Purification and characterization of a leucine aminopeptidase from the bovine filarial parasite Setaria cervi

Available online at www.sciencedirect.com

Acta Tropica 106 (2008) 1–8

Purification and characterization of a leucine aminopeptidase from the bovine filarial parasite Setaria cervi Daya Ram Pokharel 1 , Sushma Rathaur ∗ Department of Biochemistry, Faculty of Sciences, Banaras Hindu University, Varanasi 221005, UP, India Received 15 April 2007; received in revised form 14 December 2007; accepted 18 December 2007 Available online 3 January 2008

Abstract Using synthetic peptide substrate Leu-p-NA, leucine aminopeptidase (LAP) activity was detected in both microfilarial and adult stages of a bovine filarial parasite Setaria cervi. A single protein fraction containing LAP activity was purified from the adult female S. cervi using three different chromatographic techniques. This purified enzyme was shown to be a 321 kDa zinc dependent metalloexopeptidase having maximum activity at pH 9.0 and 37 ◦ C. Its activity was significantly inhibited by aminopeptidase specific inhibitors such as 1,10-phenanthroline, ethylene diaminetetraacetic acid (EDTA), amastatin and bestatin; and activated by Co2+ , Mn2+ and Mg2+ ions. Puromycin and l-amino acids (e.g., glutamine, leucine and glycine) also showed some moderate inhibitory effects on the purified enzyme. Among various synthetic substrates tested, the purified enzyme hydrolysed Leu-p-NA at very high rate suggesting it to be a LAP. Both ELISA and western blotting analyses of S. cervi LAP revealed the presence of homologous protein in human filarial parasite Wuchereria bancrofti. The higher sensitivity of S. cervi LAP with microfilariaemic sera compared to other categories of W. bancrofti infected human sera implied its potential as a serodiagnostic marker against active filarial infection. The antigenic similarity between S. cervi LAP and W. bancrofti makes this molecule ideal for the discovery of new diagnostic marker, drugs and/or vaccine candidate for human lymphatic filariasis. © 2007 Elsevier B.V. All rights reserved. Keywords: Filarial nematodes; Lymphatic filariasis; Setaria cervi; Metalloproteases; Leucine aminopeptidase; ELISA; Western blotting

1. Introduction Lymphatic filariasis is an ancient mosquito borne parasitic disease caused by tissue dwelling human filarial nematodes and considered to be a major obstacle to the socioeconomic development in endemic countries. This disease has been identified as the second leading cause of permanent and long-term disability, with morbidity estimated at 5.5 million DALYs (Behm et al., 2005). It infects more than 120 million people throughout the world and puts another 1.1 billion people at risk of infection (WHO, 1997; Melrose, 2004). A range of inflammatory conditions are associated with the chronic disease which include recurrent attacks of adenolymphangitis, lymphoedema, hydrocele, and elephantiasis (Nutman, 2000). Since 2000, WHO has launched a global program to eliminate lymphatic filariasis by ∗

Corresponding author. Tel.: +91 542 2307323. E-mail address: [email protected] (S. Rathaur). 1 Present Address: Department of Biochemistry, Universal College of Medical Sciences, P.O. Box 53, Bhairahawa, Nepal. 0001-706X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2007.12.009

the year 2020 through the mass drug administration (MDA) in the endemic countries (Molyneux and Zagaria, 2002). Although this novel effort is now expanding in many parts of the world, initial results are not very much encouraging. These observations suggest that elimination of lymphatic filariasis through MDA program might be difficult with the existing control measures. Antifilarial drugs currently being used are largely ineffective in killing adult parasites and have very unpleasant side effects. Beside these, broad use of these drugs in endemic population might increase the possibility of drug resistance. No filarial vaccines or adulticides are yet available in the market. WHO has now realized the need of alternative mode of therapy such as vaccine or adulticides as an adjunct to the existing treatment protocols for successful elimination of lymphatic filariasis by the year 2020. Hence, there is still an urgent need for the identification of novel filarial target that can be exploited for the discovery of new vaccine and/or drug against this debilitating parasitic disease. Leucine aminopeptidases (LAP; EC 3.4.11.1) are a group of metalloexopeptidases which hydrolyse short peptide fragments

