Comparative studies of the effects of Naja ashei venom-derived proteins on model and native lipid membranes

TL;DR

This study explores how proteins from Naja ashei venom affect cancer cell membranes, showing that leukemia cells are more sensitive due to their lipid composition.

Contribution

The study provides new insights into the membrane-level effects of less-studied SVMP and CRISP proteins from Naja ashei venom.

Findings

Leukemia cell membranes were more susceptible to toxin-induced damage due to higher unsaturated lipid content.

PLA₂, CRISP, and SVMP proteins alter membrane stiffness, elasticity, and surface charge in distinct ways.

The effects of these proteins align with predictions from model lipid systems and highlight their potential for anticancer research.

Abstract

Venoms contain toxins that are increasingly recognized as valuable sources of biologically active compounds. In this study, we examined the biochemical and biophysical properties of PLA₂, CRISP, and SVMP fractions isolated from Naja ashei venom with the detailed description of their interactions with cell membranes. By integrating these results with our previous analysis of the 3FTx fractions, we provide a broad and coherent overview of the most relevant protein components in N. ashei venom. Experiments were performed on two cancer cell lines with distinct membrane architectures: HL-60 (leukemia) and SK-N-SH (neuroblastoma). These lines differ particularly in membrane cholesterol content and in the saturation level of hydrophobic lipid parts. Our results enabled a comparative assessment of how each protein fraction modulates the mechanical and electrostatic properties of both model and native membranes. In agreement with predictions derived from model lipid systems, leukemia cell membranes were more susceptible to toxin-induced damage than neuroblastoma membranes, likely owing to their higher proportion of unsaturated lipids. Physicochemical analyses confirmed that the isolated PLA2, CRISP, and SVMP fractions alter key membrane parameters, including stiffness, elasticity, lipid-protein interactions, and the net charge of the polar headgroup region. Importantly, this work provides new insights into the membrane-level effects of SVMP and CRISP proteins, which have been less comprehensively studied compared with well characterized 3FTx and PLA₂ families. These results reveal distinct, cell membrane–dependent responses to N. ashei venom proteins and justify further basic research to better understand their action with cells.

Animal venoms contain many proteins that may serve as promising leads for developing new anticancer therapies. In this study, we examined how specific proteins isolated from Naja ashei venom interact with cancer cell membranes. We focused on two cell lines-HL-60 (leukemia) and SK-N-SH (neuroblastoma) - which differ in cholesterol levels and lipid saturation, two factors known to influence membrane sensitivity. The interaction between lipid membranes and toxins belonging to: PLA₂, CRISP, and SVMP families have been studied with the use of complementary techniques. The Langmuir method allowed us to observe how each protein affects the behavior of lipid monolayers, zeta potential measurements showed how they modify membrane surface charge, and LDH release assays revealed the degree of membrane permeability they induce. We also compared these effects with our earlier findings on three-finger toxins, a major component of N. ashei venom. Our results show a gradation in how the tested proteins influence membrane mechanics and electrostatics. PLA2, CRISP, and SVMP each produce distinct and measurable changes, some of which are more pronounced in leukemia cells, which have more unsaturated lipids and therefore more sensitive membranes. This study highlights how venom proteins can modify cancer cell membranes and identifies key toxin families as promising candidates for future research.

Figures

Isolation and identification of Naja ashei toxins.A. Anion Exchange Chromatography of fractions C and E obtained from previously performed Size-Exclusion Chromatography of crude Naja ashei venom [11]. B. Protein composition of selected fractions identified by shotgun LC-MS/MS. LAP (Low abundant proteins) – identified proteins whose contribution was less than 1%. Detailed composition of each fraction is available in Supplementary Information (S1 Table).

Exemplary surface pressure isotherms (π) as a function of the area per lipid molecule (left side) and the relationship between static compression modulus (Cs−1) and surface pressure (π) (right side) obtained for tested Langmuir model membranes after SVMP, CRISP and PLA2 treatment. The control isotherms were obtained for model membranes without toxin fractions exposure.

Percentage changes calculated for the surface area per molecule, the surface pressure (πcoll) at the moment of monolayer collapse, and the static compressive modulus (Cs−1) obtained for Langmuir model membranes that mimic HL-60 and the SK-N-SH cell lines.Values represent the mean ± SD calculated from 3-5 counts, based on independently recorded isotherms (n = 5). Statistically significant differences between concentrations observed for the SK-N-SH model membranes were indicated by uppercase letters, whereas for the HL-60 membranes, the differences were indicated by lowercase letters.

Percentage changes in zeta potential of liposomes measured at pH 7.4 after SVMP, CRISP and PLA2 fractions treatment.The parameter was measured in three biological repetitions and converted into percentage changes in comparison to liposomes without applied fractions. A value of 100% was assigned to the zeta potential of the pure liposomes.

Membrane integrity disruption measured by lactate dehydrogenase (LDH) release from HL-60 and SK-N-SH cells treated by SVMP, CRISP and PLA2 fractions.Values represent the average ± SD (n = 5). Different letters indicate significant differences between concentrations (lower case letters for HL-60 cells, uppercase letters for SK-N-SH). The 100% reference value was defined as the enzyme leakage measured in cells that were not exposed to the tested venom proteins.

