Charge of Phospholipids Determines the Rate of Lysozyme Aggregation but Not the Structure and Toxicity of Amyloid Aggregates

Abstract

Biophysical properties of plasma membranes are determined by a chemical structure of phospholipids, including saturation of fatty acids and charge of polar heads of these molecules. Phospholipids not only determine fluidity and plasticity of membranes but also play an important role in abrupt aggregation of misfolded proteins. In this study, we investigate the role of the charge of the most abundant phospholipids in the plasma membrane on the aggregation properties of the lysozyme. We found that the charge of phospholipids determines the aggregation rate of lysozyme and the morphology of the protein aggregates. However, the secondary structure and toxicity of these protein specimens are determined by the chemical nature rather than the charge of phospholipids. These findings show that the charge of phospholipids can be a key factor that determines the stability and aggregation mechanism of amyloidogenic proteins.

Graphical Abstract

Plasma membranes perform a large number of physiologically important processes that range from cell division to transduction of ions and small molecules.^1,2 The incredible plasticity and functionality of membranes are attributed to a fine balance between carbohydrates, proteins, and lipids.³ The vast majority of lipids found in the plasma membranes are phospholipids.⁴ These molecules contain glycerol esterified to fatty acids (FAs) at sn-2 and sn-3 positions with the sn-1 position occupied by a phosphoric acid residue.^3,5 FAs can have a different number of carbon atoms and a degree of saturation.¹ The phosphoric acid residue may have no subsequent groups esterified to it (phosphatidic acid, PA) or can possess a broad variety of polar molecules such as choline (phosphatidylcholine, PC), ethanolamine (phosphatidylethanolamine, PE), serine (phosphatidylserine, PS), or phosphatidylglycerol, PG) (Scheme 1).^6,7

Scheme 1.

Molecular Structure of PC, PE, PS, PG, and CL with Their Electrostatic Potential that Illustrates Charge Distributions in These Phospholipids

Based on the net charge of the headgroup, all phospholipids can be divided into two big groups: zwitterions and anionic lipids (Scheme 1).^6,7 Zwitterions constitute around 75% of the lipid bilayer.⁸ These molecules determine membrane plasticity and fludity.^3,9 Anionic lipids occupy significantly smaller volume of the plasma membrane. However, these lipids perform a large number of important physiological functions such as signal transition and cell apoptosis.^1,2 Cardiolipin (CL) is a unique phospholipid that constitutes ~20% of inner mitochondrial membrane.⁶ It carries two negative charges playing an important role in cell respiration and energy conversion. Around 10% of the plasma membrane is occupied by PS. Under physiological conditions, negatively charged PS is primarily localized on the inner part of the membrane.^1,5,10 Its exposure to the exterior part of the plasma membrane indicates cell malfunction.¹¹ In such cases, PS is recognized by phagocytes that degrade such apoptotic and necrotic cells.³ PG is present in plasma membranes in low amounts (only 1–2% of all phospholipids). However, PG is one of the most important lung surfactants that constitutes up to 11% of the surfactant lipids.^1,2

Microscopic analysis of Lewy bodies, protein deposits that are found in midbrain, hypothalamus, and thalamus as a result of Parkinson’s disease, revealed the presence of plasma membranes.¹² This finding suggests that lipids can be directly involved in the onset and development of neurodegenerative pathologies, such as Alzheimer’s and Parkinson’s diseases. Galvagnion and co-workers found that lipids can uniquely alter rates of protein aggregation. It was found that large unilamellar vesicles of phospholipids accelerated the rates of α-synuclein (α-syn) aggregation.^13–15 Similar findings were recently reported for amyloid β peptide islet amyloid precursor protein and insulin.^16–18 Furthermore, a growing body of evidence shows that lipids not only altered the rates of protein aggregation but also drastically changed the secondary structure of oligomers and fibrils, highly toxic protein aggregates that are linked to the onset and spread of neurodegenerative diseases.^19–23 Matveyenka and co-workers recently discovered that unsaturation of FAs of both PC and CL uniquely alters rates of insulin aggregation and determines the toxicity of insulin aggregates.²⁴ Similar findings were also observed for PS.²⁵ These results helped to understand the role of saturation of FAs in the phospholipids in the stability of amyloidogenic proteins. However, the role of the charge of the phospholipids remains unclear.