2

D.R. Pokharel, S. Rathaur / Acta Tropica 106 (2008) 1–8

from N-terminal end of the polypeptide chain (Taylor, 1993). They have been detected, purified and characterized in many helminth and protozoal parasites (Rogers, 1965; Rogers and Brooks, 1978; Acosta et al., 1998; Rhoads and Fetterer, 1998; Nankya-Kitaka et al., 1998; Morty and Morehead, 2002; McCarthy et al., 2004) and shown to play important roles such as moulting, surface membrane remodeling, egg hatching and digestion for the survival of parasites within host (Davey and Kan, 1968; Rogers, 1982; Xu and Dresden, 1986; Malet and Lesage, 1987; McCarthy et al., 2004). A recent tissue localization study in Setaria cervi also suggested its important roles in parasite feeding, cuticle remodeling, egg hatching and embryogenesis (Pokharel et al., 2006). Many of these reported aminopeptidases are immunogenic in nature and have distinct structural and biochemical properties compared to their host counterparts. For this reason they have been exploited in the discovery of novel vaccine (Piacenza et al., 1999) and drug targets (Knowles, 1993; Nankya-Kitaka et al., 1998; Howarth and Lloyd, 2000) for various parasitic diseases. Despite these observations, there has been no report on the purification and characterization of LAP from S. cervi. Although some aminopeptidases have previously been detected and purified from other filarial nematodes (Richer et al., 1992; Hong et al., 1993; Harnett et al., 1999), they remain poorly characterized and are not conclusively shown as LAPs. It is of interest, therefore, to purify and characterize LAP from S. cervi since exploring its biochemical and immunological properties would be an essential step towards identification of new diagnostic markers, vaccine antigen and drug target for the effective diagnosis and control of lymphatic filariasis. 2. Materials and methods 2.1. Collection of W. bancrofti infected human sera Wuchereria bancrofti infected human blood samples were collected from filarial endemic areas of Varanasi in north India and Wardha in Central India. Filarial cases were divided into microfilariaemics, chronic elephantiasis and occult based on the clinical signs and symptoms, and history of the patients. Healthy individuals devoid of any infections living in the endemic areas were treated as endemic normals and those living in non-endemic areas as non-endemic normals. Sera were separated by centrifuging the blood samples at 10,000 rpm for 20 min in a cooling centrifuge and stored at −20 ◦ C till further use. 2.2. Parasite collection and preparation parasite materials Adult, motile S. cervi worms were collected from the peritoneal folds of freshly slaughtered Indian buffaloes. Crude soluble extracts (10%) of microfilariae (mf), male and female adult worms were prepared in ice-cold homogenization buffer (50 mM phosphate buffer, pH 7.0, 1 mM PMSF, 5 mM DTT, 10% glycerol and 0.02% NaN3 ) according to the method described elsewhere (Singh and Rathaur, 2006). ES products of adult

worms and mf were collected following the method of Sharma et al. (1998). 2.3. Leucine aminopeptidase assay LAP assay was carried out in 50 mM Tris–HCl, pH 8.0 containing 1 mM each of l-Leu-p-NA substrate and MnCl2; and appropriate amount of parasite extracts/ES products in a final volume of 1.0 ml. The reaction mixture without substrate was preincubated for 15 min at 37 ◦ C followed by the addition of substrate solution and reincubation for 30 min at the same temperature. Reaction was terminated by the addition of 50 ␮l of 40% trichloroacetic acid (TCA). The reaction mixture was then centrifuged for 10 min at 10,000 rpm and absorbance was measured at 405 nm in a Visible Spectrophotometer. The concentration of p-nitroanniline (p-NA) was determined by using its molar absorptivity of 9450 M−1 cm−1 . One unit of enzyme activity was defined as the amount of enzyme liberating 1 ␮mol of p-NA per minute at 37 ◦ C. 2.4. Purification of leucine aminopeptidase LAP was purified from adult female worms using three consecutive chromatographic steps. The crude worm extract was first applied onto a DEAE-Sephadex A50 (Pharmacia Biotech, Uppsala, Sweden) column equilibrated with buffer A (50 mM Tris–HCl, pH 8.0 containing 1 mM MgCl2 and 0.02% NaN3 ) and eluted with 0–0.4 M NaCl gradients in buffer A at a flow rate of 40 ml/h. Fractions containing LAP activity were pooled and concentrated and loaded onto Sephadex G-200 column (1 cm × 20 cm) equilibrated with buffer A and eluted with the same buffer at a flow rate of 12 ml/h. Active fractions were pooled, concentrated and applied to a Ni2+ -IDA column (Sigma–Aldrich) (0.5 cm × 3.8 cm) equilibrated with buffer B (50 mM Tris–HCl, pH 8.0 containing 0.5 M NaCl and 0.02% NaN3 ). Elution was carried out with a gradient of 0–0.1 M imidazole (Sigma–Aldrich) in buffer B at a flow rate of 60 ml/h. Active fractions were pooled, dialyzed against fresh 50 mM Tris–HCl, pH 8.0, concentrated and stored at −20 ◦ C until used. In all purification steps, protein elution was monitored as absorbance at 280 nm using Shimadzu-1201 UV–Vis Spectrophotometer. 2.5. Protein estimation and SDS-PAGE Protein concentration was measured according to dyebinding method using bovine serum albumin as standard protein (Bradford, 1976). SDS-PAGE was performed under nonreducing condition (Laemmli, 1970) and protein bands were visualized by silver stain. Prestained molecular weight markers (10–170 kDa) were used for the estimation of subunit weight of the purified LAP. 2.6. Activity staining for leucine aminopeptidase Activity staining of purified LAP (30 ␮g) was performed in 5% native PAGE using Leu-p-NA substrate in 50 mM Tris buffer,