Full text

1 Introduction

Studies on the chemistry of snake venom and the analysis of the role of these toxins in discovering their new properties show that, in recent years, venoms from various animal species have demonstrated promising cytotoxic activity against a wide range of malignant cancers [1–4]. However, despite these advances, no venom-derived anticancer drug has yet been approved by either the U.S. Food and Drug Administration or the European Medicines Agency [5].

Over the years, intensified scientific research has provided growing evidence that snake venom toxins - including phospholipases A₂, L-amino acid oxidases, three-finger toxins, and disintegrins (notably the disintegrin from Agkistrodon contortrix contortrix venom, which is currently in the preclinical stage; [6]) - exert potent cytotoxic effects on cancer cells. Their activity involves both intrinsic (mitochondrial) and extrinsic (receptor-mediated) apoptotic pathways, resulting in caspase activation, DNA fragmentation, cell cycle arrest, and altered Bcl-2/Bax protein expression. Many of these processes are further associated with enhanced oxidative stress and the generation of reactive oxygen species (ROS) [5].

Despite recent progress, there a limited number of studies that adequately explain the interaction of venom components with the cellular membrane. Such efforts are essential for understanding how these molecules integrate into membranes and for characterizing the resulting structural, mechanical, and thermodynamic alterations in both the membrane and its lipid components. In many cases, interactions with the lipid bilayer alone are sufficient to induce damage, modifications, or dysfunction of membrane constituents.

This approach not only helps to explain and predict their toxic properties following envenomation but also facilitates the identification of new agents capable of selectively damaging cancer cell membranes. Furthermore, it advances our understanding of the cell-mediated and membrane-specific functions of cobra venom proteins and supports the design of innovative peptide-based therapies inspired by Naja venom proteins (e.g., [7,8]).

Naja ashei, an African spitting cobra, is characterized by its predominantly cytotoxic venom, which makes it a valuable model for investigating toxin-membrane interactions and a promising reservoir of anticancer compounds. The venom is composed mainly of two protein families -three-finger toxins (3FTx) and phospholipases A_₂_ (PLA_₂_) - that together constitute more than 95% of the total protein content. It also contains several less abundant but biologically active components, including snake venom metalloproteinases (SVMP), venom nerve growth factors (VNGF), and cysteine-rich secretory proteins (CRISP) [9,10].

The aim of this study was to determine which fractions of Naja ashei venom most effectively modify the lipid components of biological membranes. Purified proteins from three families- PLA₂, SVMP, and CRISP - were examined and their effects compared with existing data on the membrane activity of another major protein family from Naja ashei venom - three-finger toxins. The analyses were carried out at three levels of complexity: interactions with model membranes assessed by the Langmuir technique, interactions with liposome-based model cell surfaces evaluated through zeta potential measurements, and the influence on native membranes determined by measuring lactate dehydrogenase (LDH) release from cancer cells as an indicator of membrane permeability.

Both model lipid systems and cancer cell lines were employed in the study, specifically SK-N-SH (neuroblastoma, representing the cells from nervous system) and HL-60 (leukemia, representing the cells from immune system). These cell lines were chosen not only because they represent malignancies of the nervous and immune systems-two of the most difficult cancers to treat, characterized by very low survival rates - but also because they differ in lipid saturation and cholesterol content, which are key determinants of the mechanical and physicochemical properties of membranes. This diversity provided an opportunity to obtain broader insights into the effects of venom-derived proteins on biologically distinct membrane types.

2 Methods

2.1 Toxin purification with ion exchange chromatography

The toxin samples for Ion Exchange Chromatography (IEX) were obtained by previously performed Size Exclusion Chromatography (SEC) using crude Naja ashei venom [11]. After SEC, we buffer-exchanged samples into 50 mM acetate bufer (pH 5.0) and applied them in separate runs to a strong cation exchange column (Cytiva Resource S column, 6 mL; GE Healthcare, Little Chalfont, UK) using the NGC Chromatography System (Bio-Rad, Hercules, USA). Proteins were eluted from the column with increasing concentration of the eluent (50 mM acetate buffer containing 1 M NaCl). Eluted toxins were monitored at 215 nm, with 1 mL fractions being collected. Samples belonging to the same chromatographic peak were concentrated using Vivaspin 2 centrifugal filters with 3000 MWCO PES membrane (Sartorius Stedim Lab Ltd., Stonehouse, UK). Protein concentration in obtained fractions was measured using the Pierce BCA Protein Assay Kit according to the manufacturer’s instructions.

2.2 Identification of proteins by LC–MS/MS

The protein composition of purified toxins after Ion Exchange Chromatography separation was determined using Liquid Chromatography coupled with tandem mass spectrometry (LC-MS/MS). Detailed sample preparation methodology was described in our previous publication [12]. Briefly, protein reduction and alkylation were conducted using dithiothreitol and iodoacetamide solutions, respectively and the proteins were digested using trypsin enzyme, with resulting peptides purified on StageTips C18 resin following protocol [13].

