Peptide-Mediated Membrane Transport of Macromolecular Cargo

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Peptide mediated membrane transport of macromolecular cargo driven by membrane asymmetry XIN LI, Jing Huang, Matthew A Holden, and Min Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03421 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 21, 2017

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Analytical Chemistry

Peptide mediated membrane transport of macromolecular cargo driven by membrane asymmetry

Xin Li, Jing Huang, Matthew A. Holden, and Min Chen§

Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States

§

To whom correspondence should be addressed. Department of Chemistry, University of Massachusetts

Amherst,

Amherst,

Massachusetts

01003,

United

States.

Email:

[email protected]

Abstract Pep-1 is a cell-penetrating peptide that represents a powerful strategy for delivering large, hydrophilic therapeutic molecules into cells. Model membranes, such as lipid vesicles and planar bilayers, have been useful for investigating the direct translocation of cell-penetrating peptides. Here, we present a droplet interface bilayer–based approach to quantify pep-1– mediated β-galactosidase translocation. We found that β-galactosidase translocation is driven only by the negative transmembrane potential resulting from the asymmetric bilayers. The asymmetric droplet interface bilayer method may be generally applicable for high-throughput screening the efficacy of cell-penetrating peptides.

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INTRODUCTION Lipid bilayers constitute the biological membranes that pose a hydrophobic barrier to the penetration of large therapeutic molecules, such as proteins or nucleic acids, into cells. Cellpenetrating peptides (CPPs), highly cationic peptides usually rich in arginine and lysine amino acids, are characterized by their ability to translocate quickly into almost any live cells.1-3 Since their discovery in the late 1980s, the use of CPPs has become a popular strategy for carrying proteins, DNA, nanoparticles, and other cargo across the biological membrane for intracellular delivery.4-6 The mechanism(s) by which CPPs are able to cross biological membranes are complex and not fully understood.7-9 Two general pathways have been suggested: endocytosis and direct translocation. Some of the most commonly used CPPs, such as the HIV Tat protein, are actively taken up into cells by one or more types of endocytosis,10-12 while others may gain access to the cytosol by direct translocation.13,14 Recent findings indicate that both translocation pathways could coexist, and the mechanism could depend upon the interaction of the CPPs with specific cell membrane components.15,16 In the endocytosis pathway, it is unclear how the peptide or cargo escape from the endosome. Changes in the endosomes, such as decreasing pH, may trigger the CPPs to translocate the cargo across the membrane directly or may disrupt the endosomes and thus allow the cargo to be released.17 Pep-1

(KETWWETWWTEWSQPKKKRKV-cysteamide)

is

an

amphipathic

CPP

containing 21 amino acids. It is usually acetylated at its N-terminal and carries a cysteamide group at its C-terminus.18,19 The C-terminal cysteamide is a essential to the transduction mechanism and probably also increases the stability of the peptides. Pep-1 comprises three domains: a hydrophobic, tryptophan-rich motif (KETWWETWWTEW), followed by a linker domain (SQP) and a positively charged, lysine-rich domain derived from the nuclear localization sequence (NLS) of SV40 large T antigen (KKKRKV). While most CPPs must be covalently linked to the cargo protein, pep-1 can form a stable, noncovalent complex with cargoes.20,21


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Pep-1 has been used to deliver cargoes of small peptides, large proteins, and nanoparticles to cancer, neuronal and primary cell lines.18,19,22-24 The major pep-1 cell-translocation mechanism is independent of the endosomal pathway, although the exact mechanism(s) by which pep-1 is able to cross biological membranes remains unknown25. The process may involve transient membrane disorganization associated with pep-1 folding into a helical structure within the phospholipid membrane. Further elucidating the mechanism of direct translocation of pep-1 will provide useful knowledge for developing efficient methods of intracellular delivery. In vitro methods are useful for analyzing the direct translocation of CPPs and the interaction between CPPS and bilayers. Model membranes, such as vesicles and planar bilayers, make it possible to precisely control the chemical composition of the membrane’s environment, and thus avoid the complexities introduced by biological systems.26 Vesicles, in particular, have been used by several groups to study the cell-penetration mechanism of pep1.27,28 Recently, we used a model membrane based on a droplet interface bilayer (DIB) to investigate pep-1 translocation.29 A DIB membrane is formed between two lipid monolayer– encased aqueous droplets that are brought together under an oily hydrocarbon.30 The two droplets can be pulled apart mechanically, allowing the contents of each droplet to be assayed separately. This feature makes it possible to precisely quantitate molecule translocation across the DIB membrane.29 Our DIB-based method relied on applied electric potential across the membrane to drive translocation. The approach required the use of a patch clamp amplifier, and limited experiments to one per amplifier. In parallel, we discovered that membrane asymmetry may also drive pep-1–assisted translocation. In the present work, we investigate pep-1–assisted translocation using asymmetric bilayers as the only energy source. This method of quantifying CPP-mediated protein translocation is easily scalable and may potentially represent a highthroughput method for screening the efficacy of CPPs.



