Journal of Applied Sciences Research 5(7): 905-913, 2009 © 2009, INSInet Publication

Fasciola gigantica: Purification and Characterization of a Leucine

1,2 Saleh A. Mohamed, 2Mohamed O. El-Badry, 3Soha M. Hamdy, 2Somia S. Abdel-Ghany, 2Hala A. Salah, 2Afaf S. Fahmy

1Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia. 2Molecular Biology Department, National Research Centre, Cairo, Egypt. 3 Biochemistry Division, Chemistry Department, Faculty of Science, Fayoum University, Fayoum, Egypt.

Abstract: Leucine aminopeptidase, LAP2, was purified from Fasciola gigantica. LAP2 was purified by DEAE-Sepharose and Sephacryl S-200 column chromatographies. The relative molecular mass of F. gigantica LAP2 was estimated to be 30 kDa by gel filtration on Sephacryl S-200 column and electrophoresis on SDS-polyacrylamide gel. The Km value of F. gigantica LAP2 was estimated to be 0.13 mM L-leucine â-naphthylamine as substrate. The relative hydrolysis rates of amino acid-naphthylamines were highest with L-leucine â-naphthylamine (100%) and moderate with L-lysine â-naphthylamine (62%) and L-arginine â-naphthylamine (45%). The showed high activity in the pH range 7.5–9.0, with optimum activity in Tris-HCl buffer at pH 8.0. F. gigantica LAP2 had maximal activity at 30oC and was stable up to 40o C. The effects of various standard inhibitors against the F. gigantica LAP2 were demonstrated. Sulphahydryl reagent, PCMB, and inhibitor, PMSF, had no effect on the enzyme activity. F. gigantica LAP2 was inhibited by 1,10-o-phenanthroline, EDTA, sodium oxalate and sodium citrate indicating that the enzyme is metaloaminopeptidase. Except for Fe3+ which had no effect on F. gigantica LAP2, all metal cations tested inhibited the enzyme activity.

Key words: Fasciola gigantica, leucine aminopeptidase, purification, properties, protease inhibitors.

INTRODUCTION of amino acids from the N-terminus of peptides, most effective Leu, generated in the process of hemoglobin Fascioliasis is an important freshwater snail-borne degradation[36] . Amino acids derived from hemoglobin helminthiasis caused by the trematode parasites are essential to protein synthesis required for growth Fasciola hepatica and Fasciola gigantica that produces and development of the parasite[35]. a chronic liver infection of cattle and sheep, inflicting Since their initial characterization by Dalton and substantial productive loses on affected animals. While Heffernan[12] , gut-associated cysteine F. hepatica has been recorded in all continents, F. released by adult F. hepatica, later demonstrated to be gigantica is restricted to Africa and Asia. In addition, cathepsin L-like cysteine proteinases and termed FhCL1 human fascioliasis caused by F. hepatica has been and FhCL2, have attracted considerable attention due recently recognized as an emerging/re-emerging to their predominant presence in excretory/secretory zoonotic disease in many countries with an estimated products of the mammalian stages[14]. Moreover, their prevalence of up to 17 million people infected and 180 proteolytic activity on natural polypeptide targets shown million at risk of infection worldwide[1,24]. in vitro[6,5] has led to the consideration of CLs as Successful invasion of host tissue and nutrient vaccine candidates and different trials have evaluated uptake are essential for survival and maturation of the their potential either as native or recombinant parasite into an adult worm. Tissue invasion utilizes proteins[13,32]. gut-associated proteolytic that digest host In addition, leucine aminopeptidase (LAP, E.C. proteins including hemoglobin as a source of 3.4.11.1), FhLAP, a protein , was nutrition[11,9,10] . The initial cleavage events of characterized from the detergent soluble extract of hemoglobin are mediated by endopeptidases, which adult worms, based on its activity against different generate short peptides that are in turn degraded into amino acids coupled to 7-amido-4-methylcoumarin available peptide and free amino acids for the (AMC)[2]. This metalloenzyme was purified using a a b s o r p t i o n b y e x o p e p t i d a s e s i n c l u d i n g combination of gel filtration and bestatin-affinity aminopeptidase[37,33,26]. M17 leucine aminopeptidase is chromatography. Histochemistry analysis revealed the a cytosolic metallo-exopeptidase catalyzing the removal presence of FhLAP preferentially localized inside the