For each LC-MS/MS analysis, 0.86 μg of digested peptides were analyzed. Peptide separation was conducted on a Dionex Ultimate 3000 RSLC NanoLC system (Thermo Fisher Scientific, Waltham, MA, USA) using an Acclaim PepMap RSLC nanoViper C18 column (75 μm × 25 cm; 2 μm granulation (Thermo Fisher Scientific, Waltham, MA, USA)) with a 180 min ACN gradient (4–60% in 0.1% formic acid). Ion signals were detected on a Q Exactive Orbitrap mass spectrometer (Termo Scientifc, Waltham, MA, USA) operating on-line with the LC system during data-dependent acquisition (DDA) with survey scans acquired at a resolution of 70000 at m/z 200 in MS1 mode, and 17500 at m/z 200 in MS2 mode. Spectra were recorded over the scanning range of 350–2000 m/z in positive ion mode. The 15 most intense precursor ions per scan were selected for higher-energy collisional dissociation (HCD) with an isolation window of 2.0 m/z and normalized collision energies set to 28%. Both MS1 and MS2 scans used an Automatic Gain Control (AGC) target of 10^6^ ions and a maximum injection time of 100 ms.

MaxQuant software (ver. 1.6.7.0) was used to analyze the acquired MS/MS raw data files. UniProtKB database (Taxonomy: Serpentes; release 9/2019) was used for protein identification. Carbamidomethylation of cysteines was set as fixed modification, while oxidation of methionine and protein N-terminal acetylation were selected for variable modifications in the search. Other parameters used by the search engine were: precursor mass tolerance 20 ppm; main MS search tolerance 4.5 ppm; and MS/MS fragment ions tolerance 20 ppm. Enzyme properties: Trypsin with full specificity; missed cleavages: max. 2. PSM and protein False Discovery Rate (FDR): 1%. iBAQ (intensity-based absolute quantifcation) values of razor and unique peptides were used for the calculation of the amount of particular protein in the sample. The proteomics data has been deposited to the the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD033540 [14].

2.3 Lipid composition for model membranes

Based on the membrane composition of HL-60 and SK-N-SH cell lines, solutions for model lipid membranes (used in the Langmuir technique) and for liposome preparation (used in zeta potential measurements) were prepared using high-purity synthetic lipids (Avanti Polar Lipids, USA; for cholesterol - Sigma - Aldrich, USA). The following lipids were selected:1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (PC Brain), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), lysophosphatidylcholine (lyso-PC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), L-α-phosphatidylethanolamine (PE Brain), Sphingomyelin (SM Brain), Cholesterol (Chol).

DOPC, DPPC, DOPE, PE Brain, SM Brain, and cholesterol were dissolved in chloroform. DPPS was dissolved in a 9:1 (v/v) chloroform:methanol mixture. For SK-N-SH, the final membrane mixture [15–17] included: 64% phosphatidylcholine (PC; a mixture of 1,2-dioleoyl- and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, lyso-PC, and PC Brain), 19% L-α-phosphatidylethanolamine (PE Brain), and 18% sphingomyelin (SM Brain). For HL-60 [18–20], the membrane model was composed of 15.96 mol% DPPS, 18.64 mol% DPPC, 32.42 mol% DOPC, and 32.98 mol% DOPE. Lipid-to-cholesterol ratios were adjusted to reflect native membrane compositions: 79% phospholipids (PL) and 21% cholesterol for SK-N-SH, and 65% PL with 35% cholesterol for HL-60. The saturated-to-unsaturated fatty acid ratios were 48% to 52% for SK-N-SH, and 34.6% to 65.4% for HL-60.

All solvents were of chemical purity and purchased from Avantor or Pol - Aura (Poland).

2.4 Modeling cell membrane monolayers via Langmuir technique: lipid packing and compressibility analysis

The membrane study was initiated with Langmuir monolayer measurements, representing the primary and essential step for characterizing membrane lipid–protein interactions. The formation of a monolayer from a lipid bilayer is a widely accepted experimental model for studying membrane lipid interactions with various components [21]. The protein-free model allows us to directly determine whether the lipid component of the membrane is a target for the proteins under study. In contrast, cellular models inherently contain proteins within the membrane, making it difficult to isolate lipid-specific effects. The Langmuir model membrane allows us to demonstrate and observe whether venom proteins in a membrane-like environment can alter the membrane’s properties, thereby disrupting its homeostasis. Monolayers composed of lipid mixtures mimicking the membranes of the examined cell lines were prepared using the Langmuir monolayer technique, according to the standard protocol previously performed [11,12,22,23]. Lipid solutions were spread onto a phosphate-buffered saline (PBS, C = 0,01M, pH 7,4) subphase using a Hamilton micro syringe. To exclude possible catalytic activity of PLA_2_, the subphase did not contain Ca^2+^ ions. The spreading process was conducted in a Langmuir trough (Minitrough, KSV, Finland) equipped with a platinum Wilhelmy plate for precise surface pressure measurements. After deposition, the films were allowed to equilibrate on the subphase for 15 minutes. In the experiments, pure monolayer model lipid membranes were used as control systems. In the experimental systems, the monolayers were individually supplemented with CRISP, SVMP, and PLA_2_ proteins at concentrations of 10 and 40 ng/mL. These concentrations were determined based on previous studies on proteins isolated from the same source [11]. The selection of concentrations was made to not only allow the observation of changes in the π–A isotherms but also to enable an empirical comparison of the activity of the analyzed proteins with the previously characterized 3FTx fractions.

All measurements were conducted in a thermostatic experimental room. The Langmuir apparatus is equipped with an independent temperature control system (25 °C ± 1°C). Surface pressure-area (π-A) isotherms were recorded and controlled using KSV NIMA software. Each measurement was repeated three to five times to ensure reproducibility, with variation in molecular area ranging from ±0.1 to 0.3Ų. The standard deviation for surface tension measurements was maintained at ±0.1 mN/m.