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MATERIALS AND METHODS Materials. Pep-1 (Ac-KETWWETWWTEWSQPKKKRKV-Cya) (No. 218110, >99% purity) was obtained from BiomatiK (Carlsbad, CA). Recombinant β-galactosidase (β-gal) (No. G3153) purified from Escherichia coli was obtained from Sigma-Aldrich (St. Louis, MO). Resorufin-β-Dgalactopyranoside (RG) (No. R1159) was obtained from Life Technologies (Carlsbad, CA). 1,2Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3-[phospho-rac(1-glycerol)]

(sodium

salt)

(DPhPG),

and 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine

(sodium salt) (DPhPS) were purchased from Avanti Polar Lipids (Alabaster, AL). 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and hexadecane (99%, anhydrous) were purchased from Sigma Aldrich. Sodium Chloride (99.8%) was purchased from Fisher Scientific (Hampton, NH). Vesicle preparation. The large unilamellar vesicles were prepared by extrusion using the protocol provided by Avanti Polar Lipids. All lipid powder was dissolved in pentane to a final concentration of 10 mg/mL. Lipid mixtures of different compositions were dried by air flow and subsequently hydrated by adding 1 mL buffer, resulting in a 2mM lipid suspension. The suspension was mixed in a vortex mixer for 30 minutes to fully hydrate the lipids. Afterwards, the hydrated lipid suspension was subjected to five freeze-and-thaw cycles by alternately placing the sample vial in a liquid nitrogen bath and a 37°C water bath. Finally, the sample was extruded through a 100-nm pore size membrane 21 times. Dimple chip fabrication. The DIB experiments were performed in homemade plastic chips derived from polystyrene tissue culture plates. A standard 24-well plate was cut into individual plastic wells (Figure S1). Next, a 10 × 10 array of dimples (spaced 0.7 mm apart on center) was machined into the bottom of each well using a 0.5-mm-radius ball-end mill controlled by a desktop CNC instrument (computer numerical control; Roland MDX-40). The depth of each well


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was approximately 250 µm. Finally, the wells were polished (to remove residual burrs and flatten machining marks) using a silica-based toothpaste and cotton swab. Protein translocation experiments in dimple chips. Droplets were prepared by immersing a small volume of aqueous solution in a hexadecane bath containing 1.8 mM dissolved lipids of the desired composition. It was found that the monolayer formation was accelerated in the presence of the dissolved lipids compared with a hexadecane-only bath. When asymmetric membranes were created, hexadecane baths with different dissolved lipids were used for each droplet. The capture droplet solution consisted of 1.8mM lipid vesicles and 50µM RG in buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). To prepare the source droplet solution, 20 µM pep-1 and 0.25 µM β-gal were incubated in HPLC-grade deionized water for 30 minutes to allow the pep1/protein complex to form. The complexes were then diluted with lipid vesicles to a final concentration of 1.8 mM lipids, 2 µM pep-1, and 25 nM β-gal in a 10 mM HEPES, 150 mM NaCl buffer at pH 7.4. A 0.3 µL droplet of each droplet solution was pipetted into the oil bath. After a 30-minute period to allow for monolayer formation around the droplet, the source and capture droplets were brought into adjacent dimples on the dimple chip to form a DIB. The ionic strength of both droplets were kept the same at 0.077 M to prevent the creation of a chemical potential across the bilayer. The fluorescence signal of the capture droplet was monitored by fluorescence microscopy for 2 hours to observe production of the fluorescent resorufin. Differential curves were derived from the fluorescence signal using Origin 2017 to obtain the turnover rates. As a control, the fluorescence increase in a capture droplet that was in the same dimple chip but not in contact with a source droplet provided a baseline representing the spontaneous breakdown of RG. Calibration of β-gal turnover rate. To calibrate the β-gal turnover rate, various concentrations of β-gal were mixed with 50µM RG or 1.8mM DPhPC vesicles in buffer consisting of 10mM HEPES and 150mM NaCl at pH 7.4. A 0.3-µL of the β-gal mixture was submerged in 5 ACS Paragon Plus Environment