D.R. Pokharel, S. Rathaur / Acta Tropica 106 (2008) 1–8

pH 8.5 following the method described elsewhere (Bozi´c and Vujci´c, 2005). 2.7. Characterization of leucine aminopeptidase 2.7.1. Determination of pH and temperature optima Optimum pH was determined by assaying purified LAP in 50 mM sodium acetate buffer (pH 4.0–6.0), 50 mM Tris–HCl (pH 6.5–9.0) and 50 mM glycine–NaOH (pH 9.5–10.5) whereas optimum temperature was determined by incubating the routine assay mixture at 10, 20, 30, 37, 40, 50 and 60 ◦ C, respectively. 2.7.2. Determination of native molecular mass A Sephadex G-200 column equilibrated with 0.1 M Tris–HCl, pH 8.0 containing 1 mM MnCl2 and 0.02% NaN3 and calibrated by passing a mixture of standard proteins [thyroglobulin (669 kDa), ferritin (443 kDa), catalase (232 kDa) and aldolase (158 kDa) (Pharmacia Biotech, Uppsala, Sweden)] was used for the estimation of native molecular mass of LAP. Elution volume (Ve ) for LAP was measured by passing it through the calibrated column. Void volume (V0 ) was estimated as the elution volume of blue dextran (Mr = 2,000,000). The native molecular mass of LAP was then estimated by plotting relative elution volume (Ve /V0 ) against the logarithm of native molecular mass (Mr ) of standard proteins. 2.7.3. Substrate specificity and enzyme kinetics Substrate specificity of the purified LAP was determined by using l-Leu-p-NA, l-γ-Glut-p-NA and l-Ala-p-NA (Sigma–Aldrich) as substrates. Enzyme assays were carried out with all these substrates using the method described above in Section 2.3. Michaelis–Menten constants (Km ) and maximum velocity (Vmax ) for LAP were determined using Leu-p-NA substrate at concentrations ranging from 0.25 to 10.0 mM. Their values were calculated respectively from x and y intercepts of the double reciprocal plot of substrate concentration (mM) versus LAP activity (U/ml) using GraphPad Prism software. 2.7.4. Effect of protease inhibitors, divalent metal ions and l-amino acids Effects of a range of protease inhibitors, divalent cations and l-amino acids on LAP activity were assessed by incorporating them into the routine assay buffer. Where necessary compounds were dissolved in organic solvents prior to incorporation into assay medium and appropriate controls and reaction blanks were

3

performed concurrently. Purified LAP was preincubated with activators or inhibitors for 15 min at 37 ◦ C prior to addition of Leu-p-NA substrate. The rest assay method was similar to those described above in Section 2.3. 2.7.5. Antigenic cross-reactivity with W. bancrofti infected human sera Antigenic cross-reactivity between purified S. cervi LAP and various categories of W. bancrofti infected human sera were checked by ELISA (Rathaur et al., 1987) and western blotting (Lunde et al., 1988). Briefly, ELISA was performed by coating polystyrene microplate wells (Nunc, USA) with 1 ␮g/ml solution of purified LAP in 60 mM carbonate buffer, pH 9.6, followed by blocking with a solution of 5% dry milk in PBS, addition of different categories of filarial sera as primary antibodies (1:100 dilution) and detection of bound antibodies with peroxidase-conjugated rabbit anti-human IgG solution (1:5000 dilution) (Bangalore Genei, India). Color was developed by using o-phenylenediamine (OPD) and H2 O2 (Sigma–Aldrich); and absorbance was measured at 490 nm using ELISA plate reader (Bio-Rad). Western blotting was carried out by electrotransferring purified LAP (15 ␮g) separated on 10% SDS-PAGE to PVDF membrane (Sigma–Aldrich). Blotted membrane strips were incubated in blocking buffer (5% skimmed milk in PBS) followed by washing and incubation in various categories of filarial sera (diluted 1:100 in wash solution) for 1 h at 37 ◦ C. After washing, they were incubated with peroxidase-conjugated rabbit anti-human IgG (1:5000 dilutions) for 1 h at 37 ◦ C. Blots were developed by using substrates 4-chloro-1-naphthol and H2 O2 (Sigma–Aldrich). 3. Results 3.1. Detection and purification of LAP Presence of LAP was detected in crude extracts and ES products of both microfilarial and adult stages of S. cervi. This was higher in crude extracts of both the life stages than their ES products (Table 1). Female crude extract was found to have the highest LAP activity followed by mf and male crude extracts. Purification of adult female LAP using ion exchange, gel filtration and Ni2+ -IDA affinity chromatographies resulted in a single enzyme protein fraction (Fig. 1a, lane 5). The overall enzyme purification was 701-fold with the specific activity of 18,187.7 U/mg and recovery of 37.4% (Table 2).

Table 1 LAP activity in ES products and somatic extracts of microfilariae and adult worms using Leu-p-NA as substrate Life stages of S. cervi

Microfilarie Adult males Adult females a b

Assay pH

8.0 8.0 8.0

ES productsa

Crude soluble extract Activity ± S.D. (U/mg)

Specific activity ± S.D. (U/ml)

Activity ± S.D. (U/mg)

Specific activity ± S.D.b (U/ml)

297.7 ± 17.7 198.4 ± 11.4 595.4 ± 24.0

1417.5 ± 48.8 82.7 ± 2.4 220.5 ± 8.9

55.0 ± 15.0 49.6 ± 5.0 59.5 ± 8.2

717.2 ± 27.2 4961.0 ± 59 4961.0 ± 681.0

One unit of enzyme activity was defined as the amount of enzyme liberating 1 ␮mol of p-NA per minute at 37 ◦ C. S.D. = standard deviation.

