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Membrane pore-formation correlates with the
hydrophilic angle of histidine-rich amphipathic peptides with multiple biological activities
Morane Lointier, Christopher Aisenbrey, Arnaud Marquette, Jia Hao Tan, Antoine Kichler, Burkhard Bechinger
To cite this version:
Morane Lointier, Christopher Aisenbrey, Arnaud Marquette, Jia Hao Tan, Antoine Kichler, et al..
Membrane pore-formation correlates with the hydrophilic angle of histidine-rich amphipathic peptides with multiple biological activities. Biochimica et Biophysica Acta:Biomembranes, Elsevier, 2020, 1862 (8), pp.183212. �10.1016/j.bbamem.2020.183212�. �hal-03020613�
Membrane pore-formation correlates with the hydrophilic angle of histidine-rich amphipathic peptides with multiple biological
activities
Morane Lointier
1, Christopher Aisenbrey
1, Arnaud Marquette
1, Jia Hao Tan
1, Antoine Kichler
2, and Burkhard Bechinger
1,*1Université de Strasbourg / CNRS, UMR7177, Institut de Chimie, 4, Rue Blaise Pascal, 67070 Strasbourg, France
2Université de Strasbourg/CNRS, UMR7199, Laboratoire de Conception et Application de Molécules Bioactives, Faculté de Pharmacie, 67401 Illkirch, France.
* Corresponding author: Burkhard Bechinger, ORCID 0000-0001-5719-6073 4, rue Blaise Pascal, 67070 Strasbourg, France
Tel.: +33 3 68 85 13 03, bechinge@unistra.fr
Running title: correlation between pore formation and hydrophilic angle
Key words: antimicrobial peptide, amphipathic helix, cell penetrating peptide, membrane partitioning, transfection, lentiviral transduction
*REVISED Manuscript (text UNmarked) Click here to view linked References
ABSTRACT
The LAH4 family of amphipathic peptides exhibits pronounced antimicrobial, cell penetrating and nucleic acid transfection activities. Furthermore, variants were designed with potent lentiviral transduction enhancement. When viewed along a helical wheel the four histidines are arranged to form an amphipathic arrangement. In order to optimize some of these biological activities the number of leucine and alanine residues exposed to the hydrophilic surface was systematically varied which resulted in the design of vectofusin a peptide with strong lentiviral transduction enhancement activities. Here the series of peptides with varying numbers of alanine or leucine residues framed by the histidines was tested for their calcein release activity. Interestingly, the membrane pore formation and DNA transfection activities show a clear correlation with the hydrophilic angle. In contrast the membrane partitioning and the propensity to adopt helical conformations was hardly affected as long as the hydrophilic angle did not exceed a limiting value of 150 degrees.
ABBREVIATIONS USED CD circular dichroism DLS dynamic light scattering DNA deoxyribonucleic acid
EDTA ethylenediaminetetraacetic acid LUV large unilamellar vesicles NMR nuclear magnetic resonance
MALDI matrix assisted laser desorption ionization
MS mass spectrometry
POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPE 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine
RP HPLC reversed phase high performance liquid chromatography siRNA small interfering ribonucleic acid
SUV small unilamellar vesicle VF-1 vectofusin-1
INTRODUCTION
The LAH4 peptides were designed using membrane-active linear cationic amphipathic antimicrobial peptides such as magainins as a template [1]. The parent peptide consists of a hydrophobic core made of leucines and alanines, which is interrupted by four histidines.
These are arranged in such a manner along the primary sequence to be positioned on one face of an amphipathic helix [1]. Two lysines at each terminus assure good solubility in aqueous environments and can act as anchors at the bilayer interface. Because the pK of histidine is in the physiological range the LAH4 peptides exhibit a pH-dependent amphipathic character which results in transmembrane orientations at neutral pH and a membrane surface orientation when the histidines are charged at acidic conditions [1, 2]. This pH-dependent membrane alignment allowed the evaluation of different membrane interaction models that have been suggested to explain their antimicrobial action [3]. Furthermore, to better understand the energetic contributions that determine their membrane topology derivatives have been designed and investigated [4, 5].
In accordance with their design LAH4 peptides exhibit antimicrobial activities [3, 6, 7]. They have later on been found to be efficient for DNA and siRNA transfection [8-11], to help in the membrane passage of proteins, peptides, nanodots [12-14] and adeno-associated viruses [15, 16]. Furthermore, they have been optimized to enhance lentiviral transduction activities [17]. In order to improve these activities many variants of LAH4 have been created and tested for these different biological activities [11, 13, 18]. In particular, the distribution of alanine and leucine residues relative to the four histidines was systematically varied to
optimize and rationalize the various biological effects of the peptides [11, 17]. Thus, LAH4- L1 was identified to be a highly potent nucleic transfection agent [10, 11] while LAH4-A4, commonly called vectofusin-1 (VF-1), is very active in enhancing lentiviral transduction [17, 18]. Notably, these two peptides exhibit the same overall composition of amino acids but their distribution along the sequence and three-dimensional structure is different.
