Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Summary

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Abstract

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU₅₀ (11.1 ± 0.3 µg/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Introduction

Marine animals are a rich source of biologically active compounds. In recent decades, the composition of sea anemone venom has attracted scientific attention since it comprises a diversity of polypeptides with hemolytic, cytotoxic, enzymatic (phospholipase, protease, chitinase), and neurotoxic activity and inhibitory effects on proteolytic activity¹. In addition, these polypeptides are potential sources for the development of molecular tools in biotechnological and therapeutical use^2,3.

There are few reports about sea anemone venom and its molecular components due to the complexity of obtaining the venom, even isolation, and characterization of toxins. The extraction methods used in the reports involved lysis and emptying the contents of cells that are related and unrelated to the venom production¹.

A particular characteristic in all cnidarians is the absence of a system for production and release of the venom centralized in a single anatomical region. Instead, the nematocysts are structures that keep the venom^4,5. Other types of cells, called epidermal gland cells, also secrete toxins and are also distributed throughout the body of sea anemones⁶.

The first and most crucial challenge in obtaining the venom is the generation of an extract with sufficient manipulation in subsequent processes, without the inactivation or degradation of labile proteins. Next, the cells must be lysed, and the components-in this case, polypeptides must be efficiently and quickly extracted, avoiding proteolysis and hydrolysis while eliminating other cellular components⁷.

Different methods are used to obtain the crude extract of a sea anemone; some involve sacrificing the organism while others allow it to be kept alive. Methods that imply the use of the organism´s whole body allow for the release of most toxins from the venom⁸, compared to methods that keep organisms alive, which extract only some components of the venom⁹. The preparation of an extract requires evaluating the presence and potency of a substance of interest through a specific bioassay, which includes strategies to observe the pharmacological effects by in vivo or in vitro methods¹⁰.

Sea anemone venom contains cytolytic polypeptides, pore-forming toxins (PFTs)¹¹, and phospholipases¹²; these molecules are models in the study of protein-lipid interaction, molecular tools in cancer therapy, and biosensors based on nanopore³. The classification of sea anemone PFTs is carried out according to their size or molecular weight, from 5 kDa to 80 kDa. The 20 kDa PFT, the most studied and known as actinoporins¹¹, is of particular interest for its biomedical potential in the development of molecular tools for possible applications as anticancer, antimicrobial, and nanopore-based biosensors. Another cytolysin, including phospholipases, specifically phospholipase A2 (PLA2)¹³, releases a fatty acid due and hydrolyzes phospholipids, destabilizing the cell membrane. Due to this mechanism of action, PLA2 promises to be an essential model for the study and applications in inflammatory diseases. It could serve as a model for studies of lipid behavior in the cell membrane¹⁴.

Here, we describe an efficient protocol for obtaining the crude extract from sea anemone Anthopleura dowii Verrill, 1869, and detecting hemolysins and phospholipases. Both are relevant toxins that could be used as a template to design new molecular tools.

Protocol

The sea anemones were collected according to guidelines of the National Commission for Aquaculture, Fisheries, and Food of the Federal Government of Mexico (permit number PPF / DGOPTA 07332.250810.4060). Bioethics Committee of the Institute of Biotechnology, National Autonomous University of Mexico approved all the experiments with sea anemones. The sheep blood sample was purchased at the Center for Practical Teaching and Research in Animal Production and Health (CEPIPSA, National Autonomous University of Mexico).

1. Organism collection

1. Collect sea anemone A. dowii.
   NOTE: Here, the organisms were collected during low tide periods in the intertidal zone off the coast of Ensenada Baja, California, Mexico (Figure 1A,B).
2. Transport the organisms to the laboratory in containers with seawater (Figure 1C).
3. In the laboratory, clean the organisms with distilled water by hand to remove the big substrate particles adhering to the body.
4. Immediately freeze the organisms at -80 °C in an ultra-freezer for 72 h.
5. Place the organisms in special glasses for freeze-drying, with lyophilization conditions of -20 °C, 0.015 psi, for 48 h.
6. Store the lyophilized organisms at -20 °C until use.

2. Tissue hydration

1. Reconstitute 20 g dry weight of lyophilized organisms with 60 mL (1/3 w/v) of 50 mM sodium phosphate buffer, pH 7.5, and 1 inhibitor cocktail tablet (Table of Materials).
2. In a magnetic stirrer, continuously stir the sample at ~800 rpm, 4 °C, for 12 h.