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hexadecane and the fluorescence image of the droplet was recorded over 1 hour using a Nikon Eclipse T3i microscope with an excitation wavelength of 561 nm. The β-gal turnover rate remained constant over the 2 hour recording time. The β-gal turnover rate vs. concentration was plotted as a standard curve, which was used in later experiments to quantify the translocated βgal enzyme (Figure S2). The standard curve was determined periodically to verify that the activity of the enzyme was unchanged. Blue-native polyacrylamide gel electrophoresis. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) was performed with 4% to 15% Mini-Protean TGX Stain-Free Protein Gels (BioRad, Hercules, CA). Pep-1 was incubated with β-gal in a ratio of 20:1, 40:1, 60:1, 80:1, or 160:1 (mol:mol) respectively in water for 30 minutes. The mixture was then diluted with 5M NaCl to a final concentration of 2.5µM β-gal and 150mM NaCl. Each sample had a pH of approximately 7.0 as measured by a plastic pH indicator strip. Gels were run at 180 mV in a 4°C cold room for 1 hour using dark cathode (0.02% w/v Coomasie Blue G250, 15mM bis-tris, 50 mM tricine, pH 7.0) and anode (50mM bis-tris, pH 7.0) buffers. The dark anode buffer was then replaced by light cathode buffer (50mM tricine, 15mM bis-tris, pH 7.0) and the gel was run for another 2 hours at 300 mV. Size exclusion chromatography with in-line multi-angle static light scattering. A size exclusion chromatography with in-line multi-angle static light scattering (SEC-MALS) facility was bought from Wyatt Technology Corporation, equipped with a silica particle–filled column (No. 08542, Tosoh) that has an appropriate fractionation range for globular protein samples up to 7 million Da. Fifty micrograms of β-gal was sampled in 1× phosphate-buffered saline (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, pH 7.0) with a flow rate of 0.5 mL/min.

RESULTS


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Pep-1 translocation driven by bilateral asymmetry.

Figure 1: Fluorescence measurement of pep-1–assisted β-gal translocation. (a) Pep-1 and βgal were mixed to form complexes. (b) A source droplet containing peptide–enzyme complexes and a capture droplet containing substrate were prepared by mixing each solution with vesicles separately. (c) The droplets came into contact in a dimple chip under hexadecane and formed a bilayer. (d) Multiple droplet interface bilayer pairs were arranged on a dimple chip for parallel translocation experiments. Lipid vesicles within each droplet supplied lipids to form monolayers that annealed to create a bilayer membrane at the contacting region. The white dashed lines highlight one row of dimples on the dimple chip. (e) The fluorescence intensity of the capture droplet was monitored over time. (f) The derivative curve of figure (e). The derivative translocation curve was fitted with the dose response function.