For this study, we analysed different peptides of the “LAH4-L” and the “LAH4-A”
series. These peptides are named by the number of leucine or alanine residues which are framed by two pairs of adjacent histidine residues when the peptide is viewed in the Schiffer- Edmundson helical wheel representation [18, 19] (Figure 1). The amino acid sequence was systematically changed to vary the number of these leucine or alanine residues, respectively, and thereby also the hydrophilic angle between 60° and 180° (Figure 1, Table 1). Whereas the helix content of the peptides was determined by circular dichroism spectroscopy their pore-
forming activities were characterized by calcein release from lipid model membrane systems.
On the one hand POPC/POPS (3/1 mole/mole) was studied to mimic the plasma membrane and POPE/POPG (3/1 mole/mole) as a model for bacterial membranes. Interestingly, these measurements reveal a correlation between the hydrophilic angle of the peptides and their effectiveness in forming pores in the membranes which is suggestive that the peptides differ in their depth of insertion into lipid bilayers. This correlation was also tested for biological function.
Figure 1: Schiffer-Edmundson helical wheel representations of the core region (residues 6–
23) of selected LAH4-A and LAH4-L peptides investigated in this paper. The Figures were created using the Heliquest software [20].
Table 1: Peptide sequences investigated in this paper and the corresponding hydrophilic angle when the peptide is represented as Schiffer-Edmundson helical wheel projection (cf. Figure 1).
Name Sequences Hydrophilic angle
LAH4-L0 NH2-KKALLAHALAHLALLALHLALHLKKA-Amide 60 LAH4-L1 NH2-KKALLAHALHLLALLALHLAHALKKA-Amide 80 LAH4-L2 NH2-KKALLALALHHLALLALHLAHALKKA-Amide 100 LAH4-L3 NH2-KKALLALALHHLALLAHHLALALKKA-Amide 120 LAH4-L4 NH2-KKALLHLALLHAALLAHHLALALKKA-Amide 140 LAH4-L5 NH2-KKALLHLALLHAALLAHLAALHLKKA-Amide 160 LAH4-L6 NH2-KKALLHLALLLAALHAHLAALHLKKA-Amide 180
LAH4-A1 NH2-KKALLAHALHLLAALALHLAHLLKKA-Amide 80 LAH4-A2 NH2-KKALLLAALHHLAALALHLAHLLKKA-Amide 100 LAH4-A3 NH2-KKALLLAALHHLLALAHHLAALLKKA-Amide 120 LAH4-A4 NH2-KKALLHAALAHLLALAHHLLALLKKA-Amide 140 LAH4-A5 NH2-KKALLHALLAHLAALLHALLAHLKKA-Amide 160 LAH4-A6 NH2-KKALLHALLAALLAHLHALLAHLKKA-Amide 180
MATERIALS AND METHODS
The LAH4 peptides were purchased from Pepceuticals (Leicestershire, UK). To exchange the counterions the peptides were three times solubilized in 2 mM hydrochloric acid and
lyophilized. The phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was from Sigma (Saint-Louis, MO, USA) or Larodan (Solna, Sweden), 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) from Avanti Polar Lipids (Birmingham, AL). 3,3-bis[N,N-bis(carboxymethyl)-aminomethyl]
fluorescein (calcein) was from Sigma.
Circular dichroism spectroscopy: The peptides were dissolved at 0.1 mg/ml (36 µM) in 10 mM phosphate (pH 7) or acetate (pH 5) buffer and CD spectra recorded at 25°C using a J-810 spectropolarimeter (Jasco, Tokyo, Japan). The path length of the quartz cuvette was 1 mm, and 7 acquisitions were accumulated for each spectrum. The step resolution was 1 nm at a scanning speed of 50 nm/min with 1 s integration time. The spectra were recorded between λ
= 190 and 250 nm. Note the absolute values of the residual molar ellipticity is often
systematically underestimated due to errors in weighing small amounts of sample, an increase
in sample weight due to hygroscopic effects, by the presence of salt or the underestimation of the number of counterions.
Membrane partitioning by CD spectroscopy: For the preparation of small unilamellar vesicles the lipids were mixed in the ratio POPC/POPS = 9/1 mole/mole. A homogenous solution of lipids was prepared in chloroform-methanol (3/1 v/v). Thereafter, the solution was dried under a stream of nitrogen and by exposure to high vacuum overnight in order to
remove all traces of solvent. The lipid film was rehydrated in 1 ml 10 mM acetate buffer at pH 5 to yield 13 mM lipid and sonicated 3 times for 15 sec to obtain small unilamellar
vesicles of approximately 50 nm diameter. Stock solutions of 36 µM peptide were prepared in the same buffer at pH 5. Each sample was prepared 3 times with the following lipid-to-
peptide molar ratios: 0/1, 2/1, 5/1, 10/1, 20/1, 30/1, 50/1, 60/1, 70/1 etc. The secondary structure composition was estimated using the DICROPROT software [21] or directly from the molar ellipticity at 222 nm [22].