Corresponding Author: Saleh A. Mohamed, Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Kingdom of Saudi Arabia. E-mail: [email protected] 905 J. App. Sci. Res., 5(7): 905-913, 2009 epithelial cells that line the alimentary tract of the adult extract. The crude extract was then applied directly to worm, hence, a participation in the last stages of host a DEAE-Sepharose column (10×1.6 cm i.d.) protein was proposed. FhLAP showed broad equilibrated with 50 mM Tris-HCl buffer, pH 7.0. The amidolytic activity against fluorogenic substrates at exchanged material was eluted with a stepwise gradient optimum pH 8.0 and its activity was enhanced by the ranging from 0.0 to 0.4 M NaCl prepared in the same divalent metal cations Zn2+ , Mn2+ and Mg2+. When used buffer at a flow rate of 60 mL/h. Fractions of 3 mL as immunogen, either alone or in combination with were collected. Protein fractions exhibiting leucine native CL1 and CL2, very high level of protection (89 aminopeptidase activity were pooled in four peaks and 76%, respectively) were obtained against challenge (LA1–LA4). LA2 DEAE-Sepharose fractions was infection in ovine fasciolosis[32]. These results, applied to a Sephacryl S-200 column (90×1.6 cmi.d.) subsequently confirmed in another vaccine trial equilibrated with the same buffer and developed at a (unpublished results), represent the highest protection flow rate of 20 mL/h. Fractions of 3.5 mL were level achieved in ruminants by a natural antigen, collected. The leucine aminopeptidase was eluted with therefore positioning FhLAP as one of the leading the same buffer. vaccine candidates in ruminant fasciolosis[18]. Indeed, most studies of including Protein Determination: Protein was determined by the endopeptidases and were concentrated method of Bradford[8] using bovine serum albumin as on the F. hepatica. Previously, we purified and a standard. characterized of serine and cysteine proteases from F. gigantica[28]. The present study focused on the Polyacrylamide Gel Electrophoresis: For examining purification and characterization of F. gigantica leucine the homogeneity of the enzymatic preparations, aminopetidase to integrate the profile of F. electrophoresis under nondenaturing condition was gigantica. performed in 10% (w/v) acrylamide slab gel according to the method of Davis[15] using a Tris-glycine buffer, MATERIALS AND METHODS pH 8.3. Protein bands were located by staining with Coomassie Brilliant Blue R-250. Fasciola Material: Fasciola gigantica was obtained from sheep livers from the Cairo slaughter house. F. Molecular Weight Determination: Molecular weight gigantica was saved directly into an ice box for was determined by gel filtration technique using transportation to the laboratory and transferred to Sephacryl S-200. The column (90×1.6 cm i.d.) was frozen storage at -70 °C. calibrated with cytochrome C (12,400), carbonic anhydrase (29,000), bovine serumalbumin (67,000), L e u c i n e A m i n o p e t i d a s e A s s a y : L e u c i n e alcohol dehydrogenase (150,000) and â-amylase aminopeptidase was assayed according to Arant et al. (200,000). Dextran blue (2,000,000) was used to (1984). The reaction mixture contained 1350µl 0.1M determine the void volume (Vo). Subunit molecular Tris-HCl pH 8.0, 100µl 6 mM L-leucine â- weight was estimated by SDS-polyacrylamide gel naphthylamide hydrochloride, 50µl enzyme (total electrophoresis under denaturing conditions (Laemmli, volume 1.5ml) and incubated at 37°C for one hour. 1970). SDS-denatured -lactoalbumin, soybean trypsin After incubation, 0.5ml Fast Garnet GBC dye (1mg inhibitor, carbonic anhydrase, ovalbumin, bovine serum dye + 1ml 1M acetate buffer, pH 4.2 contained 0.1 ml albumin and phosphorylase b (M. Wt.14,400–97,000) tween 20) and the hydrolysis of the substrate was were used for the calibration curve. recorded at O.D. 525 in spectrophotometer. The enzyme activity was determined by using the standard RESULTS AND DISCUSSION curve with â-naphthylamine. O ne unit o f aminopeptidase activity was calculated as the amount P u r i f i c a t i o n o f F . g i g a n ti c a L e u c i n e of enzyme liberated 1µmol of â-naphthylamine per Aminopeptidase: The specific aminopeptidase activity hour under standard assay conditions. observed with leucine–â naphthylamine as the substrate at each purification step is summarized in Table 1. Purification of F. gigantica Leucine Aminopetidiase: Leucine aminopeptidase (LA P 2), from four Unless otherwise stated all steps were performed at 4–7 aminopeptidase isoenzymes, was purified to 18.4-fold °C using 50 mM Tris-HCl buffer, pH 7.0. Crude with a recovery of 37.7% from the crude extract by extract was prepared by homogenizing 1 g of F. chromatography using DEAE-Sepharose (Fig. 1) and gigantica in 5 ml of Tris-HCl buffer, pH 7.0 using a Sephacryl-S200 (Fig. 2) columns. The enzyme obtained Teflon Pestle homogenizer. The homogenate was from the final chromatography step showed a single centrifuged at 10,000 g for 10 min to remove insoluble protein band upon PAGE electrophoresis examination debris and the supernatant was designated as crude (Fig. 3A).