4

D.R. Pokharel, S. Rathaur / Acta Tropica 106 (2008) 1–8

Fig. 1. (a) Protein staining of S. cervi LAP in SDS-PAGE. (1) Molecular marker, (2) adult female extract, (3) DEAE-Sephadex purified fraction, (4) Sephadex G-200 purified fraction and (5) LAP purified from Ni2+ -IDA column. (b) Estimation of native molecular mass of LAP using Sephadex G-200 column. (c) Activity staining of LAP in 5% native PAGE. (d) Determination of pH optimum of LAP.

3.2. Characterization of LAP Subunit and native molecular masses of S. cervi LAP were estimated to be 54 and 321 kDa by using SDS-PAGE and Sephadex G-200 column, respectively (Fig. 1a and b). Analysis of these results suggested a homohexameric quaternary structure for the purified enzyme. Determination of its optimum pH at 37 ◦ C produced a sharp pH profile: enzyme was active only in the range between pH 7.0–9.0 with a maximal activity at pH 9.0. There was a rapid decline in the enzyme activity outside this range (Fig. 1c). Similarly, the enzyme activity was found to

be a maximum at 37 ◦ C when assayed in a temperature range of 0–70 ◦ C. Enzyme activity at 60 ◦ C was only 6.15% of maximal activity and was completely denatured at 70 ◦ C and above. Enzyme staining in a native PAGE using l-Leu-p-NA as the substrate yielded a single reddish brown band corresponding to the LAP activity (Fig. 1d). The substrate specificity of LAP towards synthetic amino acyl-p-NAs was shown to be affected by amino acid residues of the peptide substrates. As shown in Fig. 2a, LAP hydrolysed Leu-p-NA at very high rate but not other amino acyl-p-NAs such as Glu-p-NA. The enzyme followed Michaelis–Menten kinetics

Table 2 Purification table for S. cervi leucine aminopeptidase Purification steps

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Yield (%)

Purification fold (n)

Crude extract DEAE-Sephadex A 50 Sephadex G-200 Ni2+ -IDA sepharose

12.30 0.60 0.13 0.024

1790.4 1041.8 583.4 218.2

145.6 551.2 4593.3 18,187.7

100 58.1 56.0 37.4

1.0 3.8 8.3 701.1

D.R. Pokharel, S. Rathaur / Acta Tropica 106 (2008) 1–8

5

Fig. 2. (a) Determination of substrate specificity for S. cervi LAP. (b) Michaelis–Menten plot for the hydrolysis of Leu-p-NA by LAP. (c) Determination of Km and Vmax values for LAP using Lineweaver–Burk plot. (d) Effects of divalent metal ions on LAP activity.

with Leu-p-NA substrate. Km and Vmax values were calculated from a double reciprocal plot of enzyme activity versus Leu-pNA concentration (Fig. 2b and c) and found to be 8.8 mM and 2976.2 U/ml, respectively. Effects of potential inhibitors and activators of S. cervi LAP were investigated using Leu-p-NA substrate (Table 3). Mn2+ ion was added to reaction mixture while assaying LAP inhibitors and activators except with metal chelators 1,10-phenanthroline and EDTA. Inhibitors of serine proteinases such as p-methyl sulphonyl fluoride (PMSF) (data not shown) and cysteine proteinases such as p-hydroxymercuric benzoate (p-HMB) and iodoacetic acid (IAA) had little effect on aminopeptidase activity, while metal-ion chelators (EDTA and 1,10-phenanthroline) strongly inhibited the LAP activity. Bestatin (0.1 mM) and amastatin (0.1 mM) abolished 64.7 and 81.26% of original LAP activity. The enzyme was also mildly inhibited by thiol group reagents dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and l-cysteine. LAP exhibited enhanced activity in the presence of certain metal ions with the order of preference: Co2+ > Mn2+ > Mg2+ . Activation of LAP with these three metal ions was concentration dependent and the maximum activity due to these metal ions was obtained at 1, 2 and 4 mM, respectively. Concentrations higher than these were proved to be inhibitory for the enzyme (Fig. 2d). On the other hand, Cu2+ , Fe2+ , Cd2+ , Ni2+ , Ba2+ , Hg2+ and Pb2+ ions were always highly inhibitory, abrogating more than 50% activity when tested at 1 mM concentration. Among various l-

amino acids and peptide tested, glutamine, leucine and glycine significantly inhibited the enzyme where as mild activation was observed with tyrosine, phenylalanine, proline, alanine and the tripeptide glutathione. Both ELISA and western blotting analyses of S. cervi LAP revealed its strong cross-reaction with microfilariaemic human sera. However, there was a very weak or no cross-reaction with non-endemic normal, endemic normal and occult human sera (Fig. 3a and b). 4. Discussion A stage specific screening for LAP in secretions and somatic extracts clearly demonstrated its presence both in mf and adult stages of S. cervi. However, its activity was relatively higher in adult female crude soluble extract than in the secretions and somatic extracts of mf and adult males. This was the reason for choosing crude soluble extract of adult females as the starting material for the purification of LAP. Analyses of subunit and native molecular masses of S. cervi LAP suggest that it exists as a homohexameric protein complex in native forms. A wide structural similarity has been shown to exist among LAPs expressed by seemingly unrelated organisms. For example, similar homohexameric structures for LAPs have been reported from S. mansoni and S. japonicum (McCarthy et al., 2004), bovine lens (Carpenter and Harrington, 1972), rabbit kidney (Oliveira et al.,