Preparation of calcein-loaded vesicles: Large unilamellar vesicles (LUVs) loaded with calcein were prepared by dissolving lipid mixtures of POPC/POPS (3/1 mole/mole) or
POPG/POPE (3/1 mole/mole) in chloroform/methanol 3/1 (v/v). The solution was evaporated under a flow of nitrogen gas and dried under the vacuum of a lyophilizer for at least 24h. The lipids were hydrated with 50 mM phosphate (pH≈7) or acetate buffer (pH≈5) complemented with 50 mM of calcein disodium salt. The mixture was agitated for an hour before undergoing several freeze-thaw cycles and extrusion (21 times) through membranes with pores of 400 nm diameter (Whatman, Maidstone, UK). The dye outside the calcein-loaded vesicles was
removed by gel filtration through a Sephadex G-50 column (2.5 x 3.5 cm) equilibrated with the corresponding buffer (50 mM) and supplemented with 50 mM NaCl to compensate for the change in osmolarity induced by the presence of calcein and its sodium counter ions. The concentration of the vesicles emerging from the column was measured by Static Light Scattering [23]. Typical lipid concentrations in the calcein release experiments were ranging from 1 to 3 M.
Calcein release experiments: The efflux of calcein from the vesicles was monitored by fluorescence measurements using a spectrofluorometer (Fluorolog-3.22, HORIBA Scientific, Kyoto, Japon). In a typical experiment, 10 µl of LUV suspension was added to 1.5 ml of buffer with salt supplemented with 1 mM EDTA in a quartz cuvette at room temperature. An
aliquot of peptide solution at 1 mg/mL was injected at t = 100 s into the cuvette to obtain a final peptide-to-lipid ratio of 1/50. The sample was continuously excited at λex = 485 nm, and the intensity of fluorescence (I) was recorded at λem = 515 nm with 1.5/1.5 nm resolution. The percentage of calcein released from the vesicles (I%) was calculated according to:
where Io is the intensity of fluorescence before adding the peptide to the solution and Imax is the maximum intensity observed after fully disrupting the vesicles with addition of 10 µl of 10% Triton X-100.
Cell culture and Preparation of peptide/DNA complexes: The human glioblastoma cell line U87 was cultured in RPMI medium supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin and 10% of fetal calf serum. Transfection complexes were prepared as follows: for a duplicate, 1.5 g of plasmid DNA and the desired amount of peptide were each diluted into 25 L of acetate buffer pH 5 and gently mixed. DNA complexes were generated using increasing peptide/DNA w/w ratios. After 20 min of incubation at room temperature, the mixture was diluted with culture medium to obtain a final volume of 0.5 mL.
Transfection experiments: In vitro cell transfection experiments were performed using the plasmid p-Luc (7.6 kb) which is an expression plasmid encoding the firefly luciferase gene under the control of the human cytomegalovirus (CMV) immediate-early promoter. 55,000 U87 cells were plated in 48-well plates one day before the experiment was performed. For transfection, 0.25 mL/well of serum-free culture medium containing the DNA complexes were deposited into each well of the duplicate. After incubation for approximately 2.5 h at 37°C the medium was replaced with fresh one containing serum. Luciferase activity was measured as described below in the cell lysate one day after transfection.
Luciferase expression: For luciferase activity, cells were harvested in 100 L of lysis buffer (8 mM MgCl2,1 mM DTT, 1 mM EDTA, 0.6% Triton X-100, 15% glycerol, and 25 mM Tris- phosphate buffer pH 7.8). The cell lysate was then transferred into Eppendorf tubes and centrifuged for 7 min at 10,000 g to pellet debris. Luciferase light units were measured in a 96-well plate format with a Centro LB luminometer (Berthold) from an aliquot of the supernatant (2 L) with 1 second integration after automatic injection of 50L assay buffer (lysis buffer without Triton X-100 but supplemented with 2 mM ATP) and 50 L of a
luciferin solution (167 M in water). Luciferase background was subtracted from each value and the protein content of the transfected cells was measured by Bradford dye-binding using the BioRad protein assay (Bio-Rad, Marnes-la-Coquette, France). The transfection efficiency was expressed as light units/s/mg protein (light units measured over a period of 1 second and the values are then normalized after measurement of the amount of protein present in each well) and the reported values are the mean of duplicates. Error bars represent the standard deviation of the mean.
RESULTS
The peptide sequences investigated in this paper were initially designed to improve the parent LAH4 and LAH4-L1 peptides for better nucleic acid transfection and lentiviral transduction activities [18]. They all consist of 26 residues, a central core composed of eight alanines, ten leucines and four histidines to form amphipathic helical arrangements (Table 1). Two charged lysine residues each on both extremities assure better solubility and anchoring at the
membrane surface [1]. When the core residues are represented as Schiffer-Edmundson helical wheel projections two pairs of histidines frame a given number of leucines or alanines,
respectively (Figure 1 adapted from reference [18]). The positioning of the histidines in the helical wheel defines a ‘hydrophilic angle’ which ranges between 60° and 180° depending on the peptide sequence (Table 1 and Figure 1). The residues between the histidines consist either of alanine residues for the “LAH4-A” series or of leucines for the “LAH4-L” series, where the nomenclature directly reflects the number of framed alanine or leucine residues, respectively (Figure 1). For example, there are four alanine residues between the histidine pairs in LAH4-A4, the peptide that proved to be most active in lentiviral transduction experiments, also named Vectofusin-1 [18].