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Table 1: Purification scheme of F. gigantica Leucine aminopeptidase. Purification step Total protein Total activity Specific activity Fold Recovery (mg) (unit*) (units mg-1 protein) Purification % Crude extract 59 2603 44.1 1 100 Chromatography on DEAE-Sepharose 0.0 M NaCl (LAP1) 8.9 509 57.1 1.3 19.5 ------0.1 M NaCl (LAP2) 2.61 1331 511 11.6 51 ------0.2 M NaCl (LAP3) 1.74 303 174 3.1 11.6 ------0.3 M NaCl (LAP4) 1.606 197 123 2.79 7 Gel filtration on Sephacryl S-200 (LAP2) 1.21 982 811.5 18.4 37.7 *One unit of aminopeptidase activity was calculated as the amount of enzyme liberated 1 µmol of â-naphthylamine per hour under standard assay conditions.

Fig. 1: A typical elution profile for the separation of F. gigantica leucine aminopeptidase on DEAE-Sepharose column (10×1.6 cm i.d.). The column was equilibrated with 50 mM Tris-HCl buffer, pH 7.0. The flow rate was 60 mL/h and 3 mL fractions were collected.

Fig. 2: A typical elution profile for F. gigantica leucine aminopeptidase LAP2 DEAE-Sepharose fraction on Sephacryl S-200 column (90×1.6 cm i.d.). The column was equilibrated with 50 mMTris-HCl buffer, pH 7.0. The flow rate was 20 mL/h and 3.5 mL fractions were collected.

Molecular Mass: The relative molecular mass of F. enzyme with a molecular mass of 30 kDa under gigantica LAP2 was estimated to be 30 kDa by gel denaturing conditions (Fig. 3B). These results suggested filtration on Sephacryl-S200 column. Electrophoresis on that the enzyme is composed of one subunit. SDS-polyacrylamide gel showed one band for the

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Table 3 shows the effects of various inhibitors and metal ions on the enzyme activity of F. gigantica LA2. PCMB and PMSF caused no effect. The enzyme was inhibited by 1,10-o-phenanthroline. At 15 mM, EDTA inhibited the enzyme completely, whereas sodium oxalate and sodium citrate exert 95% and 80% of activity, respectively. All metal ions caused a decrease of F. gigantica LA2 activity, except for Fe3+ which did not have any effect. The enzyme was completely inhibited by Hg2+ and Co2+.