3. Toxin release

1. To induce cell lysis and nematocyst discharge, freeze the sample at -20 °C for 12 h, and then place the container in water at room temperature (RT) until it thaws up to 90%. Repeat this procedure three times.
   NOTE: The sample does not thaw 100%; it is crucial to maintain the sample at ~4 °C to prevent protein degradation.
2. Observe the nematocyst discharged under a confocal microscope. Take 10 µL of the sample on a slide and place a coverslip over it. Observe under a confocal microscope at 100x and 60x magnification. Here a dye was not necessary.
3. Process the images captured in the confocal microscope in ImageJ software (version 1.53c, Wayne Rasband, National Institutes of Health, USA, https://imagej.nih.gov/ij).
4. If the tubule inside the nematocyst uncoils and is outside, the nematocysts are discharged; then, continue with the next step. Otherwise, only conduct one more freeze-thaw cycle.
5. To clarify the extract, centrifuge the sample at 25,400 x g for 40 min, 4 °C, recover the supernatant, and centrifuge it again. Repeat this step 3-4 times.
6. Recover the supernatant and discard the pellet. The supernatant or crude extract contains the venom components (toxins) and other cell molecules, such as lipids, carbohydrates, and nucleic acids.

4. Quantitation of total protein

1. Determine the protein concentration of the crude extract using a commercial Bradford colorimetric assay¹⁵ kit (Table of Materials).
2. Dilute the dye reagent (one part of dye with four parts of distilled water).
3. Prepare bovine serum albumin (BSA) Fraction V (Table of Materials) solution at 1 mg/mL concentration in phosphate buffer (50 mM sodium phosphate, pH 7.5).
4. Prepare the solutions listed in Table 1 in triplicate.
5. Vigorously mix each solution in a shaker for 4 s.
6. Incubate the samples for 5 min at RT, without shaking.
7. Measure absorbance (AU) at 595 nm.
8. Plot the averages of the sample BSA absorbances (AU vs. protein concentration), and determine the equation of the graph (Equation 1).
        Equation 1
   where y is the absorbance, m is the slope of the line, x is the protein concentration, and b is the y-intercept. To calculate crude extract concentration, solve for the x as follows:
        Equation 2

5. Determine polypeptide venom complexity

1. Assemble the glass container for vertical gel electrophoresis according to the manual.
2. Prepare acrylamide mix (30%): 29.2 g of acrylamide, 0.8 g of bis-acrylamide, the final volume of 100 mL. Filter the solution using a 0.45 µm membrane. Store the solution in a dark bottle at 4 °C. Then, prepare the mixture for the resolving gel (Table 2).
3. The SDS-PAGE gel consists of a resolving gel and a stacking gel. Prepare the gels as per the solution volumes defined in Table 2. During preparation, ensure to maintain each mix at 4 °C to reduce polymerization time.
   NOTE: The volume of each mixture varies depending on the brand of equipment used; for this protocol, the volumes correspond to the preparation of 15% acrylamide gel, 0.75 mm thick, for a vertical electrophoresis chamber (Table of Materials).
4. After the acrylamide has polymerized, ~10 min, add the mixture of stacking gel.
5. Prepare the mixture for the stacking gel, immediately add it to the glass plates, and place a comb to form the wells for loading the samples.
6. Once the stacking gel has polymerized (~15 min), remove the comb and place the glass container in a chamber for vertical electrophoresis.
7. Place 200 mL of electrode buffer (0.25 M Tris, 0.192 M glycine, 0.1% SDS) in the electrophoresis chamber.
8. Analyze the crude extract: Mix 30 µg of crude extract with 5 µL of protein loading buffer (25% glycerol, 15% sodium dodecyl sulfate, 25% 2-mercaptoethanol, 0.125 mM Tris pH 7, bromophenol blue 0.1 mg/mL) to denature proteins. The final volume must be <50 µL.
9. Heat at 90 °C for 5 min, cool, and centrifuge for 3 s at 1,400 x g.
10. Load the samples in the wells of the stacking gel. Load a standard molecular weight ladder.
11. Run electrophoresis at 25 mA constant for 1 h 30 min.
12. Remove the glass containers from the electrophoresis chamber to proceed with the staining of the gel proteins.

6. Protein staining

1. Rinse the gel in 50 mL of distilled water to remove debris from the buffer electrode.
2. Discard the water.
3. To avoid the diffusion of the gel proteins, add the fixation solution (60 mL of ethanol, 20 mL of acetic acid, and 20 mL of distilled water, final volume 100 mL). Incubate at RT for 40 min with shaking at 80 rpm. Decant the solution.
4. Add the protein crosslinking solution (15 mL of ethanol, 0.25 mL of glutaraldehyde, 50 mL of distilled water, final volume 65.25 mL), and incubate for 30 min at RT with shaking at 80 rpm. Remove the solution.
5. Wash the gel with 100 mL of distilled water for 5 min, and remove the water. Repeat this procedure four times.
6. Carry out protein staining with 50 mL of Coomassie brilliant blue R-250 filtered solution (40% methanol, 10% acetic acid, 50% distilled water, 0.1% dye, final volume 100 mL), stirring at 60 rpm in a laboratory rotary oscillator at RT for 15 min.
7. Recover the solution, which can be used to stain other gels.
8. To eliminate the excess dye, add a destaining solution (40 mL of methanol, 10 mL of acetic acid, 50 mL of distilled water). Keep stirring (80 rpm) at RT until the bands (stained proteins) visualize.
9. Keep the gel in 50 mL of distilled water and scan the image in a Gel documentation system.