Based on the π– A isotherms, the following physicochemical parameters of the lipid monolayers were determined: Limiting area per molecule (A) – the minimum molecular area reached at high surface pressures; Collapse pressure (π_coll_) – the surface pressure at which the monolayer collapses, indicating the maximum molecular packing; Static compression modulus (C_s_⁻¹) – a measure of the monolayer’s resistance to compression, calculated using the equation: $[eqn]$ , π is surface pressure, A_m_ – is a value characterizing the surface area occupied by a molecule in the membrane [24,25].

All recorded data were processed using SigmaPlot 15.0 software to determine the compression modulus and other physicochemical parameters. Data records for isotherms can be seen in Supplementary Information (S1 Dataset).

2.5 Liposome preparation and zeta potential measurement

Liposomes were prepared as described in previous studies [12,22,26]. A lipid film was formed by evaporating 0.4 mL of a 1 mg/mL chloroform lipid mixture solutions (separately for SK-N-SK and HL-60 model membrane) under constant argon flow in a round-bottom tube. Ultraclean water was added, and the suspension was ultrasonicated and vortexed to form liposomes. Freshly prepared liposomes were treated with CRISP, SVMP, and PLA_2_ venom fractions (10 and/or 40 ng/mL) and used for analysis. Measurements were performed at pH 7.4 and 25°C using Dynamic Light Scattering (Malvern Zetasizer Nano ZS). Each sample was equilibrated to the target temperature using the device’s built-in temperature control system. Zeta potential was calculated from electrophoretic mobility using the Smoluchowski equation, with results averaged over 10 replicates (±SD).

2.6 Cell cultures

To assess the effects on the entire cell membrane, rather than only its lipid component, complementary biochemical experiments were conducted on cell cultures. The SK-N-SH (ECACC) and HL-60 (ATCC) cell lines were cultured in DMEM and RPMI 1640 media, respectively, each supplemented with 10% FBS and 1% penicillin-streptomycin (CytoGen GmbH). Cells were maintained at 37 °C in a humidified incubator with 5% CO_₂_. Cell lines were used to perform the LDH assay.

2.7 Lactate dehydrogenase (LDH) assay-based analysis of membrane permeability

The lactate dehydrogenase (LDH) assay was used to determine the damage of the membrane. Cells (in an amount of 0.1 million cells per well) were incubated in the presence of CRISP, SVMP, and PLA_2_ fractions, (each applied individually at the concentration from 0 to 80 ng/mL) for 24 h. One hundred microliters of supernatants were added to the mixture containing 10 μl of 0.14-mM NADH and 0.5 mL of 0.75-mM sodium pyruvate. After incubation for 30 min at 37°C, 0.5 mL of 2,4-dinitrophenylhydrazine was added to the solution. After 1 h, the absorbance of the formed hydrazone was measured spectrophotometrically at 450 nm. One hundred percent activity was defined as the mean value obtained for cells not treated with the tested toxins, and the changes observed after exposure to the toxins were expressed as percentages relative to this reference value.

2.8 Statistics

Statistically significant differences were indicated as follows: significant differences between fractions and their concentrations (10 ng/mL and 40 ng/mL) were marked using uppercase letters for SK-N-SH cell line, and lowercase letters for HL-60 cell line.

The standard deviation was calculated as a measure of dispersion from the mean. Duncan’s multiple range test was used for statistical analysis between different treatments (p < 0.05).

3 Results

3.1 Ion Exchange Chromatography

We used Ion Exchange Chromatography followed by shotgun LC-MS/MS to purify and identify Naja ashei toxins for the functional analyses (Fig 1).

Isolation and identification of Naja ashei toxins.A. Anion Exchange Chromatography of fractions C and E obtained from previously performed Size-Exclusion Chromatography of crude Naja ashei venom [11]. B. Protein composition of selected fractions identified by shotgun LC-MS/MS. LAP (Low abundant proteins) – identified proteins whose contribution was less than 1%. Detailed composition of each fraction is available in Supplementary Information (S1 Table).

Two fractions (Fraction C and Fraction E), obtained after initial Size-Exclusion Chromatography on crude Naja ashei venom [11], were further separated using Ion Exchange Chromatography to obtain homogenous fractions, each belonging to a single toxin family. From Fraction C, we further purified the C3 fraction that contained representatives of the SVMP family with a purity above 95% as estimated by quantitative mass spectrometry analysis. Identified peptides within this fraction were assigned to P-III type SVMPs, which is expected as P-III SVMPs are the only SVMP type present in Elapidae venoms [27]. Fraction E was subsequently separated into three major fractions of high purity. The first peak predominantly (98.7%) contained Cysteine-rich secretory proteins (CRISP), while the subsequent two fractions consisted almost solely of proteins belonging to the catalytically active variants of PLA_2_ family. Within the PLA_2_ fractions, peaks E2 and E3 contained proteins from the same family but differed in their surface charge distribution, eluting at distinct ionic strengths. For functional analysis, we selected C3, E1, and E2. Fraction E2 was chosen over E3 due to its higher purity and the absence of nucleases, which were present in fraction E3.

