Isolation, characterization, and ELISA applications of alkaline phosphatase and acetylcholinesterase from Moniezia expansa

1. Reproducible purification of alkaline phosphatase (ALP) and acetylcholine esterase (AChE) enzymes from tape worms, Moniezia expansa worms was performed as a new biological available source
2. A possible future application of such enzymes in the production of ALP and AChE-coated ELISA plates to use for research purposes.
3. The prepared ALP and AChE-coated ELISA plates will be less charged than other imported ones to use for research purposes.
4. Production of both expansa ALP and AChE enzymes on large scale will allow their use in various applications.

Introduction

Tapeworms Moniezia expansa (M. expansa) is considered one of the substantial sheep parasites that causes monieziasis and is a fundamental problem in sheep breeding (1,2). The electron microscopy and cytochemical studies of M. expansa revealed that the tegumental tissues and the interproglottidal glands of the worms are responsible for secreting alkaline phosphatase and acetylcholinesterase enzymes, signifying the endocrine pathway of its secretion (3,4). The M. expansa worms have various cell organelles, secretory vesicles, and glands responsible for secretions of varied enzymes (5). Alkaline phosphatase catalyzes the hydrolysis of various phosphate monoesters to give the phosphate group and the corresponding alcohol, phenol, or sugar (6). The ALPs are membrane-bound metalloenzymes consisting of different isoenzymes encoded by four gene loci: tissue-nonspecific, placental, intestinal, and germ-cell isoenzymes. The liver, kidney, and bone express comparatively large amounts of the same ALP isoform that designated tissue-nonspecific ALP. Intestinal (IntALP), placental (PALP), and germ cell (GCALP) are designated tissue-specific ALPs (7,8). The tissue-nonspecific ALP widely distributed in nature in virtually all species with high extents of homology and implicated in different biochemical operations (9). Several ALPs have purified from tissues of many mammalians like the liver, placenta, kidney and intestine and commercially used for various purposes (10). The alkaline phosphatase used extensively in molecular procedures as a biological reagent like (i) Removal of the phosphate group from ends of DNA or RNA fragments, (ii) Hybrid protein formation for studying gene expression, (iii) Enzyme attachment to antibodies for ELISA and Immunoblotting assays, (iv) Activation of phosphorescent substrates for DNA and RNA non-radioactive detections. Enzyme-linked immunosorbent Assay (ELISA) and different immunoassays are substantial in quality control and monitoring various biotechnological industries such as pharmaceuticals (11,12). Cholinesterases (ChEs) are specialized carboxylic ester hydrolases that break down the neurotransmitter acetylcholine into choline and acetate at a synaptic cleft. This terminates neurotransmission, allowing another signal to receive. Two ChEs types have specified in mammal tissues, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), based on their substrate specificities and sensitivity to inhibitors (13). AChE is a membrane-bound enzyme in muscles, the brain, cholinergic nerves, and erythrocytes in various molecular isoforms. A significant neuron enzyme exists in vertebral and invertebrate animals' neuromuscular junctions and cholinergic synapses. Acetylcholinesterase hydrolyzes the acetylcholine to restore the synapse excitability and transmission of the following nervous signals (14). Acetylcholinesterase targets different neurotoxic substances like organophosphates and their nerve agents. Acetylcholine is clinically significant in many neuronal diseases like Alzheimer's and myasthenia gravis (MG), characterized by a sharp decrease in acetylcholine content and diminished cholinergic functions (15). MG is an autoimmune disease characterized by skeletal muscle weakness due to the antibody-mediated processes in which antibodies manufactured having a tropism for acetylcholine receptors or their related proteins situated in postsynaptic membranes of the neuromuscular junctions (16). AChE activity is a suitable biomarker for exposure to many types of pollution in the surrounding environment. In fish, AChE activity is a suitable biomarker for exposure to many pollutants, such as organophosphates and polycyclic aromatic hydrocarbons. Firstly, the excess pollutant molecules trigger the gene expression of AChE till the pollutant concentration is high enough to inhibit AChE (17).

