleaved AMA1, as evidenced by detection of the cleaved form in media by anti-GFP Western analysis, but failed to do so when their active site serines were mutated to alanine (SA). BB1101 was used to inhibit AMA1 shedding by endogenous cellular metalloproteases in all lanes in (A) and (B) except those denoted MP, which served as positive controls.
(B) Region of AMA1 important for substrate recognition by rhomboid proteases. Mutation of the top region of the AMA1 transmembrane domain (white region in diagram) ASSAA to VLVVV strongly reduced AMA1 cleavage by TgROM5 and PfROM1, but not by cellular metalloproteases (MP). Molecular weight standards in kDa are denoted to the right of each panel.
Figure 4.
Cleavage of P. falciparum EBLs by Parasite Rhomboid Enzymes
TgROM5 and PfROM4, as well as PfROM1, to a lesser extent, were able to cleave EBA-175 (A), JESEBL (B), and BAEBL (C), as assessed by anti-GFP Western analysis of media from transiently transfected COS cells. (D) Mutating the active site serine of PfROM4 to alanine (SA) abolished EBA-175 and BAEBL cleavage. The importance of the top of the transmembrane domain of EBA-175 (E) and BAEBL (F) for cleavage by parasite rhomboid enzymes was assessed by Western analysis of media fractions from COS cells transiently transfected with the wildtype (WT) and mutant (mut) adhesins. In all cases, mutation of the corresponding transmembrane sequences to AITALVVVIS from TGFα (white region in diagram) abolished adhesin cleavage by TgROM5, PfROM1, and PfROM4. Molecular weight standards in kDa are denoted to the right of each panel.
Figure 5.
Cleavage of Rh Proteins by Parasite Rhomboid Enzymes
GFP-tagged Rh1 (A), Rh2a (B), Rh2b (C), and Rh4 (D) were tested for cleavage by rhomboid enzymes by analyzing conditioned media with anti-GFP from transiently transfected COS cells. TgROM5 and PfROM4 readily cleaved each Rh protein, while PfROM1 could not cleave Rh2a but cleaved other Rh proteins at a lower efficiency. Note that in many cases smaller GFP-Rh protein breakdown products were readily detected in conditioned media (unpublished data). Molecular weight standards in kDa are denoted to the right of each panel.
Figure 6.
Specificity of PfROMs 1 and 4 for the Transmembrane Domains of AMA1 versus that of BAEBL
(A) Cleavage of GFP-tagged BAEBL (drawn in black) and GFP-tagged BAEBL harboring the transmembrane domain of AMA1 (in red) were tested for cleavage by anti-GFP Western analysis of media from transiently transfected COS cells. While TgROM5 could cleave both proteins efficiently, PfROM1 could only cleave BAEBL + AMAtm, while PfROM4 could only cleave BAEBL.
(B) Cleavage of GFP-tagged AMA1 (drawn in red) and GFP-tagged AMA1 harboring the transmembrane domain of BAEBL (in black) were tested for cleavage by anti-GFP Western analysis of media from transiently transfected COS cells. While TgROM5 could cleave both proteins efficiently, PfROM1 could only cleave AMA1 while PfROM4 could only cleave AMA + BAEBLtm.
(C) Other rhomboid enzymes including YqgP (from Bacillus subtilis) and RHBDL2 (from Homo sapiens) also cleaved BAEBL more efficiently when it contained the transmembrane domain from AMA1, like PfROM1 but not PfROM4. Molecular weight standards in kDa are denoted to the right of each panel.
Figure 7.
Cleavage of TRAP Adhesins by Parasite Rhomboid Enzymes
GFP-tagged full-length TRAP (A), full-length MTRAP (B), truncated PFF0800c (C), and full-length PTRAMP (D) were co-transfected with each rhomboid, and conditioned media was analyzed by anti-GFP Western. Molecular weight standards in kDa are denoted to the right of each panel.
Figure 8.
Cleavage of MAEBL and CTRP Adhesins, Which Are Required for Invasion of Mosquito Cells, by Parasite Rhomboid Enzymes
GFP-tagged MAEBL (A) and CTRP (B) were cotransfected with each rhomboid, and conditioned media were analyzed by anti-GFP Western. Molecular weight standards in kDa are denoted to the right of each panel.