3.2 Langmuir multicomponent monolayers

The Langmuir monolayer modeling technique allows for the precise reconstruction of the lipid part of the membrane, effectively mimicking the lipid layer of native cell membranes. π-A isotherms and, accordingly, static compression modulus was obtained for monolayers simulating neuroblastoma and leukemia cell membranes.

Regarding the shape of the isotherms, a noticeable change is visible, particularly after treatment with protein fractions - especially after the application of the PLA_2_ fraction (green lines, Fig 2). These changes were more pronounced in the case of model lipid membranes representing the composition of the SK-N-SH cell membrane.

Exemplary surface pressure isotherms (π) as a function of the area per lipid molecule (left side) and the relationship between static compression modulus (Cs−1) and surface pressure (π) (right side) obtained for tested Langmuir model membranes after SVMP, CRISP and PLA2 treatment. The control isotherms were obtained for model membranes without toxin fractions exposure.

The most significant changes in the shape of the curves, indicating lipid reorganization in the forming membrane, were observed in the initial stages of compression - during monolayer formation, at pressures up to 25 mN/m. These changes were documented as deviations from the ‘control’ curve.

Changes in membrane’s resistance to mechanical compression were also recorded and was manifested by a decrease in the C_s_⁻¹_max_ value, visible as a decrease and flattening of the curves.

Based on the isotherms, the following physicochemical parameters were calculated using the SigmaPlot software: surface area occupied by the molecule (A), collapse pressure (π_coll_) and static compression modulus (C_s_⁻¹) (Fig 3). The percentage changes in these parameters, caused by the action of the tested protein fractions, were referred to the control without the presence of venom proteins.

Percentage changes calculated for the surface area per molecule, the surface pressure (πcoll) at the moment of monolayer collapse, and the static compressive modulus (Cs−1) obtained for Langmuir model membranes that mimic HL-60 and the SK-N-SH cell lines.Values represent the mean ± SD calculated from 3-5 counts, based on independently recorded isotherms (n = 5). Statistically significant differences between concentrations observed for the SK-N-SH model membranes were indicated by uppercase letters, whereas for the HL-60 membranes, the differences were indicated by lowercase letters.

An increase in parameter that characterize the surface area occupied by a lipid was observed following the application of toxin fractions in both tested membrane models - SK-N-SH and HL-60, in a dose-dependent manner. The most pronounced change in this parameter was recorded after treatment with the PLA_₂_ fraction, showing an increase of up to 14.3% at the highest tested concentration. In contrast, the effects of SVMP and CRISP were less significant (Fig 2 red and blue lines). At the highest concentrations tested, the increase in discussed parameter was: SVMP: 5.2% (SK-N-SH) and 10.2% (HL-60), CRISP: 4.8% (SK-N-SH) and 9.2% (HL-60).

In the HL-60 membrane model, the collapse pressure at a concentration of 10 ng/mL showed little to no change or a slight increase (ranging from 0.4% to 2.4%), with statistically significant differences observed.

In the SK-N-SH cells (at both 10 and 40 ng/mL), and in HL-60 cells at the higher concentration, a faster collapse (decrease in π_coll_) was recorded, indicating increased membrane destabilization. The most pronounced effect was again observed following PLA_2_ treatment: 5.2% in HL-60 and 13.5% in SK-N-SH.

In all cases, a decrease in membrane stiffness and an increase in lability were noted, as indicated by a reduction in the C_s_⁻¹ parameter (static compression modulus). These changes were evident in both models, with PLA_2_ inducing the strongest effect – a greater than 50% reduction in C_s_⁻¹ in SK-N-SH cells, and a 33.5% reduction in HL-60 at the highest tested concentration.

The SVMP and CRISP fractions also induced changes in this parameter, albeit to a lesser extent. Percentage changes relative to the control reached up to: 17% in HL-60, and 12.5% (SVMP) and 14.2% (CRISP) in the SK-N-SH model.

3.3 Zeta potential

Calculation of changes in zeta potential parameters of liposomes revealed a varied effect of the tested protein fractions (Fig 4).

Percentage changes in zeta potential of liposomes measured at pH 7.4 after SVMP, CRISP and PLA2 fractions treatment.The parameter was measured in three biological repetitions and converted into percentage changes in comparison to liposomes without applied fractions. A value of 100% was assigned to the zeta potential of the pure liposomes.

The most consistent direction of changes was observed for the SVMP fraction in both cell models tested - charge stabilization towards positive values (compared to the control systems).

For the HL-60 model, a charge shift towards positive values was observed in almost all cases, ranging from 20.4% to 32.6%. The exception was the PLA_₂_ fraction, which caused a more negative charge shift of 15.4% compared to the surface of the control liposome (without added toxins).

In the liposome model representing the SK-N-SH cell line, only the CRISP protein showed a charge reduction effect compared to the zeta potential values. The remaining fractions tested showed an increase in zeta potential, from 9.6% (PLA_₂_) to as much as 50.8% (SVMP; compared to the zeta potential of the SK-N-SH model liposome).

It is worth noting the general trend: as protein concentration increases, charge changes tend to be more negative. Even if a clear increase in the positive direction was observed at lower concentrations (e.g., 10 ng/mL), at higher doses this effect may be less pronounced or even reversed (e.g., for CRISP, at 40 ng/mL, a 55.2% charge reduction).

3.4 Lactate dehydrogenase (LDH) release

Notably, the cell lines showed differential responses to the treatments.