6

D.R. Pokharel, S. Rathaur / Acta Tropica 106 (2008) 1–8

Table 3 Effect of protease inhibitors and divalent metal ions on S. cervi LAP activity Inhibitors/activators None

Concentration (mM) –

Metalloprotease inhibitors Amastatin 0.001 0.01 Bestatin 0.001 0.01 EDTA 10.0 1,10-Phenanthroline 5.0 Cysteine protease inhibitors p-HMB 1.0 Iodoacetic acid 0.1 Other Puromycin

1.0

–SH and S–S group reagents DTT 5.0 2-ME 1.0 Divalent metal ions Co2+ Mn2+ Mg2+ Pb2+ Zn2+ Ni2+ Fe2+ Cu2+ Ba2+ Cd2+

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

% Relative activity (U/ml) 100.00 25.0 ± 4.08 19.0 ± 1.1 42.0 ± 1.9 36.5 ± 2.02 7.0 ± 3.03 45.0 ± 5.34 84.6 ± 5.4 102.7 ± 4.68 35.2 ± 5.10 71.0 ± 20.02 90.8 ± 8.90 528.3 ± 22.95 385.7 ± 13.93 471.4 ± 21.41 40.4 ± 4.14 44.1 ± 10.84 45.0 ± 21.41 27.40 ± 2.0 25.1 ± 4.6 26.4 ± 6.4 4.0 ± 3.74

1999), tomato (Gu et al., 1996) and E. coli (Vogt, 1970). The purified enzyme hydrolysed Leu-p-NA at very high rate compared to other aminoacyl-p-NA substrates. But its hydrolytic activity towards other aminoacyl-p-NA substrates, although at lower rates, suggests its broad and overlapping substrate profile with other aminopeptidases (Sanderink et al., 1988). This kind of property might be useful in the final breakdown of peptide fragments obtained from initial hydrolysis of the proteins involved in various biological functions assigned to this enzyme. Dependence of initial enzyme activity on substrate concentrations and appearance of hyperbolic curve when initial enzyme activities are plotted against different substrate concentrations clearly suggest that the purified enzyme follows Michaelis–Menten kinetics. This has also been the case with LAPs reported from many other organisms. Study of effects of specific peptidase inhibitors, metal ions and l-amino acids on LAP activity was proved to be crucial in confirming the mechanistic class of this enzyme. Inhibition of enzyme activity with known aminopeptidase inhibitors such as bestatin and amastatin confirmed its identity as a LAP. Bestatin and amastatin are compounds that mimic the LAP catalytic transition state and are strong inhibitors of animal aminopeptidases (Aoyagi et al., 1978; Umezawa et al., 1976). Inhibition by chelating agents such as 1,10-phenanthroline and EDTA indicated that LAP had at least one divalent zinc cation associated with its active site and should be considered as a zinc-aminopeptidase. It does not seem to have a free sulfhydryl residue close to

Fig. 3. (a) ELISA using purified S. cervi LAP as antigen and various categories of W. bancrofti infected human sera as primary antibodies. (b) Western blotting showing antigenic cross-reactivity between S. cervi and W. bancrofti using (i) purified S. cervi LAP and (ii) adult female crude extract using various categories of W. bancrofti infected human sera. MW, molecular weight markers; NEN, nonendemic normals (n = 12); EN, endemic normals (n = 17); MF, microfilariaemic (n = 18); EL, elephantiasis (n = 10); OC, occult (n = 11).

or at the active site since there was very slight or no inhibition by p-HMB and IAA. It rather seems to have disulphide bonds involved in the molecular integrity of an active enzyme as it was inhibited by thiol group reagents like DTT, l-cysteine and 2-ME. Enzyme inhibition with serine protease inhibitor PMSF was also insignificant and this was the reason why PMSF was added to the tissue homogenization buffer to inhibit other classes of proteases. Lack of enzyme inhibition with cysteine and serine protease inhibitors suggests that purified aminopeptidase does not have any endopeptidase activity. Interestingly, puromycin also inhibited S. cervi LAP, although at higher concentration, possibly suggesting its similarity to human cytosolic puromycin sensitive aminopeptidase (PSA). Recently, Brooks et al. (2003) showed that PSA orthologus to human cytosolic PSA is expressed throughout development in intestinal and nerve cells of free living nematode C. elegans and plays important roles in embryo development and reproduction. Metalloaminopeptidases exhibit a broad range of metal-ion dependence. Mammalian M13 LAPs typically utilize Zn2+ ion (Carpenter and Vahl, 1973), whereas other aminopeptidases are reported to utilize Mn2+ (Cottrell et al., 2000), Fe2+ (D’Souza