The current study isolates and characterizes two essential enzymes, ALP and AChE, from a new biological source, the helminthic parasite M. expansa. Preparation of ALP and AChE antibodies in rabbits and determination of ALP and AChE protein levels in different sheep, human and fish samples by ELISA test carried out.

Material and methods

Ethical approval

The experiment carried out according to the institutional guidelines of the National Research Centre’s Animal Research Committee under registration number 19-145: project number 1201034 NRC, Egypt.

Samples collection

Adult worms of M. expansa acquired from the intestine of lambs immediately after slaughter, then washed in 30% salted water at 37 ºC and used immediately (18).

Chemicals

Acetylthiocholine iodide (AcSChI), molecular weight marker kits for gel filtration. benzoylthiocholine iodide (BzSChI), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), disodium p-nitrophenyl phosphate (p-NPP). Sephacryl S-300, bovine serum albumin (BSA), glucose 6-phosphate, creatine phosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, phosphoenolpyruvate, propionylthiocholine iodide (PrSChI), 1,4 dithiothritol, butyrylthiocholine iodide (BuSChI), β-mercaptoethanol and o-phenylenediamine dihydrochloride (OPD) were purchased from Sigma Chemical Co. Molecular weight marker kits for SDS polyacrylamide gel electrophoresis were products of Pharmacia Fine Chemicals, Uppsala, Sweden. The other chemicals were of analytical grade.

Alkaline phosphatase activity assay

ALP assay mixture is 500µl of 1M diethanolamine-HCl pH 10.0, including 1 mM MgCl₂, 4 mM p-NPP, and enzyme solution was incubated for 20 min at 37° C and terminated with 500µl 1 N NaOH. The released p-nitrophenol monitored at 405 nm, and one ALP unit represents 1µ mol p-nitrophenol produced per minute (19).

Acetylcholinesterase activity assay

In the AChE activity assay, 1.0 mL of 60 mM Tris-HCl buffer pH 8.0, comprising 1 mM AcSChI, 1 mM DTNB, and enzyme solution, incubated for 1 hr at 37 °C and the OD was read at 412 nm. One AChE unit was the enzyme amount hydrolyzing 1μmol substrate per hour (20).

Protein measurement

Determination of protein contents achieved by the dye-binding assay utilizing BSA as a standard (21).

Purification of M. expansa ALP and AChE enzymes

All purification proceedings carried out at 4˚ C unless reported otherwise. 20 gm of Moniezia expansa worms homogenized by Teflon-pestled homogenizer with one volume of 0.05 M Tris-HCl buffer pH 8.0 and centrifuged for 30 min at 12.000 xg and 4˚ C to acquire the supernatant that designated crude extract. The crude extract was gradually saturated to 80% (NH₄)₂SO₄ with stirring for 30 min at 4 °C and centrifuged for 30 min at 12,000 xg to acquire the pellet, which was dissolved and extensively dialyzed in 0.05 M Tris-HCl buffer pH 8.0. The concentrated (NH₄)₂SO₄ fraction was fractionated utilizing a Sephacryl S-300 resin (142 cm x 2.4 cm) that previously equilibrated with the Tris-HCl buffer pH 8.0 and run with the same buffer at 30 ml / h flow rate. This chromatographic step repeated two times, the first for separating M. expansa ALP and the second for M. expansa AChE. In each chromatographic step, the column fractions tested for the activity of the desired enzyme (14).

Determination of the native molecular mass

For estimation of the native molecular weights of M. expansa ALP and M. expansa AChE, the Sephacryl column calibrated with the protein ferritin, catalase, alcohol dehydrogenase, bovine serum albumin, and myoglobin of the known molecular weights 440, 240, 150, 67 and 17 kDa respectively as standard markers (4).

Electrophoretic analysis

Electrophoresis analysis of M. expansa ALP and AChE was carried out on 12% SDS-PAGE, the subunit molecular weights of both enzymes were determined, and the protein staining was achieved utilizing 0.25% Coomassie BBR-250 (22).