7. Prepare red blood cell solution

1. Extract 1 mL of blood from the jugular vein of a sheep.
2. Remove the needle from the syringe and immediately drain blood into a tube with 50 mL of Alsever solution (0.002 M citric acid, 0.07 M NaCl, 0.1 M dextrose, and 0.027 M sodium citrate as an anticoagulant, pH 7.4).
3. Slowly invert the solution three times.
4. Keep at 4 °C for transfer to the laboratory.
5. Centrifuge at 804 x g for 5 min at 4 °C and discard the supernatant.
6. Resuspend the pellet in 30 mL of Alsever solution. Add the solution by sliding it along the tube's inner walls and, using a Pasteur plastic pipette, slowly resuspend the cells. Centrifuge again at 804 x g for 5 min, 4 °C. Repeat this procedure until the supernatant is clarified.
7. Add 15 mL of Alsever solution; resuspend the pellet slowly with a Pasteur plastic pipette.
8. Maintain the final volume of Alsever solution to achieve a concentration of 2 x 10⁶ cells/mL, approximately. Then, use a Neubauer chamber or hemocytometer to count the cells.
   NOTE: Count the erythrocytes in the hemacytometer slide (Improved Neubauer). The slide is a 30 mm x 70 mm x 4 mm slide. The center has two silver footplates (Figure 2A) (one top and one bottom grid), each 3 mm x 3 mm, and two channels on the sides of the grid. Counting cells is in the corner and center squares (red squares in Figure 2B). The optimal concentration of cells for counting should be in the order of 10⁶ cells. The central box has a surface area of 0.1 cm², which is equivalent to 0.0001 mL.
9. Place a coverslip over the slide.
10. Place 10 µL of the sample between the slide and the coverslip and wait for the sample to disburse.
11. In an optical microscope (magnification 60x), perform the count in the first square box. Count only the cells in the center grid and those that touch the upper or the left margin of the box.
12. Determine cell concentration using the following equation:
    Equation 3
    If the sample is very concentrated and dilution is required, then use the following equation:
    Equation 4
    NOTE: When taking the erythrocyte solution for the hemolysis test, slowly homogenize the solution with a plastic Pasteur pipette.
13. Perform the steps in triplicate to reduce error.

8. Hemolysis assay

1. Prepare the solution mixtures (in triplicates) indicated in Table 3 in conical 96-well plates.
   NOTE: Mix the erythrocytes slowly to maintain a homogeneous suspension.
2. Incubate for 1 h at 37 °C, without shaking.
3. Centrifuge the plate at 804 x g for 5 min at 4 °C.
4. Recover the supernatant in another 96-well plate with a flat bottom.
5. If the crude extract contains cytolysins, erythrocytes will lyse and release hemoglobin. Read the absorbance at 415 nm.
6. Calculate hemolysis percentage, using the following equation:
   % Hemolysis = ((Acrude extract-AAlsever buffer) / (AH₂O-AAlsever)) x 100
   NOTE: Acrude extract, AAlsever buffer, and AH₂O correspond to the absorbance of erythrocytes with the crude extract, absorbance of Alsever solution, and absorbance of distilled water, respectively.
7. Perform a hemolysis test before and after each freezing and thawing cycle with 50 µg of total protein from the crude extract.
8. Determine the number of freezing and thawing cycles required to reach 100% hemolytic activity.
9. Calculate the amount of extract that produces hemolysis in 50% of the erythrocytes (HU₅₀); follow the same protocol described in Table 3, increase the amount of sea anemone crude extract, and design the plot with sigmoidal adjustments using appropriate software (e.g., Origin, Table of Materials).

