Two Plasmodium Rhomboid Proteases Preferentially Cleave Different Adhesins Implicated in All Invasive Stages of Malaria
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).