Plasmodium food vacuole plasmepsins are activated by falcipains

Abstract

Intraerythrocytic malaria parasites use host hemoglobin as a major nutrient source. Aspartic proteases (plasmepsins) and cysteine proteases (falcipains) function in the early steps of the hemoglobin degradation pathway. There is extensive functional redundancy within and between these protease families. Plasmepsins are synthesized as integral membrane proenzymes that are activated by cleavage from the membrane. This cleavage is mediated by a maturase activity whose identity has been elusive. We have used a combination of cell biology, chemical biology, and enzymology approaches to analyze this processing event. These studies reveal that plasmepsin processing occurs primarily via the falcipains; however, if falcipain activity is blocked, autoprocessing can take place, serving as an alternate activation system. These results establish a further level of redundancy between the protease families involved in Plasmodium hemoglobin degradation.

FIGURE 1.

Plasmepsin processing can occur through two redundant activities. A, schematic depicting the activation of the plasmepsins by proteolytic processing. Shaded box, transmembrane domain. B, in vivo processing assay in which metabolically labeled parasites were washed and chased in the presence of various protease inhibitors (10 μm) followed by immunoprecipitation with anti-PM II antibody and visualization by autoradiography. Only treatment with ALLN or with the combination of E-64d/pepstatin (pep) inhibited processing activity. The double band seen in the E-64d lane likely represents alternative autoprocessing activity (see “Discussion”). C, cell-free processing assay confirms in vivo assay. ³⁵S-labeled pro-plasmepsin (pro) was synthesized by metabolic labeling of cells in the presence of brefeldin A followed by production of total parasite lysate. Inhibitors (10 μm) and additional unlabeled parasite lysate were then added, and the reaction was incubated at pH 5.0 at 37 °C for 1 h, followed by immunoprecipitation as above.

FIGURE 2.

Cleavage site specificity of E-64 and pepstatin processing activities by radiosequencing. A, plot of [³⁵S]methionine activity released for each sequencing cycle of in vitro translated pro-PM II, which had been incubated with E-64 (dotted line) or pepstatin treated parasite lysate (solid line). B, mapping of the N terminus of processed PM II generated by each treatment. Radiolabeled methionine is indicated by an asterisk, and endogenous cleavage site is indicated by arrowhead.

FIGURE 3.

Alternative processing has slowed kinetics. Pulse-chase experiment demonstrating altered kinetics of pro-PM2 (pro) autoprocessing in the presence of E-64d. The parasites were metabolically labeled for 5 min and then chased in complete medium without (control) or with (+E-64d)25 μm E-64d for indicated times followed by immunoprecipitation with anti-PM II antibody and visualization by autoradiography. t_½ values were calculated as 15.0 and 9.5 min for control and E-64d, respectively. E-64d was added to both pulse and chase media.

FIGURE 4.

Profile of E-64d targets. Competitive labeling in which parasites were incubated in the absence or presence of E-64, or its more membrane-permeant analog E-64d for 15 min is shown. The parasites were then purified, and the lysates were incubated with E-64-bio to label remaining active sites. E-64 (post lysis) represents untreated parasite lysate that was incubated for 5 min at 37 °C with E-64 prior to the addition of E-64-bio.

FIGURE 5.

Falcipain-2 and 3 both cleave a synthetic peptide at the PM II processing site. A, peptide HHDG comprising the eight amino acids spanning the pro-PM II processing site flanked on the N terminus with dabcyl-GABA and on the C terminus with edans (molecular weight = 1453.6). Predicted masses for both N- and C-terminal fragments after cleavage at the endogenous G/S peptide bond are shown. B, matrix-assisted laser desorption ionization spectra of either uncleaved peptide or cleavage products after 15 min of incubation with FP-2 or FP-3. Predominant ion species of FP-2 cleavage products are 823.8 [M+Na]⁺, 845.8 [M+2Na]⁺ (N-terminal fragment, N) and 691.8 [M+Na]⁺, 713.8 [M+2Na]⁺ (C-terminal fragment, C). Predominant ion species of FP-3 cleavage products are 801.8 [M+H]⁺, 823.8 [M+Na]⁺ (N-terminal fragment, N) and 691.8 [M+Na]⁺ (C-terminal fragment, C). Ions corresponding to alternative peptide bond cleavage were not detected.

FIGURE 6.

Model of proplasmepsin processing. Upon delivery to the food vacuole, membrane-bound pro-plasmepsin (pro-PM) is processed to its mature, active form (mature-PM) by the E-64d sensitive cysteine protease falcipains (FPs). If, however, falcipain activity is inhibited, then plasmepsins (PMs) can undergo autoprocessing that is sensitive to pepstatin. The previous observation that ALLN (typically referred to as a cysteine protease inhibitor) can block pro-PM processing is explained by its ability to inhibit both the falcipains and the plasmepsins, thus blocking both pathways of pro-PM processing.