9. Phospholipase assay

1. Wash one chicken egg with 1% SDS in distilled water.
2. Separate the egg yolk from the egg white under sterile conditions.
3. Prepare 50 mL of 0.86% NaCl solution, and filter through a 0.22 µm filter. Then, prepare Solution A: Add 12 mL of egg yolk and 36 mL of 0.86% NaCl solution.
4. Prepare Solution B: Mix 300 mg of agarose in 50 mL of buffer (50 mM Tris, pH 7.5), filter through a 0.22 µm filter. Heat the solution in a microwave until boiling. Cool down in warm water to reach 43-45°C.
5. Prepare Solution C: Prepare 10 mM CaCl₂ and filter it with a 0.22 µm filter.
6. Prepare Solution D: 10 mg of rhodamine 6G in 1 mL of distilled water.
7. Under sterile conditions (laminar flow hood), add 500 µL of solutions A and C to solution B and 100 µL of solution D, mix, and pour 25 mL into each Petri dish (90 x 15 mm).
8. Wait for the solution to solidify under sterile conditions (30 min).
9. Make wells (~2-3 mm diameter) with a thin tube.
10. Add a total of 20 µL of phosphate buffer (negative control) in one well and 20 µL of a determined phospholipase (positive control) in another well.
11. Place different amounts of the crude extract protein, 5, 15, 25, 35, 45 µg, in the remaining wells, each in a final volume of 20 µL.
12. Wait for the agar to adsorb all the samples (30 min).
13. Incubate at 37 °C for 20 h.
14. If a halo forms around the well, this indicates phospholipase activity. Measure the halo formed with a vernier caliper.
    NOTE: Perform the experiment in triplicate (steps from 9.1-9.14).

Results

The representative results of the protocol used to obtain the crude extract of sea anemone showed that combining two techniques (agitation and cycles of freezing and thawing) produced an efficient discharge of nematocysts, and the total amount of protein was 500 mg (8 mg/mL) (Figure 3).

The crude extract's protein complexity could be observed from 10 kDa and greater than 250 kDa through SDS-PAGE electrophoresis. In addition, cytolysins were detected in the mol...

Discussion

The high demand for new compounds with applications in different fields of science and industry has led to the study of venom. Venom represents a rich source of molecules that serves as a template for generating new molecular tools. However, the complexity of these venoms requires the implementation and combination of various methods to obtain and study them.

Here, we show a method for obtaining and analyzing the venom of the sea anemone Anthopleura dowii, Verrill 1869, which can be u...

Materials

Name	Company	Catalog Number	Comments
15 mL conical centrifuge tube	Corning	430766
2-Bromophenol blue	Sigma	B75808
2-mercaptoetanol	Sigma-Aldrich	M6250-100ML
50 mL conical centrifuge tubes	Corning	430828
Acetic Acid Glacial	J.T. Baker	9515-03
Acrylamide	Promega	V3115
Agarose	Promega	V3125
Bisacrylamide	Promega	V3143
Bovine Serum Albumin Fraction V	Sigma	A3059-100G
Bradford Protein Assays	Bio-Rad	5000006
Calcium chloride	Sigma-Aldrich	C3306
Cell culture plates 96 well, V-bottom	Corning	3894
Centrifuge	Eppendorf	5804R
Centrifuge tubes	Corning	CLS430829
ChemiDoc MP system	Bio-Rad	1708280
Citric acid	Sigma-Aldrich	251275
Clear flat.bottom 96-Well Plates	Thermo Scientific	3855
Coomassie Brilliant Blue G-250	Bio-Rad	#1610406
Coomassie brilliant blue R-250	Bio-Rad	1610400
Dextrose	J.T. Baker	1916-01
Ductless Enclosure	Labconco	Vertical	https://imagej.nih.gov/ij ImageJ 1.53c
Gel Doc EZ	Bio Rad.		Gel Documentation System
Glycerol	Sigma-Aldrich	G5516-4L
Hemocytometer	Marienfeld	650030
ImageJ (Software)	NIH, USA	Version 1.53c
Incubator 211	Labnet	I5211 DS
Methanol	J.T. Baker	9049-03
Mini-PROTEAN tetra cell	Bio-Rad	1658000EDU
Na2HPO4	J.T. Baker	3824-01
NaCl	J.T. Baker	3624-01
NaH2PO4.H2O	J.T. Baker	3818-05
Origin software		version 9	To design the plot with sigmoidal adjustments
Petridish	Falcon	351007
Pipetman kit	Gilson	F167380
Precast mini gel	BioRad	1658004
Prestained Protein Ladder	Thermo Scientific	26620
Protease Inhibitor Cocktail	Roche	11836153001
Protein Assay Dye Reagent Concentrate	Bio-Rad	5000006
Rhodamine 6G	Sigma-Aldrich	252433
SDS	Sigma-Aldrich	L4509
Sodium citrate dihydrate	JT Baker	3646-01
Spectrophotometer	THERMO SCIENTIFIC	G10S UV-VIS
Tris Base	Sigma-Aldrich	77-86-1
Volt Power Supply	Hoefer	PS300B