D.R. Pokharel, S. Rathaur / Acta Tropica 106 (2008) 1–8

and Holz, 1999), and Zn2+ ions (Walker and Bradshaw, 1998). The metal-ion-binding sites of bovine lens and plant LAPs have been identified and shown to consist of two zinc binding sites in their active sites (Kim and Lipscomb, 1994; Gu and Walling, 2002). Site 1 readily exchanges Zn2+ ions for other divalent metal cations including Mn2+ , Mg2+ , and Co2+ , and site 2 binds Zn2+ ions much more strongly and retains them under conditions that allow exchange of the Zn2+ ions in site 1. S. cervi LAP activity is also maximally enhanced by 1, 2 and 4 mM concentrations of Co2+ , Mn2+ and Mg2+ ions, respectively. Thus, it is most likely that S. cervi LAP also contains two such zinc binding sites at its active site and activation of LAP activity observed in this study with Co2+ , Mn2+ and Mg2+ ions might have resulted from substitution of site 1 Zn2+ ion with these metal ions. Smith and Spackman (1955) have reported that incubation of bovine lens LAP with Zn2+ , Pb2+ Hg2+ Cu2+ , Fe2+ , Ni2+ and Cd2+ ions inhibited enzyme activity, although by different extent. Others have also reported complete inhibition of LAP by Cd2+ ion (Himmelhoch, 1969; Kettman and Hanson, 1970). S. cervi LAP was also significantly inhibited by Zn2+ , Pb2+ Hg2+ Cu2+ , Fe2+ , Ni2+ , Ba2+ and Cd2+ ions. The later metal ion was found to be highly effective inhibitor of LAP as it alone inhibited more than 90% enzyme activity at 1 mM concentration. The inhibition of aminopeptidase activity by l-amino acids has been demonstrated earlier in various studies (Garner and Behal, 1975; Gilboa et al., 2001). We too observed marked inhibition of S. cervi LAP by l-glutamine, leucine, glycine and to some extent with cysteine. Other amino acids such as tyrosine, phenylalanine, proline, alanine, valine and a tripeptide glutathione rather activated the enzyme instead of inhibiting it. Unlike earlier studies, here we did not observe any differences in inhibition pattern due to size, hydrophobicity and aromatic nature of the amino acids. This inhibition, as shown for other aminopeptidases, might result due to binding of the amino acids to the two active site zinc ions via their free carboxylate group while displacing the zinc-bound water/hydroxide, which is present in the native enzyme. Beside this, the strength of inhibition of LAP by these three amino acids may be in part due to their differential ability to chelate Mn2+ ions used as an activator of LAP. However, complete insight into the mechanism of inhibition and activation by these l-amino acids can be obtained only when this LAP is crystallized and its structure is determined at high resolution. The biochemical and biophysical properties of other LAPs which belong to M17 family have been widely studied. They are hexameric enzymes having two zinc binding sites and one substrate binding site in a single molecule (Kim and Lipscomb, 1994). These two different sites are well conserved in this family of enzyme and consequently its members from prokaryotes, plants and animals display similar properties (Strater and Lipscomb, 1998). Hence on this ground, it can be inferred that S. cervi LAP also belongs to M17 family of aminopeptidases, as it displayed a marked substrate preference for N-terminal leucine residue, a requirement for divalent metal ions for activity, a weakly alkaline pH optimum and similarity to inhibition patterns by many LAP specific inhibitors and metal ions. However, it still remains to be determined the precise locations and

7

numbers of the zinc binding sites and substrate binding sites present in the native enzyme molecule by amino acid sequencing and X-ray crystallography. Preliminary analyses using ELISA and Western blotting supported many earlier observations that show antigenic similarity between S. cervi and W. bancrofti (Kaushal et al., 1987; Sharma et al., 1998). These observations suggest that a protein homologous to S. cervi LAP is expressed by W. bancrofti and thus it could be used for the screening of possible vaccine antigen, drug target and diagnostic marker against human lymphatic filariasis. Moreover, these analyses also indicated the potential of S. cervi LAP as possible diagnostic marker for the detection of active filarial infection. Functional analysis of LAP in S. cervi and several other nematode parasites suggests that this enzyme is involved in final digestion of the partially hydrolysed peptide fragments within gastrodermal cells, moulting, tissue remodeling, egg hatching, embryogenesis (Richer et al., 1992; Hong et al., 1993; Joshua, 2001; McCarthy et al., 2004; Pokharel et al., 2006). Hence, given its multidimensional roles in parasite biology, S. cervi LAP is worthy of investigation as a vaccine candidate and/or drug target against human lymphatic filariasis. Moreover, it is also imperative to carry out further studies to confirm its diagnostic potential using large number of sera samples from endemic area. Acknowledgements Daya Ram Pokharel is grateful to University Grants Commission, Nepal for partial fellowship for this work. Authors are also grateful to CSIR, Delhi Scheme No. 37(1189)/04/EMR-II for financial assistance. References Acosta, D., Goni, F., Carmona, C., 1998. Characterization and partial purification of a leucine aminopeptidase from Fasciola hepatica. J. Parasitol. 84,1–7. Aoyagi, T., Tobe, H., Kojima, F., Hamada, M., Takeuchi, T., Umezawa, H., 1978. Amastatin, an inhibitor of aminopeptidase A, produced by actinomycetes. J. Antibiot. 31, 636–638. Behm, C.A., Bendig, M.M., McCarter, J.P., Sluder, A.E., 2005. RNAi-based discovery and validation of new drug targets in filarial nematodes. Trends Parasitol. 21, 97–100. Bozi´c, N., Vujci´c, Z., 2005. Detection and quantification of leucyl aminopeptidase after native electrophoresis using leucine-p-nitroanilide. Electrophoresis 26, 2476–2480. Bradford, J., 1976. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brooks, D.R., Hooper, N.M., Isaac, R.E., 2003. The Caenorhabditis elegans orthologue of mammalian puromycin-sensitive aminopeptidase has roles in embryogenesis and reproduction. J. Biol. Chem. 278, 42795–42801. Carpenter, F.H., Harrington, K.T., 1972. Intermolecular cross-linking of monomeric proteins and cross-linking of oligomeric proteins as a probe of quaternary structure: application to leucine aminopeptidase (bovine lens). J. Biol. Chem. 247, 5580–5586. Carpenter, F.H., Vahl, J.M., 1973. Leucine aminopeptidase (bovine lens). Mechanism of activation by Mg2+ and Mn2+ of the zinc metalloenzyme, amino acid composition, and sulfhydryl content. J. Biol. Chem. 248, 294–304. Cottrell, G.S., Hooper, N.M., Turner, A.J., 2000. Cloning, expression and characterization of human cytosolic aminopeptidase P: a single manganese (II)-dependent enzyme. Biochemistry 39, 15121–15128.