Because the peptides of the series exhibit the same amino acid composition they are all nominally charged +5 at neutral pH and +9 when the pH is below the pK of the histidines (pK = 6.2 for an isolated histidine). The histidine allows one to manipulate the polarity and the hydrophobic moment of the helices by changing the pH [1]. Previous studies showed that in aqueous buffer the parent LAH4 peptide adopts α-helical conformations at neutral pH but is mostly unstructured when the pH is below 6.2 when the histidines are charged [2, 3]. Thus, LAH4 peptides exhibit properties that are strongly pH-dependent, and it is interesting to investigate their activities and secondary structure as a function of pH [3, 7].
In order to test the capacity of this series of peptides to form membrane openings, fluorescence dye release experiments were performed (Figure 2). The experiments were conducted at pH 5 and 7 using vesicles either made from POPC/POPS or from POPE/POPG at molar ratios of 3/1 (mole/mole). The total peptide-to-lipid ratio was adjusted to 1/50 for all experiments. The peptide was injected at 100 seconds and the percentage of calcein released is recorded 250 seconds thereafter (Figure 2). Each experiment was repeated at least twice and up to 12 times, and the percentage of calcein release is the average at the 350 seconds time point of the different curves obtained (i.e. 250 s after the addition of peptide). The percentage of calcein released depends on the lipid composition of the vesicles and the pH. While for POPC/POPS at pH 5 calcein release varies between 10 % and 70% (Fig. 3A) at pH 7 the variation ranges between only 10% and 40% (Fig. 3B) indicating that the LAH4 peptides are more efficient at pH 5 in releasing calcein from POPC/POPS vesicles. In contrast, for
POPE/POPG liposomes at pH 5 the percentage of calcein released varies between 10% and 40% (Fig. 3C) and is more efficient at pH 7 where 10 - 70 % of the fluorophore escapes from the vesicles (Fig. 3D). Notably, the percentage of calcein release depends in a systematic manner on the number of residues framed by the two histidines. When the number of residues between the histidines and thereby the hydrophilic angle increases a decrease in calcein release activity is observed. For example, after 350 seconds 66% of calcein has escaped from POPC/POPS at pH 5 in the presence of LAH4-L0, but only 40% for LAH4-L1, 35% for LAH4-L2, and decreases further when the ‘hydrophilic angle’ increases and thereby the number of leucines between the histidines (Fig. 3A). Similar dependences of the calcein release activity and the hydrophilic angle are observed for the two series of LAH4 peptides at both pH and lipid compositions investigated here (Figs. 2 and 3). When the hydrophilic angle reaches 180°, the activity of the peptides increases again for LAH4-A6 and LAH4-L6 (Fig.
3).
Figure 2: Percentage of calcein released over time from LUVs made of POPC/POPS 3/1 mole/mole the presence of A. peptides from the LAH4-L at pH 5 or B. peptides from the LAH4-A series at pH 7. The peptide-to-lipid ratio was 1/50 and the temperature was stabilized at T = 25 oC. The release activities at t = 250 s after addition of peptide are summarized in Figure 3.
Figure 3: Percentage of calcein released 250 s after peptide addition and measured from POPC/POPS 3/1 mole/mole (A,B) or POPE/POPG 3/1 LUVs (C,D) in the presence of peptides from the LAH4-L or LAH4-A series at pH 5 (A,C) or at pH 7 (B,D). Typical traces over time are shown in Figure 2.
The correlation between hydrophilic angle and pore forming activity may reflect differences in membrane interaction such as insertion depth or alignment but also on the secondary structure preferences and the number of peptides associating with the lipid bilayers when the amphipathicity (hydrophobic moment) of the peptide changes in a systematic manner. Therefore, circular dichroism spectra of the peptides were recorded for the peptides in solution and upon titration with POPC/POPS (9/1 mole/mole) sonicated vesicles (Figure 4).
In these experiments the fraction of negatively charged lipids was reduced when compared to the calcein release experiments because considerable agglutination concomitant with light scattering [24] hampered a quantitative lipid titration study of the POPC/POPS (3/1 mole/mole) SUVs.
Figure 4: Circular dichroism of LAH4-A3, -A6, -L3 and -L6 in the presence of increasing amounts of POPC/POPS 9/1 mole/mole small unilamellar vesicles at pH 5, temperature T = 25 oC. The corresponding lipid-to-peptide ratios are indicated next to the corresponding panels.
In a first step the secondary structure of the peptides in solution, a parameter, which may influence their activity in lipid membranes, was estimated from CD spectra using the dicroprot software [21] (Tables 2, S1). In the absence of lipid, the helical contribution within
the LAH4-L series is around 20% at pH 5 (Table 2). For the LAH4-A series at pH 5 similar observations were made for LAH4-A1 to LAH4-A4, however a much higher helicity of about 55% was observed for LAH4-A5 and LAH4-A6 (Table 2). At pH 7 all peptides of the LAH4- A and LAH4-L series adopt about much higher helical conformations (Table S1).