Substrate specificity and enzyme Kinetics

The substrate specificity of M. expansa ALP screened toward the substrates, p-nitrophenyl phosphate (p-NPP), glucose 6-phosphate, creatine phosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, thiamine pyrophosphate, and phosphoenol pyruvate. The Km value of M. expansa ALP derived from the plot constructed for the reaction velocity (V) versus the substrate (p-NPP) concentrations (0.1 - 16 mM). In addition, the substrate specificity of M. expansa AChE screened toward the substrates, AcSChI-, PrSChI-, BuSChI-, and BzSChI iodides. The Km derived from the plot constructed for the reaction velocity (V) versus the substrate (AcSChI) concentrations (0.1 - 3.0 mM) (23).

Preparation of M. expansa ALP and AChE antibodies in rabbits

Enzyme antibodies were prepared according to the technique adopted by Toaleb et al. (24). Briefly, four rabbits (two for each enzyme of an average weight of 1.25 Kg/each) were subcutaneous immunized using ~40 µg from each of M. expansa ALP and AChE proteins in Freund’s complete adjuvant. A second ~ 40-µg protein booster dose in Freund's incomplete adjuvant was provided on day 15. Two further doses of about ~ 40-µg protein each without adjuvant also given on days 21 and 28. Serum samples were collected four days after the last immunization. These sera were stored at -20 ^oC until used in ELISA to determine ALP and AChE protein levels in random serum samples.

Collection of different serum samples

Different serum samples from different hosts (sheep, human, and fish) subjected to different circumstances were collected and stored at -20^oC until analysis was used for detection of ALP and AChE (2)

Enzyme-linked immunosorbent assay (ELISA)

1. expansa ALP and AChE protein levels determined by a semi-quantitative ELISA test (25). The optimum antigen, serum, and conjugate concentrations previously determined by check board titration (26). Enzyme antisera diluted to ~30 µg/ml concentration in 0.05 M Na-bicarbonate buffer pH 9.5 and 100 µl, which adsorbed to a polystyrene 96-well microtiter plate overnight at 4^oC. The plates subjected to three washes using Tween-20 phosphate-buffered saline (TPBS) pH 7.4, leaving the last wash in the wells for about 5 min as a soaking step. The wells then blocked for unspecific binding using 200 µl of 5% non-fat dry milk in PBS for 1h at room temperature. After washing, 100 µl of fish, sheep, and human sera at dilution 1:50 in PBS were added to the wells and incubated at 37^oC for 2h. After washing, 100-µl secondary antibody (Horseradish peroxidase-conjugated Anti-sheep IgG for sheep samples, Anti-human for human samples, and protein A for fish samples at 1:1000 dilution in PBS) added to the wells and incubated at 37 ^oC for 1h. The last washing step was repeated 5 times and followed by adding 100 µl OPD 60 mg tablets in 0.15 M Na-phosphate + 0.05 M Na-citrate buffer, pH 5.7 + 0.012% H₂O₂. The reaction was developed for about 30 min, terminated by adding 100 µl 1N NaOH, and absorbance was monitored at 450 nm utilizing an ELISA plate reader (27).

Results

Purification of M. expansa ALP and AChE

The starting specific activity of the ALP in the crude extract of the M. expansa worms found to be 178.1 units/mg protein (Table 1). This crude extract brought to 80% (NH₄)₂SO₄ saturation, stirred, and centrifuged for 30 min at 12,000-x g, and the obtained pellet dissolved and dialyzed in 0.05 M Tris-HCl buffer pH 8.0. Most of the ALP activity acquired in this fraction, and 80.1% enzyme activity retrieved, yielding a specific activity of 248.3 units/mg protein. The 80% (NH₄)₂SO₄ fraction was fractionated through a Sephacryl S-300 column, resulting in one peak exhibiting ALP activity resolved and designated M. expansa ALP (Figure 1a). The M. expansa ALP-specific activity was increased to 1070.8 units/mg protein, representing 6 purification folds with recovery of 53.6% (Table 1).