In this study, we investigate the effect of the charge of phospholipids on lysozyme aggregation. Abrupt aggregation of this protein is observed upon systemic amyloidosis.^26,27 We utilized a thioflavin T fluorescence approach to monitor the rate of lysozyme aggregation in the presence of two zwitterionic lipids, PC and PE, as well as anionic lipids with charges of negative one (PG) or negative two (CL). It should be noted that at the pH value of 3.0 used in our experiments, the carboxyl group of PS is protonated; therefore, this lipid possesses net positive charge (cationic lipid). Lysozyme was mixed with lipids at 1:5 molar rations. We also utilized atomic force microscopy (AFM) and the innovative nanoscale infrared spectroscopy, known as AFM-infrared (AFM-IR), to examine the morphologies and secondary structure of protein aggregates grown in the presence of these lipids.^28–30 Finally, we use mice midbrain N27 cells to investigate the extent to which zwitterionic, cationic, and anionic lipids altered the toxicity of lysozyme aggregates.²⁵

In the lipid-free environment, lysozyme aggregation is characterized by a well-defined lag-phase (t_lag = 5.1 ± 0.6 h) that is followed by a rapid increase in the thioflavin T (ThT) fluorescence signal, which indicates the formation of protein aggregates (Figure 1). We found that both PS and PG drastically shortened t_lag of lysozyme aggregation. Specifically, Lys:PS t_lag was found to be 3.2 ± 0.2 h, whereas Lys:PG t_lag=4.1 ± 0.3h. We also found that in the presence of CL, lysozyme aggregated instantaneously with the shortest (t_lag = 0.2 ± 0.01 h) lag-phase. At the same time, our results showed that PC and PE strongly inhibited lysozyme aggregation. These findings point to the direct correlation between the net charge of the phospholipid and the t_lag of lysozyme aggregation. Specifically, CL with two negative charges enabled the strongest acceleration of lysozyme aggregation compared to PS and PG, which, in turn shortened the t_lag of lysozyme aggregation compared to the lipid-free environment. Finally, zwitterionic lipids fully inhibited protein aggregation.

Figure 1.

Negatively charged phospholipids drastically accelerate, whereas zwitterionic phospholipids strongly inhibit lysozyme aggregation. (A) ThT aggregation kinetics with (B) corresponding values of t_lag and t_1/2 of lysozyme in the lipid-free environment (gray), as well as in the presence of PG (red), PS (brown), CL (green), PC (blue), and PE (light blue). Each kinetic curve is the average of three independent measurements.

We have also found that CL, PS, and PG enhanced the rate of lysozyme aggregation. Specifically, CL t_1/2 was 0.7 ± 0.1 h, whereas t_1/2 of PS and PG was 3.3 ± 0.2 h and 4.6 ± 0.2 h, respectively (Figure 1). It should be noted that t_1/2 of lysozyme in the lipid-free environment was 6.1 ± 0.5 h. Finally, we found that the intensities of Lys:CL, Lys:PG, and Lys:PS were greater than the intensity of lysozyme aggregates formed in the lipid-free environment. This finding suggests that insulin aggregation in the presence of cationic and anionic lipids yields more ThT active protein aggregates.