Table 2: Content of helical secondary structure of the LAH4-A and -L peptides at different L/P ratios at pH 5. SUVs made from POPC/POPS 9/1 were added during the titration experiments to reach the indicated lipid-to-peptide ratios. The data shown are averages from three independent measurements. See Table S2 for more details on the deconvolution analysis using the DICROPROT software [21]. Due to systematic errors and potential light scattering artefacts we estimate the uncertainties in the secondary structure in the 5-10% range although the statistical error of most of the reported experiments are well below this value.
Upon addition of SUVs made of POPC/POPS 9/1 mole/mole at pH 5 almost all peptides adopt a more helical secondary structure notable exceptions being A5, A6, L5 and L6, where further changes are small or absent (Fig. 4). Therefore, these latter four peptides were excluded from the following discussion. A line fit analysis of the CD spectra in the presence of high concentrations of lipid are indicative of about 70-80 % helix for peptides of the A-series and LAH4-L0 to -L4 and around 30 % for -L5 and -L6 (Figure 4, Table 2).
Notably the CD spectral titrations exhibit an isosbestic point indicating that the membrane association equilibria are characterized by a two-state transition from predominantly random coil to predominantly helical conformations (Figure 4A,C). Only a few spectra deviate from this behaviour at the highest L/P ratios probably due to extensive light scattering at lipid concentrations > 1 mM (e.g. Fig. 4D).
L/P RATIO PEPTIDE
0 2 5 10 20 30 50 60 70 80
LAH4-A1 20 28 36 42 61 67 71 77 80
LAH4-A2 17 22 27 35 47 60 70 74 73 78
LAH4-A3 21 19 21 41 55 65 75 82 82
LAH4-A4 22 26 32 37 51 62 71
LAH4-A5 53 47 47 43 55 62 61
LAH4-A6 53 53 57 59 66 66 80 75
LAH4-L0 22 22 27 32 40 52 66 62
LAH4-L1 23 30 34 38 50 65 71
LAH4-L2 17 20 24 32 43 52 63 70
LAH4-L3 19 20 26 31 42 48 78 79 81 82
LAH4-L4 19 21 23 32 38 41 74 76
LAH4-L5 24 24 29 25 24 26 26
LAH4-L6 31 27 27 30 29 30 31 29
When the helix content is used to evaluate the amount of membrane-associated peptide as a function of lipid concentration the fraction of membrane-associated peptide increases in a close to linear fashion up to the point when all peptide is bound at L/P ratios of about 40 (Figure 5A,B). This behaviour has previously been observed in more elaborate binding studies of LAH4-L1 to POPC/POPS membranes using CD spectroscopy and ITC [22]
and shall only be discussed briefly here. In this previous work utilising different POPC/POPS molar ratios it has been found that one LAH4-L1 associates with four POPS molecules when a Langmuir adsorption model has been used. The data also fit to a Gouy-Chapman/membrane partitioning model where LAH4-L1 partitions with a K = 100 M-1 and at pH 5 carries an effective charge of 4, i.e. less than its nominal charge of 8, as has been observed for other peptides during membrane association [25]. To first approximation (and within experimental error) the peptides of the LAH4-L and LAH4-A series exhibit about the same degree of membrane association (Fig. 5A,B). When the data shown in Figure 5 are analysed by a conventional partitioning equilibrium the apparent partition coefficients [25] are in the 2000 M-1 range (not shown). Because the CD spectral changes of the A5, A6, L5 and L6 peptides are small or absent upon lipid addition the titration curves of these peptides could not be used to derive membrane association constants (Fig. 5C).
Figure 5: Fraction of membrane-associated peptides from the LAH4-A and -L series as a function of L/P ratio determined from the changes in helical content in CD spectra as shown in Figure 4. A. Data from LAH4-A series peptides with hydrophilic angle < 150o, B. from LAH4-L series peptides with hydrophilic angle < 150o and C. > 150o. In panel A the lines are shown to guide the eye and represent the plateau and the approximately linear increase in bound peptide at low L/P ratios as observed in [22, 26]. Whereas in panels A and B the fraction of bound peptide is scaled for each peptide individually, panel C shows changes relative to the highest and lowest helical content of the four peptides.
An important activity of the LAH4 family of peptides with potential biomedical importance is their potency in transfecting human cell lines with nucleic acids [10, 11]. When the efficiency of the peptides of the A- and L- series to transfect human glioblastoma cells with a luciferase reporter plasmid was evaluated, a strong increase in the luminescence activity of the luciferase was reported for all peptides one day after transfection (Figs. 6, S2 and S3). The highest activity is observed for LAH4-L1 and -A1 and tends to diminish when the number of leucines in the hydrophilic window decreases (Fig. 6). The same trend is observed for LAH4-A1 to -A4 while high levels of activity are observed for -A5 and -A6 (Figs. 6 and S2).