Table 1: A typical purification scheme of M. expansa worms ALP and AChE

Concerning AChE, the starting specific activity in the crude extract of the M. expansa worms found to be 2619 units/mg protein. This crude extract brought to 80% (NH₄)₂SO₄ saturation, stirred, and centrifuged for 30 min at 12,000-x g, and the obtained pellet dissolved and dialyzed in 0.05 M Tris-HCl buffer pH 8.0. Most of the AChE activity acquired in this fraction, and 84.8% enzyme activity retrieved, yielding a specific activity of 3213 units/mg protein. The 80% (NH₄)₂SO₄ fraction fractionated through a Sephacryl S-300 column, which showed one peak exhibited AChE activity was resolved and designated M. expansa AChE (Figure 1b). The M. expansa AChE-specific activity increased to 5250 units/mg protein of 2 purification folds and recovery of 43% (Table 1).

Molecular weight determination

The native molecular weight of both enzymes deduced from their elution volumes from the gel filtration column to be 312 kDa for M. expansa ALP (Figure 1a) and 270 kDa for M. expansa AChE (Figure 1b)

Figure1: A typical elution profile for the chromatography of the M. expansa ammonium sulfate fraction on Sephacryl S-300 column (142 cm x 2.4 cm) previously equilibrated with 0.05 M Tris-HCl buffer, pH 8.0 (a): M. expansa ALP and (b): M. expansa AChE.

Electrophoretic analyses of M. expansa ALP and AChE enzymes

To determine subunit molecular weights of the separated M. expansa ALP and AChE, the denatured ALP and AChE electrophoretically analyzed on 12% SDS-PAGE compared to molecular weight marker proteins. On SDS-PAGE, M. expansa ALP was found as one band of 312 kDa (Figure 2a), while M. expansa AChE was one band of 90 kDa subunit (Figure 2b).

Figure 2: Subunit molecular weight determination by electrophoretic analysis of M. expansa ALP and AChE on 12% SDS-PAGE (a): M. expansa ALP and (b): M. expansa AChE.

Estimation of optimum pH

The impact of pH on M. expansa ALP activity examined in diethanolamine buffer, while AChE in Tris-HCl buffer of various pH values. The M. expansa ALP pH profile displayed its maximum activity at pH 9.6 (Figure 3a), while M. expansa AChE displayed its maximum activity at pH 8.0 (Figure3b).

Substrate specificity and enzyme Kinetics

The substrate specificity of M. expansa ALP and AChE screened toward various substrates. M. expansa ALP cleaved preferentially p-NPP (100% relative activity) followed by ATP 17.3% and displayed the lowest activity toward phospho-enol-pyruvate 3.8%. Furthermore, M. expansa AChE cleaved preferentially AcSChI (100% relative activity) followed by PrSChI 55.7% and displayed the lowest activity toward BzSChI 13.4%.

The M. expansa ALP incubated with increasing concentrations of p-NPP, and a definite amount of the enzyme was utilized to construct the plot for the reaction velocity (V) versus the substrate concentration [S] (Figure 3c). The M. expansa ALP obeyed the Michaelis-Menten kinetics, and a linear relationship between (V) and [S] obtained and ended with a steady state at the maximum velocity. M. expansa AChE incubated with increasing concentrations of AcSChI, and a definite amount of the enzyme utilized to construct the plot for the reaction velocity (V) versus the substrate concentration [S] (Figure 3d). The M. expansa AChE obeyed the Michaelis-Menten kinetics, and a linear relationship between (V) and [S] obtained and ended with a steady state at the maximum velocity. The Km value found to be 0.9 mM AcSChI (Table 3).

Figure 3: (a): Effect of pH on the purified M. expansa ALP using diethanolamine buffer, pH (8.0-10.8). (b): Effect of pH on the purified M. expansa AChE using Tris-HCl buffer pH (6.8 - 9). (c): Effect of the substrate p-nitrophenyl phosphate concentration in mM on the reaction velocity of the purified M. expansa ALP. (d): Effect of the substrate acetylthiocholine iodide (AcSChI) concentration in mM on the reaction velocity of the purified M. expansa AChE.

