Ov-APR-1, an aspartic protease from the carcinogenic liver fluke, Opisthorchis viverrini: functional expression, immunolocalization and subsite specificity

Abstract

The human liver fluke Opisthorchis viverrini is endemic in Thailand, Laos and Cambodia where long standing infection is associated with cancer of the bile ducts, cholangiocarcinoma. Here we describe a cathepsin D-like aspartic protease from the gut and other tissues in O. viverrini. Phylogenetic analysis indicated that Ov-APR-1 is cathepsin D-like, conforming with Clan AA, Family A1 of the MEROPS classification. Ov-APR-1 is expressed in the gut of the mature hermaphroditic parasite, in the reproductive tissues including the testis and immature spermatids, and the developing miracidium within the eggshell. The enzyme was also detected in the excretory/secretory products of cultured adult flukes, indicating a role in host-parasite relationships. A recombinant form of the enzyme expressed in Escherichia coli and refolded from denatured inclusion bodies underwent autocatalytic activation and demonstrated hydrolytic activity against the peptide substrate 7-methoxycoumarin-4-acetyl-GKPILFFRLK(DNP)-D-Arg-amide with a k(cat)/K(m)=1.7 x 10(4)M(-1)s(-1) and a pH optimum around pH 2.5-3.0. The recombinant enzyme digested hemoglobin and bovine serum albumin. Forty-six serum albumin peptides were detected after digestion with recombinant Ov-APR-1 and sequenced. Like many other aspartic proteases, Ov-APR-1 displayed promiscuous preferences for residues accommodated at the key subsites of the binding pocket although hydrophobic (Leu, Ala, Ile), positively charged (Lys) and bulky aromatic (Phe) residues, in that order, were preferred at P1. Similar residues were accommodated at P1' although even less selectivity was exerted at this position.

Figure 1

Phylogenetic tree of aspartic proteases, including Ov-APR-1 from Opisthorchis viverrini (GenBank protein accession no. AAZ39883); protozoa, Plasmodium falciparum (plasmepsin1, P39898): fungi, Laccaria bicolor (XP_001877133), Aspergillus niger (XP_001399855); flatworm, Clonorchis sinensis (AAL14708) Fasciola hepatica (ABJ97285), Schistosoma japonicum (AAC37302), Schistosoma mansoni (AAB63442); arthropod, Aedes aegypti (XP_001657556), Drosophila melanogaster (NP_652013); roundworm, Ancylostoma ceylanicum (AAO22152), Meloidogyne incognita (ABC88426); vertebrates, Homo sapiens (NP_001900), Canis familiaris (NP_001020792), Mus musculus (NP_034113), Bos taurus (BAB21620). The number on each branch represents the bootstrap value from 1000 replicates.

Figure 2

Expression of Ov-apr-1 mRNA in different developmental stages of O. viverrini, as determined by RT-PCR. The following cDNA templates were included: lane 1, eggs; lane 2, metacercariae; lane 3, 2 week old juvenile worms; lane 4, one month old juvenile worms; lane 5, 2 month old adult worms. The upper panel shows expression of the β-actin mRNA in the different developmental stages, included here as a constitutively expressed control transcript. The lower panel shows expression of Ov-apr-1 mRNA. Control reactions where reverse transcriptase was omitted did not result in detectable amplicons (not shown).

Figure 3

Expression of Ov-APR-1 in Escherichia coli as insoluble inclusion bodies and refolding of the purified denatured protein. SDS-PAGE gel stained with Coomassie Brilliant Blue showing non-induced cell pellet (lane 1), induced cell pellet (2), insoluble protein remaining in the pellet after sonication of the bacteria under native conditions (3), soluble protein after sonicating bacteria under native conditions (4), insoluble material after resuspending the pellet shown in lane 3 with 8 M urea (5), soluble material after resuspending the pellet shown in lane 3 with 8 M urea (6), flow-through from a nickel-NTA column loaded with material from lane 6 under denaturing conditions (7), eluate from the nickel-NTA column loaded with material from lane 6 under denaturing conditions (8), equal loading of soluble protein from lane 7 after refolding into PBS (9).

Figure 4

Auto-activation of Ov-APR-1 from a pro-enzyme to its mature form at acidic pH. Recombinant Ov-APR-1 (pro-enzyme) was incubated in 50 mM sodium acetate at half-unit pH increments from pH 2–5 at 37°C for 18 h. C – control Ov-APR-1 protein in PBS, pH 7.2; 2.0 – 5.0 - pH values at which Ov-APR-1 was incubated to observe auto-activation from pro- (upper band) to mature form (lower band).

Figure 5

Recognition of recombinant Ov-APR-1 and parasites extracts from different developmental stages of O. viverrini. Western blot of recombinant Ov-APR-1 (rAPR-1), soluble egg extract (egg), soluble metacercariae extract (MC), soluble adult somatic extract (adult) and adult worm excretory/secretory proteins (ES) probed with pooled serum from mice immunized with recombinant Ov-APR-1. ES products were also probed with normal mouse serum (NMS - pre-vaccination with APR-1) and did not reveal any bands.

Figure 6

Immunohistochemistry of Ov-APR-1 in adult O. viverrini and infected hamster bile ducts. Negative control using pre-immune mouse serum shows no staining in the fluke and bile duct epithelium (A). Ov-APR-1 expresses in the vitelline glands (v), gut (g) and parasite eggs (e) (B). The miracidia in the eggs show intense staining (inset). Strong expression of Ov-APR-1 is observed in the fluke testis (t) (C) and ovary (o) (D). Immunoperoxidase staining, original magnification (A–B, 100×; C-D, 200×).

Figure 7

Refolded recombinant Ov-APR-1 digests human hemoglobin and serum albumin. Cleavage of human hemoglobin (Hb) by Ov-APR-1 at pH 2.0 - 4.0 but not at pH >5.0, and inhibition of hemoglobinolytic activity by pepstatin A (A). Cleavage of serum albumin by Ov-APR-1 at pH 2.0 - 3.5 but not at pH >4.0, and inhibition of catalytic activity by pepstatin A (B). Catalytic activity of refolded Ov-APR-1 against the peptidyl substrate 7-Methoxycoumarin-4-Acetyl-GKPILF↓FRLK(DNP)-D-Arg-Amide at different pH values (C). The final substrate concentration was 1.0 μM and the final volume of each reaction was 100 μl. Enzyme (2 nM) efficiency was assessed at pH 2.5 by measuring initial rates over a range of substrate concentrations (0.2–25 μM). The catalytic constants k_cat, K_m and k_cat/K_m were established from the resulting Michaelis-Menten plot.

Figure 8

Sites within serum albumin where Ov-APR-1 digested the protein as determined by liquid chromatography tandem mass spectrometry (LC-MS-MS). Peptides detected by LC-MS-MS are shown in red – forward slash (/) denotes the cleavage sites (A). P4–P4′ subsite specificities of Ov-APR-1. MS/MS data compiled from Mascot searches were used to create an alignment of peptide cleavage sites within serum albumin which was submitted to the WebLogo program to generate the image (B). Plots depict relative entropy between the observed and background distributions of amino acids at each subsite. The larger the letter denoting each amino acid, the more frequently that residue occurred. Hydrophobic residues are black, polar are green, basic are blue and acidic are red.