Figure 6. Transfection efficiency of the LAH-A and -L peptides evaluated on human
glioblastoma U87 cells. Increasing amounts of peptide were mixed with a constant amount of reporter plasmid (1.5 g per duplicate of pDNA) and the complexes were incubated for 2.5 h with the cells plated in 48-well plates. The transfection medium was then removed and replaced with fresh culture medium supplemented with 10% serum. Luciferase activity was measured 1 day post-transfection. The transfection efficiency is expressed as light units/s/mg protein and the reported values are the mean of duplicates (and are shown on a logarithmic scale). Error bars represent the standard deviation of the mean. In the Figure, only the conditions giving the highest luciferase expression are reported. The complete assays are reported as supporting information (Figures S2 and S3).
DISCUSSION
LAH4 peptides have been shown to exhibit antimicrobial, transfection, cell penetration, AAV and lentiviral transduction activities [8, 13-16, 18, 27, 28]. During some of these processes the membranes allow for the selective passage of ions or large biomolecular structures, or they lyse completely, fuse and/or reshape in different manner. In particular during transfection experiments it has been postulated that LAH4 peptides not only form large complexes with nucleic acids but they are also responsible for a self-promoted endosomal membrane passage [13, 29]. Furthermore, the antimicrobial activity has been associated with the selective pore formation in bacterial membranes and such processes have been studied by calcein release experiments of LAH4 and LAH4-L1 [3, 7]. In order to optimize the lentiviral transduction enhancement, the hydrophobic/hydrophilic angles of LAH4 peptides have been systematically varied [18] and the same series of peptides has been investigated here for their membrane permeation activities.
Indeed, the peptides investigated exhibit considerable calcein release activities where a quite remarkable correlation between membrane pore formation and hydrophilic angle is observed (Table 1, Figs. 1-3). When studied in detail the situation is somewhat more complex as some of the release activities exhibit a very fast and a slow component thus the exact time of analysis can change the quantitative evaluation (Fig. 2B). This observation is less
pronounced at pH 5 (Fig. 2A) and we suspect that peptide oligomerization and dissociation upon membrane insertion makes a contribution.
Because the calcein release activities could merely reflect differences in membrane association or systematic changes in the structure in solution and/or when associated with the membrane we tested the secondary structure preferences and membrane association of all peptides by CD spectroscopy. Upon addition of SUVs made of POPC/POPS 9/1 mole/mole the helical content of most peptides increases and the changes in spectral line shape have been analysed by deconvolution [21] (Tables 2, S1 and S2). Importantly, the peptides all follow about the same binding isotherm (Figure 5A,B) that has already been analysed in detail for LAH4-L1 [22] indicating that differences in the number of membrane-associated peptides may only play a minor role for the very different calcein release activities. When compared to the L-series a somewhat increased membrane-partitioning is observed for the A-series (Fig.
5A,B) which exposes more of the leucines at the hydrophobic face of the helix (Fig. 1).
Notably, when the peptide solutions are titrated with SUVs membrane association is accompanied with a transition from predominantly random coil structures to predominantly
helical conformations, where the peptides with the largest hydrophobic angles namely A5, A6, L5 and L6 show a different behaviour as will be discussed later. The other LAH4 peptides show approximately 20% helical contribution in solution, a value that increases to 70-80% when associated with membranes (Table 2). Indeed, the parent peptide LAH4 adopts helical conformations from residues 9-24 when investigated in DPC micelles [2] and a very high helix content has been observed for LAH4-L1 in the presence of related lipid bilayers in a previous CD-spectroscopic investigation [22]. The helical contribution in the absence of lipid (about 20% for most conditions at pH 5 but much higher at pH 7, Table S1) could be due to the formation of oligomeric structure, in particular at neutral pH where the histidines are deprotonated and the overall cationic charge of the peptides is decreased. Oligomerization is potentially driven by hydrophobic interactions, in particularly the many leucines which in some cases form a continuous hydrophobic surface (Figure 1).
The LAH4-A5, -A6, -L5 and -L6 peptides show considerable differences when compared to those of the series with smaller hydrophilic windows. First of all, they show the lowest calcein release activities (Figs. 2 and 3). Second, in aqueous solution at pH 5 A5 and A6 exhibit a much higher degree of helicity (53 %; Table 2) with only small CD spectral changes upon addition of lipid (Fig. 3). Notably these peptides exhibit a continuous face of 8 L at the opposite side and could also be considered a L8 variant with high hydrophilic angle (Figure 1). Possibly they oligomerize through a leucine zipper structure.
Third, the helix content of the L5 and L6 peptides matches those of other members of the L-series in solution (25-30%) but does not increase in the presence of SUVs (although some scattering may relate to the increase in residual molar ellipticity < 200 nm, Fig. 3D). It should be noted that due to the extended accumulation of hydrophobic leucine residues on one face of the helix the hydrophobic moment (at pH 7) is inverted when compared to the other peptides. It thus appears that neither the alanine-rich ‘hydrophobic’ face nor the ‘hydrophilic’
face of 5 or 6 leucines framed by charged histidines (at pH 5) efficiently insert into the membrane interface.