In HL-60 cells, a concentration-dependent increase in LDH release was observed (Fig 5). At the highest concentration tested (80 ng/mL), CRISP induced the strongest effect (13.5% ± 1.4), significantly higher than the control. PLA_₂_ also showed a notable increase (12.7% ± 0.33), although with greater variability depending on the concentration. SVMP, while also elevating this parameter in a similar direction, demonstrated a more moderate effect (8.4% ± 1.05) but still effectively stimulated enzyme release.

Membrane integrity disruption measured by lactate dehydrogenase (LDH) release from HL-60 and SK-N-SH cells treated by SVMP, CRISP and PLA2 fractions.Values represent the average ± SD (n = 5). Different letters indicate significant differences between concentrations (lower case letters for HL-60 cells, uppercase letters for SK-N-SH). The 100% reference value was defined as the enzyme leakage measured in cells that were not exposed to the tested venom proteins.

In contrast, analysis of the trend from SK-N-SH cells treated with the protein fractions showed the greatest effects at the lowest concentration (PLA_2_, Fig. 5) - 16.3% reduction at 10 ng/mL (up to a maximum of 16.8% at 80 ng/mL). Additionally, CRISP exhibited a somewhat weaker but still - reduction of LDH release (6.6-14.3% reduction relative to control), as did SVMP (2.5–14.1% reduction).

4 Discussion

Studies conducted in model systems using snake venom components are particularly important for understanding the underlying mechanisms that may occur when venom proteins interact with the membrane. But the ‘goal’ is not only to identify the mechanism of action regarding specific cell lines, but also to derive a common mechanism leading to predictable action with lipid membrane components. The dominant components of many Elapidae venoms are three-finger toxins (3FTx) and phospholipase A₂ (PLA₂) proteins, which are well known for their involvement in toxic mechanisms associated with damage to, or modification of, the properties of whole cells and tissues [11,12,28–30]. It has long been known that three-finger toxins - as non-enzymatic components – exhibit high interaction with the polar part of the lipid membrane and their amino acid distribution facilitates their localization and penetration inside the lipid bilayer [31,32]. A special role in this interaction is played by zwitterionic lipids (e.g., phosphatidylcholine, phosphatidylserine), as well as unsaturated fatty acids and cholesterol [11], the content of which changes in the membranes of some types of cancer cells [33,34].

In the present study, interactions with the lipid component were first analyzed, deliberately excluding membrane target proteins in Langmuir monolayers and liposomes to eliminate their influence. This approach allowed assessment of the effect of tested toxins on the lipid component itself and the resulting changes in the mechanical and physicochemical properties of membranes. The results help to understand that not only protein targets are relevant for interactions with multiple toxins, but the lipid component of the membrane can also actively engage in such interactions. When these interactions occur and induce additional changes, they may disrupt cellular membrane function. Further analyses addressed the effects of the studied fractions on surface charge, particularly in the polar regions of lipids. Finally, these findings were translated into biochemical assays, linking molecular-level interactions to functional outcomes and bridging results from model membrane studies with in vitro analyses of cell membranes.

4.1 Effects of toxins on membrane properties

To address the extent of modification and the effectiveness of individual venom fractions on the lipid components of membranes, the current results were compared with two previous studies [11,12], which provided insights into the activity of the most abundant toxin fraction in Naja ashei venom-3FTx. Using the Langmuir technique, the model study confirmed that the two selected concentrations (10 and 40 ng/mL) effectively differentiate the effects of the tested toxins. It is worth noting that tested concentrations are relatively low, which confirms the high potency and efficacy of the studied venom toxins. Physicochemical parameters obtained for the model monolayers, indicate that of the four main classes of protein fractions compared (representing 3FTx, PLA_₂_, SVMP, and CRISP), three-fingered cytotoxins and PLA_2_ are the most effective. Overall, our studies indicate that these two protein groups interact most effectively with the lipid portion of the membrane. Since they are major toxins in the analyzed venom [9], their high availability makes them particularly advantageous candidates for further research on their role in cancer membrane development. For SVMP and CRISP, changes in membrane elasticity and lability were also observed and reached statistical significance; however, these changes suggest that the proteins may alter the mechanical properties of the lipid membrane components, albeit less intensively than observed for PLA₂. It appears that they may not insert deeply into the membrane (according to parameter A), but they still influence the organization of the lipids. The direction of changes in the model membrane is quite consistent across all tested fractions - they all have the potential to interact with the membrane (localization/electrostatic spreading of lipids), and disrupting membrane thermodynamic properties and dynamics toward increased lability and elasticity. The effects observed, particularly for the static compression modulus, indicate changes directed at membrane destabilization by the tested toxins. Furthermore, our results support the existence of a non-catalytic component to the action of PLA_2_, which manifests as a structural effect on the model membrane. Phospholipase A_2_ is a well-known toxin that disrupts cell membrane integrity through its catalytic permeabilization activity [35,36]. This disruption allows an uncontrolled influx of extracellular molecules, including Ca²⁺ ions, which in turn triggers a cascade of events ultimately leading to cell death [37–40]. Consistent with previous studies showing that PLA₂ preferentially hydrolyzes highly curved or ripple-phase lipid domains, while flat or gel-phase membranes are less susceptible [39], the Langmuir monolayer model was adjusted to allow observation of physicochemical changes in membrane elasticity by removing calcium ions, thereby preventing catalytic activity and focusing solely on lipid–PLA₂ interactions. This approach enabled monitoring of the mechanical changes in the monolayer independent of enzymatic hydrolysis. This approach was intended to shift attention away from ion-dependent activation of phospholipases and instead focus on the molecular and structural mechanisms of protein-monolayer interactions. It should also be emphasized that this conclusion neither unequivocally confirms nor excludes catalytic mechanisms that may take place in native cellular systems, but rather complements our knowledge of the action of this protein fraction at the structural level - the lipid part of membranes. There are many more mechanisms beyond the traditional catalytic mode of action of PLA_2_ toxins, particularly in the absence of 3FTx [41]. These mechanisms may also involve other molecular regions distinct from the catalytic site [42,43,44].