Next, we used AFM to investigate the morphology of lysozyme aggregates grown in the presence of zwitterionic, cationic, and anionic lipids, as well as in the lipid-free environment (Figure 2). We found that in the lipid-free environment, lysozyme formed both long and short fibrils with heights ranging from 20 to 40 nm (Figure 2). In the presence of PC, we observed predominantly small spherical aggregates that were 5–7 nm in height. We also observed a small amount of short fibril species in Lys:PC. However, only small oligomers were observed in Lys:PE. This observation is in good agreement with the above-discussed ThT results showing that PC and PE strongly inhibit lysozyme fibril formation yielding formation of only small oligomeric species. We also found that CL promotes formation of both small oligomers and prolonged fibrils that were ~20 nm in height, whereas predominantly fibrils were observed in the presence of PS. Finally, we found that in the presence of PG, lysozyme formed long fibrils that stretched to micrometers in length. Such long fibrils were not observed in any other sample. These findings show that charge of phospholipids determine the morphology of lysozyme aggregates. Specifically, in the presence of zwitterions, only oligomers are formed, whereas cationic and anionic lipids promote formation of fibrillar species. These results also show that in addition to the charge, the chemical structure of lipids plays an important role in the morphologies adopted by lysozyme aggregates. Specifically, the unique chemical structure of PG enabled lysozyme aggregation into long fibrils that were not observed for PS.

Figure 2.

Zwitterionic, cationic, and anionic phospholipids significantly alter morphologies of protein fibrils that form in their presence. AFM images of lysozyme fibrils grown in the lipid-free environment (A), as well as in the presence of PC (B), PE (C), PS (D), PG (E), and CL (F). Scale bars are 500 nm.

One may wonder whether in addition to the above-discussed morphological changes, the charge of phospholipids can also determine the secondary structure of Lys:PC, Lys:PE, Lys, PS, Lys:PG, and Lys:CL aggregates. Utilization of conventional optical techniques, such as attenuated total internal reflection Infrared spectroscopy (ATR-FTIR) and circular dichroism (CD), provides very little if any information about the secondary structure of protein aggregates. This is primarily due to the predominance of unaggregated protein and very low concentrations of protein aggregates (Figures S2 and S3). Therefore, to overcome this limitation, we used AFM-IR. This technique is based on thermal expansions in the analyzed sample that are induced by pulsed tunable IR light.^31–35 The expansions are recorded by the metallized scanning probe that can be precisely positioned on the surface of the object of interest.³⁶ Next, thermal expansions are converted into IR spectra that can be used to probe (i) secondary structure of protein specimens and (ii) composition of the analyzed aggregates.^22,28,37,38 Therefore, we used AFM-IR to examine the extent to which zwitterionic, cationic, and anionic lipids can change the secondary structure of lysozyme aggregates.

AFM-IR spectra collected from lysozyme fibrils grown in the lipid-free environment (Lys) are dominated by three vibrational bands centered around 1555 cm⁻¹ (amide II) and 1630 and 1668 cm⁻¹ (amide I). The position of amide I bands can be used to characterize protein secondary structure of the analyzed protein specimens.^29,39 Amide I band around 1630 cm⁻¹ is indicative of parallel β-sheet, whereas the vibration around 1660 cm⁻¹ is characteristic for unordered protein secondary structure. In the AFM-IR spectra of Lys, the 1630 cm⁻¹ vibration was more intense than the 1668 cm⁻¹ band (Figure S4). These results show that Lys fibrils grown in the lipid-free environment are composed of parallel β-sheet with significant contribution of unordered protein and antiparallel β-sheet present in their secondary structure (Figure S5).^39,40

Lys:PE aggregates exhibit a similar spectroscopic fingerprint. Specifically, we observed both the intense 1630 cm⁻¹ band and a weak 1668 cm⁻¹ band in the AFM-IR spectra collected from Lys:PE oligomers. Considering this result, we can conclude that the secondary structures of Lys:PE are dominated by parallel β-sheet secondary structure (Figures S4 and S5).^39,40 However, a new set of vibrational bands was evident in the AFM-IR spectra collected from Lys:PE. Specifically, we observed intense vibrational bands around 800–900 and 1000–1200 cm⁻¹. These vibrational bands correspond to C−H and PO₂⁻ vibration of the ester group of lipids³⁷ (Figures 3 and S6). The presence of these vibrational bands in the AFM-IR spectra of Lys:PE shows that PE is present in the structure of these protein aggregates. Similar vibrational bands were observed in the AFM-IR spectra collected from Ins:PC oligomers. However, we found that the intensity of 800–900 and 1000–1200 cm⁻¹ (PO₂⁻) bands was significantly higher than the intensity of these bands in the spectrum of Ins:PE. These results show that Lys:PC possesses a higher amount of lipid than Lys:PE. We also found an ester (1730 cm⁻¹) vibration in the spectra collected from Ins:PC. These findings show that PE and PC have drastically different interactions with lysozyme in such oligomers. It is important to note that the amide I band of Lys:PC had significant changes compared to the amide I of Lys and Lys:PE. Specifically, we found the vibration at 1668 cm⁻¹ to be more intense than the 1630 cm⁻¹ band. These results show that Lys:PC oligomers are primarily composed of unordered protein and antiparallel β-sheet with some parallel β-sheet with some present in their secondary structure (Figure S5).^39,40