8

D.R. Pokharel, S. Rathaur / Acta Tropica 106 (2008) 1–8

Davey, K.G., Kan, S.P., 1968. Moulting in a parasitic nematode Phocanema decipiens. IV: ecdysis and its control. Can. J. Zool. 46, 893–898. D’Souza, V.M., Holz, R.C., 1999. The methionyl aminopeptidase from Escherichia coli can function as an iron (II) enzyme. Biochemistry 38, 11079–11085. Garner, C.W., Behal, F.J., 1975. Human liver alanine aminopeptidase. Inhibition by amino acids. Biochemistry 14, 3208. Gilboa, R., Spungin-Bialik, A., Wohlfahrt, G., Schomburg, D., Blumberg, S., Shoham, G., 2001. Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism. Proteins Struct. Funct. Genet. 44, 490–504. Gu, Y.Q., Walling, L.L., 2002. Identification of residues critical for activity of the wound induced leucine aminopeptidase (LAP-A) of tomato. Eur. J. Biochem. 269, 1630–1640. Gu, Y.-Q., Pautot, V., Holzer, F.M., Walling, L.L., 1996. A complex array of proteins related to the multimeric leucine aminopeptidase of tomato. Plant Physiol. 110, 1257–1266. Harnett, W., Houston, K.M., Tate, R., Garate, T., Apfel, H., Adam, R., Haslam, S.M., Panico, M., Paxton, T., Dell, A., Morris, H., Brzeski, H., 1999. Molecular cloning and demonstration of an aminopeptidase activity in a filarial nematode glycoprotein. Mol. Biochem. Parasitol. 104, 11–23. Himmelhoch, S., 1969. Leucine aminopeptidase: a zinc metalloenzyme. Arch. Biochem. Biophys. 134, 597–602. Hong, X., Bouvier, J., Wong, M.M., Yamagata, G.Y.L., McKerrow, J.H., 1993. Brugia pahangi: identification and characterization of an aminopeptidase associated with larval moulting. Exp. Parasitol. 76, 127–133. Howarth, J., Lloyd, D.G., 2000. Simple 1,2-amino alcohols as strain-specific antimalarial agents. J. Antimicrob. Chemother. 46, 625–627. Joshua, G.W.P., 2001. Functional analysis of leucine aminopeptidase in Caenorhabditis elegans. Mol. Biochem. Parasitol. 113, 223–232. Kaushal, N.A., Kaushal, D.C., Ghatak, S., 1987. Identification of antigenic proteins of Setaria cervi by immunoblotting techniques. Immunol. Invest. 16, 139–149. Kettman, U., Hanson, H., 1970. Zur Bedeutung des Zinks in der Leucinaminopeptidase aus Rinderaugenlinsen. FEBS Lett. 10, 17–20. Kim, H., Lipscomb, W.N., 1994. Structure and mechanism of bovine lens leucine aminopeptidase. Adv. Enzymol. Relat. Areas Mol. Biol. 68, 153–213. Knowles, G., 1993. The effects of arphamenine-A, an inhibitor of aminopeptidases, on in vitro growth of Trypanosoma brucei. J. Antimicrob. Chemother. 32, 172–174. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685. Lunde, M.L., Paranjape, R., Lawley, T.J., Ottesen, E.A., 1988. Filarial antigen in circulating immune complexes from patients with Wuchereria bancrofti filariasis. Am. J. Trop. Med. Hyg. 38, 366–371. Malet, S., Lesage, M.C., 1987. Relationship between exsheathment and enzyme activity (alkaline phosphatase and leucine aminopeptidase) during ageing of Trichostrongylus colubriformis infective larvae. Ann. Res. Vet. 18, 275–278. McCarthy, E., Stacka, C., Donnelly, S.M., Doyle, S., Mann, V.H., Brindley, P.J., Stewart, M., Day, T.A., Maule, A.G., Dalton, J.P., 2004. Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum. Int. J. Parasitol. 34, 703–714. Melrose, W., 2004. Lymphatic Filariasis: A Review 1862–2002. Warwick Educational Publishing Inc., Australia. Molyneux, D.H., Zagaria, N., 2002. Lymphatic filariasis elimination: progress in global programme development. Ann. Trop. Med. Parasitol. 96, S15–S40. Morty, R.E., Morehead, J., 2002. Cloning and characterization of a leucyl aminopeptidase from three pathogenic Leishmania species. J. Biol. Chem. 277, 26057–26065.