Many other physicochemical studies highlight the important role of the toxins PLA_2_ and 3FTx in affecting model membranes and suggest a different mode of action. Gasanov [45] noted that PLA_2_ often binds to membranes only transiently (as a complex). The PLA_2_ interacts with membrane proteins or acidic phospholipids, enabling this cytotoxin to hydrolyze membrane phospholipids. This process envelops the hydrophilic regions of the cytotoxin with released fatty acids, facilitating dissociation of the complex. Recent studies confirm similar mechanisms, showing that sPLA_₂_ can act outside its catalytic site at the water-lipid interface and destabilize membranes [44,46–48].

Furthermore, the degree of modification of the model membrane correlates with the dose of the tested fractions. Higher concentrations of toxins near the membrane exacerbate the structural and mechanical disturbances in membrane fluidity, affecting its thermodynamic state. Due to differences in membrane composition-such as a higher content of saturated lipids and phosphatidylcholine (PC) in the neuroblastoma model-greater membrane physicochemical disturbances were observed in this system. This highlights the specificity of PLA_2_ for different membrane types, which is consistent with previous studies demonstrating high PLA_2_ activity on PC-rich membranes [49,50]. The proposed mechanism involves initial electrostatic attraction of proteins to the membrane surface, followed by hydrophobic interactions that stabilize protein-membrane complexes, extending their residence time. Similar results for 3FTx emphasize the role of PC lipids in promoting membrane interactions, supporting the electrostatic and facilitating the location of selected venom fractions in the lipid part of the membrane [11]. However, no clear relationship was demonstrated with respect to monolayer stability (measured by the moment of monolayer collapse as a result of toxin treatment). For the tested fractions, the changes are not directional, but in some cases, it can be observed that for higher concentrations of the administered toxin, collapse usually occurs faster, which is usually a consequence of interactions of a film-like substances with the subphase of the lipid monolayer [51,52].

4.2 Electrostatic alterations in lipid membranes induced by toxins

Model studies using Langmuir monolayers complemented liposome studies prepared from the same lipid mixture solutions for the SK-N-SH and HL-60 models. In the case of liposomes, changes in zeta potential may indicate interactions between a given protein toxin and the lipid membrane surface (particularly at the electrostatic level) or changes in the exposure of negatively charged polar part of lipids. Demonstrated changes provide evidence for interactions of the studied proteins with lipid components. In a wider context, changes in surface charge of membrane lipids (as reflected in the liposome model) can disrupt cellular homeostasis, leading to oxidative stress and related damage. The reported percentage changes illustrate the intensity of the effects related to modifications in surface charge. However, it is important to emphasize that all liposomes investigated to date [12] retained their negative surface charge after treatment with the 3FTx fraction, consistent with the results obtained in the present study on protein–lipid interactions. Among the analyzed systems, the HL-60 liposome fractions exhibited less variability in surface charge at the highest measured concentration of proteins (liposome treated SVMP: −20.2 ± 0,6 mV; with PLA₂: −29.3 ± 1,01 mV; with CRISP: −20.6 ± 1,3 mV). In contrast, SK-N-SH liposomes exhibited more heterogeneous ζ-potential values (liposome treated by SVMP –13,5 ± 0,6mV, PLA_2_ –20,6 ± 0,14mV, CRISP –44,3 ± 1,4mV). For reference, the measured zeta potentials of the proteins at pH 7.4 are: SVMP –6.2 mV ± 0.34, PLA2 –19 mV ± 0.1, and CRISP –16.9 mV ± 0.7.

The most pronounced interaction with the liposome membrane was observed for SVMP in both liposome models, resulting in the greatest change in zeta potential-approaching zero. CRISP decreased the zeta potential to more negative values in SK-N-SH, while the opposite trend was observed in HL-60. Notably, except for the HL-60 model at 40 ng/mL, the PLA_2_ fraction also shifted the zeta potential toward zero.

It is worth noting that the 3FTx fractions studied by our research team [12] caused a shift toward more negative zeta potential values in both models. Moreover, this was the only fraction that consistently reduced the surface charge of the model membranes, regardless of concentration. These findings are particularly relevant given the potential of proteins as modulators of cell membranes.

One possible mechanism that may explain the preservation of negative ζ-potential values is the phenomenon of charge reversal, reflecting the nonlinear effects of protein–membrane interactions on ζ-potential [53,54]. At low protein toxin concentrations, the ζ-potential often becomes less negative due to partial neutralization of the lipid-derived surface charge by adsorbed proteins. However, at higher protein concentrations, the ζ-potential may become more negative again, which can be attributed to the formation of a “protein shell” as additional protein molecules accumulate on the lipid surface. Under such conditions, the measured ζ-potential reflects predominantly the net charge of the adsorbed proteins rather than that of the lipids alone.