Table 2: Substrate specificity of M. expansa ALP and AChE enzymes

Effect of divalent cations and inhibitors

The purified M. expansa ALP and AChE pre-incubated with 2 and 4 mM of each cation at 37˚C, and the activity then assayed. The activity of M. expansa ALP increased in the presence of 4 mM CoCl₂, MnCl₂, NiCl₂, and MgCl₂. Contrariwise, ZnCl₂, CuCl_2, and CaCl₂ were potent inhibitors of M. expansa ALP. Further, the activity of M. expansa AChE increased in the presence of CoCl₂, ZnCl₂, and NiCl₂ and decreased with MgCl₂ and CaCl₂ ions (Table 3).

Table 3: Effect of divalent cations on the M. expansa ALP and AChE enzymes

* These values represent % of the control and the means of triplicate experiments

In addition, the purified M. expansa ALP and AChE pre-incubated with each inhibitor at 37ºC for 5 min, and the residual activity then assayed. EDTA and DL-dithiothreitol (DTT) inhibited M. expansa ALP activity at 76.6 and 93.8%, respectively, while a considerable resistance of the enzyme was detected toward the inhibitors β-mercaptoethanol and 1, 10-phenanthroline. On the other hand, EDTA, 1, 10-phenanthroline, and eserine potently inhibited M. expansa AChE activity at 92.7, 50.8 and 70.1%, respectively, while a moderate, inhibition of the enzyme detected toward β-mercaptoethanol and DTT (Table 4).

Table 4: Effect of inhibitors on the M. expansa worms ALP and AChE

* These values represent % of the control and the means of triplicate experiments

Determination of M. expansa ALP and AChE protein levels in sheep, human, and fish samples

Concerning the M. expansa ALP level in random sheep samples by ELISA, the OD values ranged from 0.201 to 0.427. In humans, the OD values of M. expansa ALP recorded higher values than sheep ones. They ranged from 0.444 to 1.218. In fish samples, OD values of M. expansa ALP recorded different values in the different treated samples; values in B-a-P treated samples ranged from 0.453 to 0.858, values in Zn treated samples ranged from 0.612 to 0.793, and the pesticides group recorded OD values ranged from 0.503 to 0.672 (Figure 4).

Figure 4: ELISA determination of M. expansa ALP protein level in different sheep, human, and fish samples.

Concerning the M. expansa AChE level in some random sheep samples by ELISA test, the OD values ranged from 0.149 to 0.499. Similarly, human samples recorded more M. expansa AChE values than sheep samples, ranging from 0.522 to 1.208. While in fish samples, the highest M. expansa AChE values recorded in B-a-P treated samples, followed by pesticides-treated samples, and finally, the Zn treated ones, at ranges of 0.708 to 0.922, 0.565 to 0.785, and 0.432 to 0.596, respectively (Figure 5).

Figure 5: ELISA determination of M. expansa AChE protein level in different sheep, human, and fish samples.

Discussion

The tegumental tissues and the interproglottidal glands of M. expansa considered a significant source of ALP and AChE enzymes (3). ALP utilized exhaustively as a reagent in various molecular biological tests such as studying gene expression, ELISA, and the non-radioactive detection of DNA and RNA (11,12). It previously purified from different sources, such as the brain of Fenneropenaeus merguiensis crustaceans (28), from hen's egg yolk (29), and bovine milk (30). The activity and inhibition of AChE have known as specific human biomarkers for pesticide toxicity. Measuring AChE inhibition or activity has been utilized as a biomarker for pesticide exposure effect on the nervous system in environmental and occupational medicine. The success of using AChE as a biomarker was due to the ease of measuring the response, the dose-dependent behavior to toxin exposure, the sensitivity, and exhibiting a link to health harm effects (31).