We chose β-gal as the cargo for pep-1 because of its robust catalytic activity, which facilitates accurate quantification of the translocated proteins. β-Gal is a tetrameric protein with a molecular weight of 465 kDa. It catalyzes the hydrolysis of the β-glycosidic bond formed between a galactose and the resorufin moiety of RG. To monitor the trafficking of β-gal across the membrane, pep-1 and β-gal were pre-incubated to form complexes before they were mixed with lipid vesicles. The mixture was pipetted into hexadecane to form the source droplets. The source droplet’s monolayer is typically composed of only neutrally charged lipids DPhPC. A similar procedure was performed to form the capture droplet, which contained 50 µmol/L of 7 ACS Paragon Plus Environment


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fluorogenic substrate RG. The source and capture droplets were brought into close contact to form the lipid bilayer. The pep-1–facilitated β-gal translocation was observed by measuring the fluorescent signal of the β-gal enzymatic product (Figure 1b,c). Several pairs of droplets can be arranged in the dimple chip (Figure 1d). In this way, multiple experiments can be performed simultaneously, thus improving throughput. We were able to observe protein translocation in each pair of droplets in real time and record time-lapse data with which to generate curves such as the one shown in Figure 1e. The slope at any point along the curve indicates the turnover rate of the substrate in the capture droplet at that time. Figure 1f, the derivative curve of the fluorescence plot (Figure 1e), shows that the turnover rate increased at the beginning of the translocation experiment and reached plateau after 80 to 100 minutes, indicating that the concentration of the enzyme became constant as the translocation ceased. Indeed, owing to the stable catalytic activity of β-gal, the record of fluorescence intensity shows that the experiment could last more than 24 hours, and the slope after translocation cessation was constant for several hours. By fitting the derivative curve with a dose-response function, the substrate turnover rate when the translocation stops can be determined (Figure 1f). The substrate turnover rate was then used to calculate the β-gal concentration at the capture droplet by the calibration curve. Effect of membrane asymmetry on cargo translocation. We first sought to determine how the asymmetry of the bilayer membrane affects β-gal translocation. As a control, symmetrical bilayer membranes were prepared from two droplets that contained either the neutral lipid DPhPC or DPhPC lipids mixed with 30% DPhPG (mol/mol). Two sets of asymmetric bilayer membrane were then generated. In the first set, the lipids in the monolayer of the source droplet were composed of 30% (mol/mol) negatively charged DPhPG and 70% (mol/mol) DPhPC, while the capture droplet contained only DPhPC. In the second set, the bilayer had the reverse asymmetry, with the source droplet containing 100% DPhPC and the capture droplet containing 8 ACS Paragon Plus Environment

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Figure 2. Translocation of β-gal across an asymmetric bilayer. (a) Four lipid conditions were tested in droplet interface bilayer systems: (I) symmetric leaflets with DPhPC, (II) symmetric leaflets with 30% DPhPG(PG), (III) asymmetrical leaflets with 30% DPhPG in the source droplet leaflet, (IV) asymmetrical leaflets with 30% DPhPG in the capture droplet leaflet, and (V) asymmetrical leaflets with 30% DPhPS in the capture droplet leaflet. The opposing leaflets in (III), (IV), and (V) contained 100% DPhPC. For all experiments, the source droplet contained 2µM pep-1, 25nM β-galactosidase in buffer 150mM NaCl, 10mM HEPES, pH 7.4 and 1.8 mM of the corresponding lipid vesicles. The capture droplet contained 50 µM RB and 1.8 mM lipid vesicles. (b) A box and whisker plot showing the translocated β-gal in the capture droplets for each lipid condition. Data presented here are from 10 independent trials. The horizontal line in the middle of each box represents the median, while the box limits represent the 25th and 75th percentiles. The whiskers represent the minimum and maximum values.

30% (mol/mol) DPhPG and 70% (mol/mol) DPhPC. No translocation was observed with the control symmetric neutral bilayers or bilayers with 30% DPhPG. When DPhPG was present in the leaflet of the source droplet, we also did not observe noticeable enzyme translocation over 2 hours. In contrast, when DPhPG was present in the leaflet of the capture droplet, approximately 0.3 pM β-gal was found in the capture droplet. Replacing the negatively charged DPhPG with DPhPS in the leaflet of the capture droplet also resulted in significant accumulation of β-gal in



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the capture droplet. A one-way ANOVA to compare means of the translocated β-gal driven by 5 different membrane asymmetry shows a p0.05 0.8 0.6 0.4 0.2 0.0 -0.2

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Figure 4


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Figure 5



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Dimple Chip Array

Asymmetric DIB

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Pep-1 Mediated Cargo Translocation

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Peptide-Mediated Membrane Transport of Macromolecular Cargo

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