Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1

Abstract

Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.

Figure 1

(A) Schematic diagram of the recombinant MBP-DPAP1-His₆ fusion. The white boxes indicate those regions of DPAP1 found in the mature protein, namely the exclusion domain (excl) and catalytic region, the latter being proteolytically cleaved to generate a heavy chain (hc) and a light chain (lc). The gray box within the DPAP1 sequence is the proregion (pro), the boundaries of which are not precisely known but were estimated from a sequence alignment with cathepsin C [5]. The black box represents the linker between MBP and DPAP1. The sequence below shows the TEV protease recognition sequence (underlined) with the cleavage site indicated by “/”. The resulting N-terminal Asp residue is shown in bold. The approximate positions of trypsin (Tr) cleavage are indicated with wavy arrows. Additional/alternate cleavage sites (not shown) give rise to size heterogeneity in the mature DPAP1 polypeptides. The hexahistidine tag is retained in the mature recombinant protein. Predicted polypeptide sizes are shown above the boxes in kDa. (B) Analysis of purified rDPAP1 by reducing SDS-PAGE. Left panel: silver stain. Contaminating proteins (probably keratins) are indicated with an asterisk. Right panels: immunoblots using antibodies that recognize the exclusion domain (“excl domain”) or the heavy chain. Sizes of markers are given in kDa.

Figure 2

(A) Elution of native DPAP1 (filled circles) and cathepsin C (open circles) from a Superdex 200 gel filtration column in 50 mM Na-MES pH 6, 200 mM NaCl and 1 mM EDTA. For each protein, activities in fractions are shown as percent relative activity with the highest activity set to 100%. (B) Activation of native (circles) and recombinant DPAP1 (squares) by chloride at pH 6.0. Ionic strength was held constant with Na₂SO₄. Activity values are expressed as relative activity with the highest activity set at 100%. Data points are the average of duplicate assays. (C) The catalytic efficiency (k_cat/K_m) of recombinant DPAP1 within the pH range 4.5 to 7.0 with the substrate Pro-Arg-AMC. Chloride concentration (30 mM) and total ionic strength (100 mM) were held constant at all pH values.

Figure 3

S1 and S2 specificity profiles of DPAP1 and cathepsin C. (A) Structure of the dipeptidyl-ACC positional scanning library. The P1 and P2 residues are indicated. In the P1 sublibrary, the each member contains a defined residue at the P1 position (the identities of which are indicated in the x-axis labels of the right panels of (B) and (C)) and the P2 position consists of an equimolar mixture of 20 amino acids. In the P2 sublibrary, the P2 position is defined (indicated in the x-axis labels of the left panels of (B) and (C)) and the P1 position is an equimolar mixture. (B) S1 and S2 specificities of native DPAP1 (black bars) and cathepsin C (gray bars). Data are reported as relative rates with those for Arg (P1 dataset) and norleucine (P2 dataset) set to 100%. The P1 or P2 amino acid is indicated with single-letter code, with n representing norleucine. Black stars indicate members of the P2 sublibrary that are substantially better substrates for DPAP1 compared to cathepsin C; a grey star indicates the P2 sublibrary member that is a much better substrate for cathepsin C than DPAP1. Each data point is the average of duplicate assays. nDPAP1, native DPAP1; catC, cathepsin C. (C) Comparison of the S1 and S2 specificities of recombinant DPAP1 with those of the native enzyme. Normalization of rates was as in (B). Values of rates that are off-scale are indicated above the graphs. rDPAP1, recombinant DPAP1. Each data point is the average of duplicate assays.

Figure 4

K_i values for inhibition of native DPAP1 (nDPAP1), recombinant DPAP1 (rDPAP1) and cathepsin C (catC) by peptide analogs containing semicarbazide (1) and nitrile (2) pharmacophores.