The chromatography of M. expansa (NH₄)₂SO₄ fraction on the Sephacryl S-300 column showed one activity peak for both ALP and AChE enzymes. The specific activity of M. expansa ALP is 1070.8 U/mg, displaying 6.0 purification folds and 53.6% yield. Furthermore, the specific activity of M. expansa AChE is 5250 U/mg, displaying 2 purification folds and 43% yield. A broad range of purification folds and recovery percentages for ALP and AChE reported. ALP was purified from non-pasteurized milk with 17.67 folds and 56% yield (32), water buffalo liver with 10.56 and 17.92 folds with 17.1 and 24.5% yields, and from lactic acid bacteria with 36.07 folds with 23.74% yield (33). The enzyme AChE was purified from human erythrocytes 658 folds with a 23.5% yield (34). Based on the protein molecular weights, M. expansa ALP molecular mass deduced from the gel filtration chromatography as 312 kDa, confirmed as a monomer protein by the visualization on the SDS PAGE. These results are different from that of 190 and 180 kDa beef brain ALPs, 172 kDa bovine kidney (35), 146 kDa human liver (36), and 188 and 194 kDa water buffalo liver (33). On the other hand, M. expansa AChE molecular mass deduced from the gel filtration chromatography as 270 kDa, while by the visualization on the SDS PAGE, it seen as 90 kDa representing a homotrimer protein. This M. expansa AChE molecular mass is more significant than 245.5 kDa quail brain AChE (37), 201.5 kDa sheep liver AChE (38), and lower than 340 kDa cattle AChE (39).

The Km value of the purified M. expansa ALP was 4.4 mM p-NPP, more significant than 0.927 mM for non-pasteurized milk ALP (32) and 2.14 mM for lactic acid bacteria ALP (40). The divalent ions Co²⁺, Mn²⁺, Ni²⁺, and Mg²⁺increased the activity of M. expansa ALP, while Zn²⁺, Cu²⁺_, and Ca²⁺ inhibited it. On the other hand, Co²⁺, Zn²⁺, and Ni²⁺increased the activity of M. expansa AChE, while Mg²⁺and Ca²⁺ hindered it. In agreement with this report, the metal ions Mg²⁺, Ca²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, and Cd²⁺ activated the green crab ALP, while Hg²⁺, Ag⁺, Bi²⁺, Cu²⁺, and Zn²⁺ inhibit the enzyme (6) and the lactic acid bacteria ALP was activated by Mg²⁺ and Ca²⁺ and inhibited with Cu²⁺ and Zn²⁺ (40). Furthermore, red palm weevil AChE was greatly enhanced with Mg²⁺ and Ca²⁺(32), and the entomopathogenic nematode AChE was strongly inhibited with Hg⁺² and Ni⁺² and stimulated by Mn⁺² (20).

ALP plays a substantial role in growth through cell signal transduction and regulation of the cell cycle and used as a clinical diagnosis biomarker in various diseases (9). It represents a high research interest enzyme since it has many industrial applications. The ALP labeling routinely used in enzyme immunoassays, such as the ELISA technique. In addition, it can used in pharmaceutical and clinical analysis by using different ALPs conjugated to Zn and Mg (40). On the other hand, AChE activity is a suitable biomarker for exposure to many types of pollution in the surrounding environment. In fish, AChE activity is a suitable biomarker for exposure to many pollutants such as organophosphates and polycyclic aromatic hydrocarbons. Excess pollutant molecules activate gene expression of AChE until the pollutant concentration is high enough to inhibit AChE (17).

Conclusion

In conclusion, this study presents an established, easy and reproducible purification procedure of alkaline phosphatase and acetylcholine esterase enzymes from tapeworms, M. expanse, as a new biological available source. Production of both M. expansa ALP and AChE enzymes on a large scale will allow their use in various applications. The antisera of the purified M. expansa ALP and AChE are effective for determining both enzymes in different unknown sera from different animal species. This provides a possible future application of purified enzyme antibodies in producing ALP and AChE-coated ELISA plates, which will less charged than those will imported for research purposes.

Acknowledgments

The authors thank the National Research Centre, Egypt, which greatly appreciated for funding this work under the 12^th Research Plan (2019-2022), research project agreement number1201034.

Conflict of interest

The authors have declared no conflict of interest.