In conclusion, when considering the peptides which exhibit hydrophobic angles < 150o (i.e. LAH4-A1 to -A4 and -L0 to L4) the systematic variations in calcein release activities are probably neither related to differences in membrane association nor their secondary structure in solution or in the membrane. However, it is likely that the peptides with a large
hydrophobic angle penetrate more deeply into the membrane interface which thereby also develops differences in curvature strain, in the disordering of the lipid fatty acyl chains [11]
and lipid-mediated interactions between the peptides [30-32].
Notably, the highest lentiviral transduction enhancement activities, which are very important for efficient ex vivo gene therapy, have been found for the LAH4-A4 peptide which exhibits a hydrophilic angle of 140° (Fig. 1) and increases this biological activity 3 to 50-fold for the GALVTR or VSV-G-LV lentiviral pseudotypes when compared to the absence of peptide (Fig. S1) [17, 18]. In contrast, within the L-peptides the differences are much less pronounced with a somewhat increased enhancement of LAH4-L3 (hydrophilic angle of 120o) for VSV-G-LV (Fig. S1). Because lentiviral transduction enhancement has been associated with fiber formation, increased lentiviral attachment to cellular membranes and promotion of membrane fusion [17, 18, 33, 34] the calcein release investigated here is probably not related to this biological activity. In contrast, it seems that within the peptides of hydrophilic angle <
150o there is a negative correlation between lentiviral transduction and pore formation reflecting the very different requirements of the two activities, namely association into fibers and membrane partitioning of monomers and/or small oligomers, respectively (Fig. S1).
However, the calcein release activity (Fig. 3A,B) correlates with the peptides’
transfection activities (Fig. 6). The data agree well with a previous study where a much smaller set of LAH4-L peptides was investigated and their membrane disordering on the fatty acyl chain correlated with transfection activities of various cell lines [11]. In this process non- covalent complexes between the peptide and the cargo have to form at neutral pH to enter an endosomal pathway. In a final step endosomal membranes need to get permeabilized for the nucleic acids to reach the cytoplasm [8, 29]. Work to characterize all biological activities in more detail of the series of LAH4 peptide presented in this paper is ongoing in our laboratory.
ACKNOWLEDGEMENTS
The financial contributions of the Agence Nationale de la Recherche (projects MemPepSyn 14-CE34-0001-01, Biosupramol 17-CE18-0033-3 and the LabEx Chemistry of Complex Systems 10-LABX-0026_CSC), the University of Strasbourg, the CNRS, the Région Alsace (grant to ML) and the RTRA International Center of Frontier Research in Chemistry and the French Foundation for Medical Research (FRM), are gratefully acknowledged. BB is grateful to the Institut Universitaire de France for providing additional time to be dedicated to
research.
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SUPPLEMENTARY INFORMATION
Membrane pore-formation correlates with the hydrophilic angle of histidine-rich amphipathic peptides with multiple biological
activities
Morane Lointier1, Christopher Aisenbrey1, Arnaud Marquette1, Jia Hao Tan1, Antoine Kichler2, and Burkhard Bechinger1,*
Table S1. The secondary structure composition of the LAH4-A and -L peptides from the CD spectra recorded in solution at pH 5 or pH 7.4. The data shown are averages from three independent measurements. The secondary structure elements were estimated using the DICROPROT software [1].
A
PH LAH4-A1 LAH4-A2 LAH4-A3 LAH4-A4 LAH4-A5 LAH4-A6ΑLPHA-HELIX 5 20 17 21 22 53 53
ΒETA 14 14 14 14 10 8
RANDOM COIL 38 38 38 35 26 30
OTHER 27 31 27 29 11 9
ΑLPHA-HELIX
7.4 89 69 92 69 56 72
ΒETA 3 7 4 8 11 7
RANDOM COIL 6 14 4 17 26 17
OTHER 2 9 1 5 7 5
B
PH LAH4-L0 LAH4-L1 LAH4-L2 LAH4-L3 LAH4-L4 LAH4-L5 LAH4-L6ΑLPHA-HELIX 5 22 23 17 19 19 24 31
ΒETA 14 15 15 13 13 13 12
RANDOM COIL 38 36 34 39 39 36 38
OTHER 27 25 31 29 28 26 19
ΑLPHA-HELIX 7 77 65 57 91 81 56 69
ΒETA 7 8 10 4 6 10 9
RANDOM COIL 10 19 25 4 10 26 17
OTHER 6 8 9 1 3 8 6
Table S2: The secondary structure composition of the LAH4-A and -L peptides from the CD spectra recorded at different L/P ratios at pH 5. SUVs made from POPC/POPS 9/1 were added during the titration experiments to reach the indicated lipid-to-peptide ratios. The data shown are averages from three independent measurements. The secondary structure elements were estimated using the DICROPROT software [1].