Nankya-Kitaka, M.F., Curley, P.G., Gavigan, C.S., Bell, A., Dalton, J.P., 1998. Plasmodium chabaudi chabaudi and Plasmodium falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin. Parasitol. Res. 84, 552–558. Nutman, T.B., 2000. In: Nutman, T.B. (Ed.), Lymphatic Filariasis. Imperial College Press, London. Oliveira, S.M., Freitas Jr., J.O., Alves, K.B., 1999. Rabbit kidney aminopeptidases: purification and some properties. Immunopharmacology 45, 215–221. Piacenza, L., Acosta, D., Basmadjian, I., Dalton, J.P., Carmona, C., 1999. Vaccination with cathepsin l proteinases and with leucine aminopeptidase induces high levels of protection against fasciolosis in sheep. Infect. Immun. 67, 1954–1961. Pokharel, D.R., Rai, R., Kumar, P., Chaturvedi, C.M., Rathaur, S., 2006. Tissue localization of collagenase and leucine aminopeptidase of a bovine filarial parasite Setaria cervi. Filaria J. 5, 7. Rathaur, S., Robertson, B.D., Selkirk, M.E., Maizels, R.M., 1987. Secretory acetylcholinesterase from Brugia malayi adult and microfilarial parasites. Mol. Biochem. Parasitol. 26, 257–265. Rhoads, M.L., Fetterer, R.H., 1998. Purification and characterisation of a secreted aminopeptidase from adult Ascaris suum. Int. J. Parasitol. 28, 1681–1690. Richer, J.K., Sakanari, J.A., Frank, G.R., Grieve, R.B., 1992. Dirofilaria immitis: proteases produced by third- and fourth stage larvae. Exp. Parasitol. 75, 213–222. Rogers, W.P., Brooks, F., 1978. Leucine aminopeptidase in exsheathing fluid of North American and Australian Haemonchus contortus. Int. J. Parasitol. 8, 55–58. Rogers, W.P., 1965. The role of leucine aminopeptidase in the molting of nematode parasites. Comp. Biochem. Physiol. 14, 311–316. Rogers, W.P., 1982. Enzymes in the exsheathing fluid of nematodes and their biological significance. Int. J. Parasitol. 12, 495–502. Sanderink, G.J., Artur, Y., Siest, G., 1988. Human aminopeptidases: a review of the literature. J. Clin. Chem. Clin. Biochem. 26, 795–807. Sharma, S., Mishra, S., Rathaur, S., 1998. Secretory acetylcholinesterase of Setaria cervi microfilariae and its antigenic cross-reactivity with Wuchereria bancrofti. Trop. Med. Int. Health 3, 46–51. Singh, A., Rathaur, S., 2006. Identification and characterization of a seleniumdependent glutathione peroxidase in Setaria cervi. Biochem. Biophys. Res. Commun. 331, 1069–1074. Smith, E.L., Spackman, D.H., 1955. Leucine aminopeptidase V. Activation, specificity, and mechanism of action. J. Biol. Chem. 212, 271–299. Strater, N., Lipscomb, W.N., 1998. Clan MF containing co-catalytic leucyl aminopeptidases. In: Barrett, A.J., Rawlings, N., Woessner, J.F. (Eds.), Handbook of Proteolytic Enzymes. Academic Press, London, pp. 1382–1389. Taylor, A., 1993. Aminopeptidases: structure and function. FASEB J. 7, 290–298. Umezawa, H., Aoyagi, T., Suda, H., Hamada, M., Takeuchi, T., 1976. Bestatin, an inhibitor of aminopeptidase B, produced by actinomyces. J. Antibiot. 29, 97–99. Vogt, V., 1970. Purification and properties of an aminopeptidase from Escherichia coli. J. Biol. Chem. 245, 4760–4769. Walker, K.W., Bradshaw, R.A., 1998. Yeast methionine aminopeptidase I can utilize either Zn2+ or Co2+ as a cofactor: a case of mistaken identity? Protein Sci. 7, 2684–2687. World Health Organization, 1997. Lymphatic Filariasis Elimination. Report of a Meeting of the Principals for the Further Enhancement of Public/Private Partnership. WHO, Geneva. Xu, Y.Z., Dresden, M.H., 1986. Leucine aminopeptidase and hatching of Schistosoma mansoni eggs. J. Parasitol. 72, 507–511.