Figure 3.

Nanoscale analysis of secondary structure of lysozyme aggregates grown in the lipid-free environment and in the presence of phospholipids. Averaged AFM-IR spectra of Lys (gray), Lys:PC (green), Lys:PE (light blue), Lys:PS (orange), Lys:PG (red), and Lys:CL (purple) aggregates.

We found that Lys:CL have similar secondary structure to Lys aggregates, Figure S3. Specifically, these fibrils were primarily composed of parallel and antiparallel β-sheet with some unordered protein present in their secondary structure.^39,40 Lys:PC and Lys:PG are dominated by parallel β-sheet, Figure S3. We also observed intense bands around 800–900, 1000–1200, and 1730 cm⁻¹ in the AFM-IR spectra collected from Lys:PS, Lys:PG, and Lys:CL. These vibrational bands show that phospholipids are present in all of these protein aggregates.³⁷ Thus, we can conclude that cationic and anionic lipids induced only minor changes in the secondary structure of lysozyme aggregates. At the same time, the effect of zwitterionic lipids on the secondary structure of lysozyme aggregates formed in their presence in not uniform. Specifically, PC strongly altered the secondary structure of lysozyme oligomers, significantly reducing the amount of parallel β-sheet in these protein specimens. However, PE did not induce these drastic changes in the structure of Lys:PE oligomers compared to Lys fibrils. Finally, we can conclude that all protein aggregates that were grown in the presence of zwitterionic, cationic, and anionic lipids possess lipids in their structure.

These results resonate with the previously reported findings on the secondary structure of insulin oligomers and fibrils that were grown in the presence of PC, CL, and PS.^23–25 Specifically, Matveyenka and co-workers found that PC, CL, and PS were present in insulin aggregates that were grown in equimolar ratio with these lipids.^23–25 Similar findings were also reported by Dou and Kurouski for α-syn oligomers that were grown in the presence of PC.²² Thus, we can conclude that lipid possession by protein aggregates that were formed in their presence can be considered a general phenomenon of amyloid biology.

It should be noted that some classes of aggregates, such as Lys:PE, Lys:PG, Lys:CL, and Lys, demonstrated very little if any structural heterogeneity of analysis aggregates (Figure S3). Lys:PC and Lys:PS exhibited a greater degree of structural heterogeneity, as can be observed in the acquired spectra. For instance, we observed two distinctly different types of AFM-IR spectra acquired from morphologically identical Lys:PS aggregates (Figure S3). These findings are in a good agreement with the previously reported AFM-IR analysis of insulin aggregates that were grown in the presence of PC and PS.^23–25 Additional studies are required to fully elucidate the observed heterogeneity of Lys:PC and Lys:PS, which are beyond the scope of the current work.

One may wonder whether the charge of phospholipids plays an important role in determination of toxicity of lysozyme aggregates that were grown in the presence of zwitterionic, cationic, and anionic lipids. To answer this question, we performed an LDH assay on mice midbrain N27 cells (Figure 4).²⁵ We found that the toxicity of Lys:PC and Lys:PE was significantly lower than the toxicity exerted by Lys fibrils. Furthermore, we found that Lys:PC was far less toxic than Lys:PE. Lys:PS, Lys:PG, and Lys:CL exhibited levels of cell toxicity similar to each other, which, in turn, were significantly lower than the toxicity of Lys aggregates. Thus, we can conclude that lipids present in the structure of Lys:PC, Lys:PE, Lys, PS, Lys:PG, and Lys:CL aggregates enabled the significant reduction of toxicity of these protein aggregates. Our results also show that there is no significant difference in the toxicity exerted by lysozyme aggregates grown in the presence of zwitterionic, cationic, and anionic lipids. It should be noted that the significantly lower cell toxicity of Lys:PC compared to all other samples can be attributed to both the unique chemical structure of PC and the low amount of parallel β-sheet present in these aggregates. Thus, the chemical structure of the lipid itself rather than the possessed charge determines the toxicity of protein aggregates that were grown in the presence of phospholipids.