4.3 Toxin-induced changes in membrane properties of native cells

The degree of membrane damage was estimated by measuring its permeability, as indicated by LDH enzyme efflux.

A common method for assessing toxin-induced cancer cell death is the detection of lactate dehydrogenase (LDH) release into the culture medium, which increases with membrane damage [55]. In our study, we detected LDH in the culture medium of HL-60 cell lines after exposure to all tested toxin fractions, indicating membrane damage in this cell line. Among the compounds tested, CRISP (at a tested concentration of 40 ng/mL) proved to be the most effective in damaging the native membrane. The apparent decrease in extracellular LDH levels at higher toxin concentrations likely reflects the kinetics of enzyme release rather than reduced cytotoxicity. At lower concentrations, partial membrane damage allows adherent cells to release measurable LDH, whereas at higher concentrations, rapid membrane disruption and cell detachment limit the number of cells capable of contributing to the assay. This is consistent with the CRISP toxin data, where maximal LDH release occurs at the lowest tested concentration, while higher doses induce extensive membrane damage, thereby reducing the amount of enzyme detectable in the supernatant. Additionally, CRISPs (specifically Css-CRiSPs) have been shown to promote the sustained release of inflammatory mediators in cell lines and also to be responsible for acute activation of innate immunity, as demonstrated in mouse models [56]. Our findings contribute to the growing body of evidence supporting the involvement of svCRISP in the damaging effects of cancer cell membranes. They also complement existing data on early inflammatory responses and the facilitation of leukocyte activation and mediator release. The remaining tested toxin fractions- such as 3FTxs (G1, G5) [12] exhibited comparable membrane-disrupting effects, indicating that these proteins also promote membrane damage in HL-60 cells. In the second cell line tested (SK-N-SH), the mentioned 3FTx (G1, G5) proteins, did not caused membrane damage. It should be noted that neuroblastoma is one of the most treatment-resistant cancers. This may be because the previously mentioned high level of membrane saturation can significantly limit protein damage, which is often associated with the oxidation of polyunsaturated fatty acids in cell membranes [57,58]. There are also reported cases of cancer cell lines responding in a variable and irregular manner to venom exposure, thereby promoting cell proliferation and viability. For example, a study by Park et al. [59] demonstrated that Vipera lebetina turanica venom toxin induces apoptosis in SK-N-SH cells through mechanisms dependent on reactive oxygen species (ROS) and disruption of mitochondrial membrane potential (healthy cells did not undergo apoptosis). On the other hand, melittin—the main component of bee venom -has demonstrated neuroprotective effects in SH-SY5Y cells (a neuroblastoma cell line) exposed to oxidative stress. In that study, melittin reduced LDH release, suggesting preservation of cell membrane integrity [60].

A similar trend was observed in the experiments conducted by Kerkkamp et al. [61] who investigated venoms from Naja species and their effects on PaTu 8988t and ZF4 cells. PaTu 8988t cells are human pancreatic cancer cells, while ZF4 cells (zebrafish embryos) serve as a model for studying cell migration and metastasis. Their LDH measurements showed no clear dose-dependent effect, and it was concluded that Naja venoms did not exhibit significant anticancer properties.

By correlating these physicochemical changes with actual cellular damage, we can gain a comprehensive understanding of how individual proteins act at the level of membrane lipids by modulating the membrane properties.

5 Conclusions

Leukemia cancer cells generally exhibit greater sensitivity to the modulatory effects of the tested venom compounds on their plasma membranes, compared to neuroblastoma cell lines. Analysis of membrane model data and biochemical studies suggests that cellular sensitivity to venom proteins may be influenced by membrane structure, particularly the proportion of unsaturated fatty acids.

These results indicate that venom fractions may be more effective against cells with elevated levels of unsaturated lipids. This represents an important step toward identifying compounds with biological potential, where the lipid composition of a given cell may serve as a key criterion for selecting the appropriate venom-derived protein.

In this context, due to specific mechanisms of action, the most effective venom fractions can be indicated for:

a) Membrane integrity disruption (in vitro):

For leukemia: CRISPFor neuroblastoma: 3FTx

b) Surface charge modification (measured by zeta potential):

Toward more negative zeta potential values:For leukemia: 3FTxFor neuroblastoma: CRISPToward more positive zeta potential values: SVMP

c) Reduction of membrane stiffness and increased membrane lability:

PLA_2_ (for both tested models)3FTx (particularly in leukemia)

Additional effects of these fractions, ranked by intensity, are presented in the accompanying S2 Table. We demonstrate that PLA₂, CRISP, and SVMP significantly alter the mechanical and electrostatic properties of cancer cell membranes. Notably, CRISP, previously understudied, shows strong membrane-disrupting activity. These venom-derived proteins are among the most promising candidates for further investigation, due to their ability to weaken membrane integrity and reduce membrane stiffness, key traits for many biological applications.

Supporting information

S1 TableComplete list of all identified proteins in venom fractions with its quantitation analysis.(XLSX)

S2 TableEffects of protein fractions on membrane parameters (ranked by intensity).(PDF)

S1 DatasetSigmaPlot data used to prepare the isotherms and calculate the physicochemical parameters.(JNB)