Purification and Characterization of Native and Recombinant Dipeptidyl Aminopeptidase 1 of Plasmodium falciparum
Wang, Flora Yinglai-Hua
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Plasmodium falciparum dipeptidyl aminopeptidase 1 (DPAP1) contributes to the degradation of hemoglobin by releasing dipeptides from globin oligopeptides in the food vacuole. The lack of success at DPAP1 gene disruption suggests that this exopeptidase is important for efficient growth during the erythrocytic asexual stage. DPAP1 is therefore an attractive target for the development of anti-malarial drugs that block the catabolism of hemoglobin. To guide the design of selective, potent DPAP1 inhibitors, it is necessary to characterize the substrate specificity of this enzyme along with its human homolog cathepsin C. Although native purification of DPAP1 is possible, the amount of purified enzyme obtained is insufficient for extensive biochemical characterization. To overcome this obstacle, a strategy was developed for the recombinant expression of soluble DPAP1 in the bacterium Escherichia coli and for its activation in vitro. The production of active recombinant DPAP1 presents three challenges: 1) expression of the protein in soluble form, 2) generation of the native N-terminus, and 3) cleavage of the pro-domain. Soluble expression of DPAP1 was achieved by fusing it to the C-terminus of maltose-binding protein (MBP). A linker sequence encoding a tobacco etch virus protease (TEVp) cleavage site was introduced between MBP and DPAP1 such that TEVp cleavage would generate the presumed native N-terminus of DPAP1. Incubation of the MBP-DPAP1 fusion with TEVp resulted in the release of free DPAP1which hydrolyzed the fluorogenic substrate proyly-arginyl-7-amido-4 methyl coumarin (Pro-Arg-AMC). Various proteases were tested for the ability to excise the pro-region. Treatment with both trypsin and papain removed the pro-region and increased DPAP1 activity two to three fold. When assayed with Pro-Arg-AMC, trypsin-treated DPAP1 had kinetic properties similar to native enzyme whereas papain-treated DPAP1 deviated from Michaelis-Menten kinetics. Using a combinational dipeptidyl substrate library, the substrate specificities of native and recombinant (trypsin-activated) DPAP1, as well as of human cathepsin C were profiled. We find that both DPAP1 and human cathepsin C accept a wide spectrum of amino acid side chains at the substrate P1 and P2 positions. Interestingly, several P2 residues show high selectivity for DPAP1 or cathepsin C. The collected data point to the feasibility of designing inhibitors that are specific for DPAP1 over cathepsin C.
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