Discussion: Aminopeptidases have the ability to liberate various amino acid residues from the N- terminal amino acid of peptides. The aminopeptidases are classified according to the preference for aminoterminal amino acids of substrates and the sensitivity to inhibitors. An leucine aminopeptidase (LAP2) from F. gigantica was purified to 18.4-fold with recovery of 37.7% from the crude extract by DEAE-Sepharose and Sephacryl-S200 columns. LAP2 had an apparent molecular weight of 30 kDa as determined by both SDS-PAGE and gel permeation Fig. 3: (A) Non-denaturing polyacrylamide gel chromatography. This strongly indicated that the electrophoresis of F. gigantica leucine enzyme exists in a monomeric form. This value was a m i n o p e p t i d a s e f r o m t h e c o l u m n lower than the 90–150 kDa observed for the cytosolic chromatography steps. 1–crude extract; 2, or membrane-bound alanyl aminopeptidases from Sephacryl S-200 LAP2. (B) SDS-PAGE for mammals[29,34,21,40] , 116 kDa for the enzyme of mollusk, molecular weight determination of F. gigentica Aplysia californica[39], 90–120 kDa for the enzyme of aminopeptidase. 1–standard proteins; 2, silkworm[19,20,41], 67 kDa for the recombinant enzyme of Sephacryl S-200 LAP2. F. hepatica[1], 58 kDa for the recombinant enzyme of the trematode parasite Paragonimus westermani[35], 57.5 Properties of F. gigantica LAP2: The Km value of kDa and 52 kDa for the enzyme of Schistosoma F. gigantica LAP2 was estimated to be 0.13 mM L- mansoni and S. japoncium, respectively[25]. leucine â-naphthylamine as the substrate (Fig. 4). Table The results of the relative hydrolysis rates of 2 shows the relative hydrolysis rates of three amino amino acid- â-naphthylamines reveled that F. gigantica acid-naphthylamine derivatives by LAP2. The LAP2 expressed a higher preference for non-polar hydrolysis rates were strikingly affected by amino acid amino acid (leucine) in comparison to basic amino acid residues of the substrates. The hydrolysis rates were (lysine and arginine). The same results had been highest with L-leucine â-naphthylamine (100%) and reported for leucine aminopeptidases from Paragonimus moderate with L-lysine â-naphthylamine (62%) and L- westermani[35], Morimus funereus[7] and S.mansoni and arginine â-naphthylamine (45%). S. japoncium[25]. In contrast, The relative specificity of The pH optimum of F. gigantica LAP2 was recombinant LAP from Haemaphysalis longicornis examined within the pH range 4-9 by monitoring the against methionine was recorded higher than against hydrolysis of L-leucine â-naphthylamine. The enzyme leucine, lysine and arginine[17]. F. gigantica LAP2 showed high activity in the pH range 7.5–9.0, with demonstrated a lower affinity for the leucine â- optimum activity in Tris-HCl buffer at pH 8.0 (Fig. 5). naphthylamine (Km 0.13 mM) in comparison to that of The enzyme was over 50% of the maximum active in Spodoptera littoralis, with Km of 75.6 ìM [23]. However the pH range 5.5-9.0 in relation to maximum. The the affinity of LAP2 was much higher than LAPs of temperature optimum of the enzyme activity was Morimus funereus (Km 0.21 mM)[7], Rhyncosiara determined at the temperature ranges 10-90oC by Americana (Km 1.55 mM)[16]. monitoring the hydrolysis of L-leucine â-naphthylamine The optimal activity of F. gigantica LAP2 was (Fig. 6). F. gigantica LAP2 had a narrow temperature obtained at pH 8. The same pH optimum was reported optimum at 30o C. The enzyme was completely for LAP from Haemaphysalis longicornis, which is a inactivated at 90o C. The enzyme was stable up to 40oC physiological pH of cytosol, as seen in other M17 and rapidly lost its activity at temperatures above 60oC cytosolic aminopeptidase family members[17]. Generally, (Fig. 7). the pH optimum is ranged from 7 to 9 for LAPs from

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Fig. 4: Lineweaver –Burk plot determine the double-reciprocal relation between the velocity of LAP2 reaction versus the substrate concentrations. The reaction mixture contained in 1.5 ml: 1350 ìl 0.1 M Tris-HCl pH 8.0, different concentration of substrate and 0.5 unit of enzyme. Each point represents the average of two experiments.

Fig. 5: pH optimum of F. gigentica LAP2. The reaction mixture contained in 1.5 ml: 100 µl 6 mM L-leucine â-naphthylamide, suitable amount of enzyme and 1350 µl 0.1 M sodium acetate buffer ( pH 3.5 – 6), sodium phosphate buffer ( pH 6–8) and Tris-HCl (7–9).

Table 2: Substrate specificity of F. gigantica LPA2. environment of the gut lumen. Therefore, P. substrate Relative activity % westermani LAP functions intracellularly and suggested L-leucine â-naphthylamide 100 L-Lysine â-naphthylamide 62 its role in the final stages of hemoglobin catabolism, L-Arginine â-naphthylamide 45 where small peptides were generated and diffused through the gut epithelium and hydrolyzed to free Morimus funereus[7], Spodoptera littoralis[23] and F. amino acids[35]. hepatica[1] . The activity of recombinant Paragonimus F. gigantica LAP2 had maximal activity at 30oC. LAP and Schistosome LAP was optimum at slightly Recombinant H. longicornis LAP activity was shown alkaline pH, indicating these trematode enzymes may to have a similar temperature optimum at 35oC. This not function efficiently in the slightly acidic temperature Indicates that the enzyme might play an

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Table 3: Effect of various compounds on F. gigantica LAP2. Compound Concentration Relative activity (%) None 100 ------p-CMB 10 mM 98 ------PMSF 10 mM 97 ------1,10-o-phenanthroline 10 mM 38 EDTA 5 mM 32 ------10 mM 15 ------15 mM 0 Sodium oxalate 5 mM 35 ------10 mM 25 ------15 mM 5 Sodium citrate 5 mM 52 ------10 mM 40 ------15 mM 20 Fe3+ 10 mM 100 ------Ni2+ 10 mM 80 ------Ba2+ 10 mM 57 ------Ca2+ 10 mM 43 ------Zn2+ 10 mM 40 ------Mg2+ 10 mM 35 ------Cu2+ 10 mM 11 ------Co2+ 10 mM 0 ------Hg2+ 10 mM 0 0p-CMP, parachloromercuribenzoate; PMSF, phenylmethyl sulphonylfluoride

Fig. 6: Temperature optimum of F. gigentica LAP2. The enzyme activity was measured at various temperatures using the standard assay method as previously described. Each point represents the average of two experiments.