LAH4-A1
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 20 28 36 42 61 67 71 77 80
ΒETA 14 13 9 10 9 8 8 10 5
RANDOM COIL 38 37 35 31 22 17 15 9 9
OTHER 27 22 23 18 8 8 6 4 6
LAH4-A2
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 17 22 27 35 47 60 70 74 73 78
ΒETA 14 14 13 11 10 9 9 12 10 13
RANDOM COIL 38 37 37 31 31 22 14 10 13 6
OTHER 31 27 23 25 12 9 7 4 4 4
LAH4-A3
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 21 19 21 41 55 65 75 82 82
ΒETA 14 7 8 13 10 8 9 9 13
RANDOM COIL 38 25 25 36 27 20 12 6 3
OTHER 27 16 12 10 8 8 4 2 2
LAH4-A4
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 22 26 32 37 51 62 71 58 61
ΒETA 14 13 9 8 11 8 9 4 18
RANDOM COIL 35 37 36 32 28 21 13 13 12
OTHER 24 23 23 10 9 7 25 9 24
LAH4-A5
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 53 47 47 43 55 62 61
ΒETA 10 11 11 12 9 8 5
RANDOM COIL 26 31 32 36 26 20 16
OTHER 11 10 11 9 8 11 19
LAH4-A6
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 53 53 57 59 66 66 80 75
ΒETA 8 10 9 9 8 6 8 9
RANDOM COIL 30 28 25 23 17 18 7 12
OTHER 9 9 9 9 9 10 4 4
LAH4-L0
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 22 22 27 32 40 52 66 62 50
ΒETA 14 14 14 11 12 9 7 7 8
RANDOM COIL 38 35 36 34 34 26 17 11 15
OTHER 27 29 22 23 15 13 10 19 27
LAH4-L1
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 23 30 34 38 50 65 71
ΒETA 15 10 9 12 9 7 10
RANDOM COIL 36 37 38 37 31 19 15
OTHER 25 23 20 12 10 9 4
LAH4-L2
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 17 20 24 32 43 52 63 70
ΒETA 15 14 13 9 13 10 9 13
RANDOM COIL 34 35 34 33 34 29 18 9
OTHER 31 32 29 30 11 9 11 8
LAH4-L3
RATIO L/P
0 2 5 10 20 30 50 60 70 80
ΑLPHA-HELIX 19 20 26 31 42 48 78 79 81 82
ΒETA 13 14 14 13 11 12 12 13 11 14
RANDOM COIL 39 41 38 38 33 31 8 6 5 0
OTHER 29 25 26 20 14 9 2 2 3 4
LAH4-L4
RATIO L/P
0 2 5 10 20 30 50 60 70 90
ΑLPHA-HELIX 19 21 23 32 38 41 74 76
ΒETA 13 14 14 12 9 8 10 17
RANDOM COIL 39 37 37 34 35 22 4 2
OTHER 28 28 26 23 17 10 12 5
Figure S1: The lentiviral transduction enhancement as reported in [2] is shown as a function of hydrophilic angle. Human CD34+ cells were transduced with the lentiviral strains VSV-G-LV or GALVTR-LV using peptides from the LAH4-A (A) or the -L series (B).
LAH4-L5
RATIO L/P
0 2 5 10 20 30 50 60 70 90
ΑLPHA-HELIX 24 24 29 25 24 26 26
ΒETA 13 13 12 24 15 14 12
RANDOM COIL 36 39 34 37 27 26 23
OTHER 26 24 25 25 35 34 38
LAH4-L6
RATIO L/P
0 2 5 10 20 30 50 60 70
ΑLPHA-HELIX 31 27 27 30 29 30 31 29 36
ΒETA 12 12 13 10 8 11 11 12 16
RANDOM COIL 38 38 39 38 32 33 30 20 19
OTHER 19 22 22 66 30 27 27 40 28
Figure S2. Transfection efficiency of the LAH-A peptides evaluated on human glioblastoma U87 cells. Increasing amounts of peptide were mixed with a constant amount of reporter plasmid (1.5 µg per duplicate of pDNA) and the complexes were incubated for 2.5 h with the cells plated in 48-well plates. The transfection medium was then removed and replaced with fresh culture medium supplemented with 10% serum. Luciferase activity was measured 1 day post-transfection. The transfection efficiency is expressed as light units/s/mg protein and the reported values are the mean of duplicates. Error bars represent the standard deviation of the mean.
Figure S3. Transfection efficiency of the LAH-L peptides evaluated on human glioblastoma U87 cells. Increasing amounts of peptide were mixed with a constant amount of reporter plasmid (1.5 µg per duplicate of pDNA) and the complexes were incubated for 2.5 h with the cells plated in 48-well plates. The transfection medium was then removed and replaced with fresh culture medium supplemented with 10% serum. Luciferase activity was measured 1 day post- transfection. The transfection efficiency is expressed as light units/s/mg protein and the reported values are the mean of duplicates. Error bars represent the standard deviation of the mean.
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[2] S. Majdoul, A.K. Seye, A. Kichler, N. Holic, A. Galy, B. Bechinger, D. Fenard, Molecular Determinants of Vectofusin-1 and Its Derivatives for the Enhancement of Lentivirally Mediated Gene Transfer into Hematopoietic Stem/Progenitor Cells, J Biol Chem, 291 (2016) 2161-2169.