Figure 4.

Toxicity of lysozyme aggregates grown in the presence of lipids is determined by the chemical structure of the lipid. Histograms of LDH (top), ROS (middle), and JC-1 (bottom) toxicity assays of Lys (brown bars); Lys:PC, Lys:PE, Lys:PG, Lys:PS, Lys:CL (orange bars); and PC, PE, PG, PS, and CL (yellow bars). Error bars represent standard errors of the mean (SEM) of three replicates. According to the t test results, asterisks (*) show significance level of differences between Lys and lysozyme aggregates grown in the presence of lipids, as well as between the control and lipids themselves. NS is a nonsignificant difference, *p ≤ 0.05; **p ≤ 0.01, and ***p ≤ 0.001.

Amyloid aggregates exert toxicities activating reaction oxygen species (ROS) production in cells.^41,42 These toxic species can also induce mitochondrial dysfunction in cells.^23–25 Therefore, we investigated the extent to which Lys:PC, Lys:PE, Lys, PS, Lys:PG, and Lys:CL, as well lysozyme fibrils grown in the lipid-free environment were engaged in ROS production and mitochondrial dysfunction of mice midbrain N27 cells (Figure 4). We found that ROS levels exerted by Lys:PC, Lys:PS, and Lys:PG were similar to the levels of ROS observed for Lys fibrils. However, Lys:PE and Lys:CL exerted significantly less ROS stress to N27 cells. Similar results were observed for level of mitochondrial dysfunction caused by these protein aggregates. Mitochondrial dysfunction can be determined using JC-1 assay.^24,25 Specifically, the presence of PC, PE, PS, and PG lipids did not significantly alter the extent to which lysozyme aggregates induce mitochondrial dysfunction. However, the presence of CL allowed for the significant reduction of the level of toxicity exerted by these protein aggregates to mitochondria. On the basis of this, we can conclude that ROS and JC-1 levels are determined by the chemical structure of the lipid rather than the charge present on its headgroup.

This conclusion is further supported by ROS levels and mitochondrial dysfunction expressed by the lipids themselves in mice midbrain N27 cells. Specifically, we found that PC, PE, and PG were inducing higher levels of ROS stress than PS and CL. We also observed that PG itself exerted significantly higher levels of mitochondrial dysfunction compared to all other lipids.

Summarizing, our results revealed a strong correlation between the rate of lysozyme aggregation and the charge of the phospholipid head. Specifically, zwitterions strongly inhibited fibril formation, whereas negatively charged PS, CL, and PG accelerated lysozyme aggregation. Furthermore, the acceleration rate was directly dependent on the net charge possessed by the lipids. We also found a direct relationship between the charge of the phospholipid head and the morphology of the protein aggregates formed in the presence of lipids. Specifically, in the presence of PC and PE, we found primarily small oligomers. However, in the presence of negatively charged phospholipids, domination of fibril-like aggregates was observed. Analysis of the secondary structure and toxicity exerted by Ins:PC, Ins:PE, Ins:PS, and Ins:PG revealed that the secondary structure and toxicity are determined by the chemical structure rather than by the charge of the phospholipid polar head. These results help to understand the importance of zwitterions in the plasma membrane. We infer that neutrally charged PC and PE stabilize proteins, preventing their fibrillization. Our findings also show that an increase in the concentration of cationic and anionic lipids in the plasma and mitochondrial membrane will promote the aggregation of misfolded proteins into fibrils, which, in turn, will activate ROS production and cause mitochondrial damage in cells.