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Fig. 7: Effect of temperature on the thermal stability of F. gigantica LAP2. The reaction mixture contained in 1.5 mL: 1350 µl 0.1 M Tris-HCl buffer, pH 8.0, 100 µl 6 mM substrate and 0.5 unit of enzyme. The reaction mixture was pre-incubated at various temperatures for 15 min prior to substrate addition, followed by cooling in ice bath and incubation the reaction mixture at 37ºC for 1 h. Activity at zero time was taken as 100% activity. Each point represents the average of two experiments. active role under on-host stage conditions (around aminopeptidase, citrate insignificantly reduced enzyme 35o C) and maintain its activity under off-host stage activity, yet EDTA and 1,10- phenanthroline resulted in (around 25o C) to continue blood digestion[17]. Slightly a large loss of activity, and EGTA slightly activated higher temperature optimum was reported for leucince the enzyme[7]. aminopeptidase from Morimus funereus (40oC)[7]. LAP2 was stable up to 40o C when it is incubated for Except for Fe3+ which had no effect on F. 15 min prior to substrate addition. The thermal stability gigantica LAP2, all metal cations tested inhibited the reached 50o C for recombinant LAPs from H. enzyme activity. Co2+ and Hg2+ completely inhibited longicornis[17] and F. hepatica[1]. LAP2 activity, while Ni2+ , Ba2+ , Cu2+ , Mg2+ , Zn2+ and The effects of various standard protease inhibitors Ca2+ inhibited LAP2 activity partially. Similar effects against the F. gigantica LAP2 were demonstrated. The of Hg2+ , Cu2+ , Zn2+ , Co2+ and Ni2+ on LAP of Morimus sulphhydryl reagent, PCMB had no effect on enzyme funereus were described[7]. Zn2+ had also inhibition activity indicating that a SH-residue is not involved in effect on LAP of Spodoptera littoralis[23]. On the the enzyme activity. The serine protease inhibitor, contrary, a several-fold increase in recombinant F. PMSF did not inhibit the enzyme. These data suggest hepatica LAP activity was detected by addition of that the enzyme may not be categorized as either a Mn2+ , Co2+ and Mg2+ . In addition, no influence was cysteine proteinase and serine proteinase. PCMB and detected by incubation with Ni2+, Fe2+, and Cu2+ , while serine inhibitors did not exert any effect on Ca2+ produced an inhibitory effect. The effect of Zn2+ aminopepetidase from mid-gut gland scallop[38]. Serine is variable, being a modest activator at low and metallo-proteinases were detected in M. funerus concentration (1 mM) and acting as inhibitor at higher leucine aminopeptidase, whereas PMSF and 1,10-o- concentration (10 mM)[1]. The recombinant H. phenanthroline inhibited proteolytic activity longicornis LAP activity was also enhanced in the significantly[7]. F. gigantica LAP2 was inhibited by presence of several divalent metal cations (Co2+, Ni2+, 1,10-o-phenanthroline, EDTA, sodium oxalate and Mg2+ , Fe2+ , Cu2+ and Ca2+ ), while Cu2+ inhibited the sodium citrate. These results indicated that the F. enzyme[17] . Mg was an activator of LAP in gigantica LAP2 is a metaloaminopeptidase. Many, but Acanthoscelides obtectus[31]. not all, aminopeptidases are metaloenzyme containing a central Zn2+ , which is essential for enzyme REFERENCES activity[20,34,38]. The apoenzyme of aminopeptidase from scallop was completely reactivated by addition of 1. Acosta, D., M. Cancela, L. Piacenza, L. Roche, excess Zn2+ indicating that the enzyme is a typical zinc C. Carmonaa, J.F. Tortb, 2008. Fasciola hepatica aminopeptidase[38]. Some aminopeptidases have been leucine aminopeptidase, a promising candidate for reported to have the metallo-binding sequence motif, vaccination against ruminant fasciolosis. Molecular HEXXHX18E[3,19,27]. For Morimus funereus leucine Biochemistry and Parasitology, 158: 52–64.

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