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Aromatic Residue Position on the Nonpolar Face of Class A Amphipathic Helical Peptides Determines Biological Activity

Aromatic Residue Position on the Nonpolar Face of Class A Amphipathic Helical Peptides Determines... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 24, Issue of June 18, pp. 26509 –26517, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Aromatic Residue Position on the Nonpolar Face of Class A Amphipathic Helical Peptides Determines Biological Activity* Received for publication, December 30, 2003, and in revised form, April 8, 2004 Published, JBC Papers in Press, April 8, 2004, DOI 10.1074/jbc.M314276200 Geeta Datta‡, Raquel F. Epand§, Richard M. Epand§, Manjula Chaddha‡, Matthew A. Kirksey‡, ¶ ¶ David W. Garber‡, Sissel Lund-Katz , Michael C. Phillips , Susan Hama , Mohamad Navab , Alan M. Fogelman , Mayakonda N. Palgunachari‡, Jere P. Segrest‡, and G. M. Anantharamaiah‡** From the ‡Departments of Medicine, Biochemistry and Molecular Genetics and the Atherosclerosis Research Unit, University of Alabama at Birmingham, Birmingham, Alabama 35294, the §Department of Biochemistry, McMaster University Health Sciences Centre, Hamilton, Ontario L8N 3Z5, Canada, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, and the Department of Medicine and the Atherosclerosis Research Unit, UCLA Cardiology, UCLA, Los Angeles, California 90095 of intense investigation in many laboratories because of its The apolipoprotein A-I mimetic peptide 4F (Ac-DW- FKAFYDKVAEKFKEAF-NH ), with four Phe residues on strong antiatherogenic properties (1–7). Two major theories on the nonpolar face of the amphipathic -helix, is strongly the role of HDL in the development of atherosclerosis are as anti-inflammatory, whereas two 3F analogs (3F and follows: (i) reverse cholesterol transport (HDL transfers choles- 3F ) are not. To understand how changes in helix non- terol from peripheral tissues to the liver for excretion) (5), and polar face structure affect function, two additional 3F (ii) the lipid oxidation theory (it has been shown that HDL analogs, Ac-DKLKAFYDKVFEWAKEAF-NH (3F-1) and 2 traps oxidized lipids that are responsible for the production of Ac-DKWKAVYDKFAEAFKEFL-NH (3F-2), were de- cytokines) (7). Several enzymes (notably platelet-activating signed using the same amino acid composition as 3F factor acetylhydrolase and paraoxonase) that are present on and 3F . The aromatic residues in 3F-1 and 3F-2 are the surface of HDL are responsible for destroying the biological near the polar-nonpolar interface and at the center of activity of oxidized lipids (6). Different strategies have been the nonpolar face of the helix, respectively. Like 4F, but used by several laboratories to support both theories (8, 9). 3 14 in contrast to 3F and 3F , these peptides effectively However, recently, it has been suggested that the two mecha- inhibited lytic peptide-induced hemolysis, oxidized nisms may be two sides of the same coin (10). phospholipid-induced monocyte chemotaxis, and scav- We have used apoA-I mimetic class A amphipathic helical enged lipid hydroperoxides from low density lipopro- peptides to understand the functions of apoA-I that may be tein. High pressure liquid chromatography retention playing a primary role in inhibiting atherosclerosis (10, 11). times and monolayer exclusion pressures indicated that With this approach, we have shown that either intraperitonial there is no direct correlation of peptide function with administration of the class A amphipathic helical peptide 5F or lipid affinity. Fluorescence studies suggested that, al- the oral administration of peptide 4F synthesized from all-D- though the peptides bind phospholipids similarly, the amino acids inhibit atherosclerosis in dyslipidemic mouse mod- Trp residue in 4F, 3F-1, and 3F-2 is less motionally re- 3 14 stricted than in 3F and 3F . Based on these results and els without altering plasma cholesterol levels (12, 13). Further- molecular modeling studies, we propose that the ar- more, investigations using apoA-I peptides support the concept rangement of aromatic residues in class A amphipathic that their ability to inhibit atherosclerosis is related to their helical molecules regulates entry of reactive oxygen spe- ability to remove “seeding molecules” (reactive lipid hydroper- cies into peptide-phospholipid complexes, thereby re- oxides formed by the oxidation of phospholipids containing ducing the extent of monocyte chemotaxis, an important arachidonic acid) from the low density lipoprotein (LDL) sur- step in atherosclerosis. face, thereby inhibiting free radical oxidation chain reactions and the subsequent propagation and release of cytokines (14). Preliminary studies suggest that oral administration of D-4F to Human apolipoprotein A-I (apoA-I), the major protein com- LDL receptor null and apoE null mice causes the rapid forma- ponent of high density lipoproteins (HDL), has been the subject tion and clearance of small HDL-like particles containing pep- tide, cholesterol, and apoA-I and paraoxonase, capable of con- * This work was supported in part by National Institutes of Health verting proinflammatory HDL into anti-inflammatory HDL Grants PO1 HL34343, 30568, and 22633 and Canadian Institutes of (15). These results, coupled with the previous observations that Health Research Grant MT-7654. M. N., S. H., A. M. F., and G. M. A. are principals in Bruin Pharma, a start-up biotech company. The costs class A amphipathic helical peptide associates with HDL (16), of publication of this article were defrayed in part by the payment of suggest that the peptide is active in a lipid-associated form. page charges. This article must therefore be hereby marked “advertise- However, not all class A amphipathic helical peptides are ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this anti-inflammatory to the same extent (17, 18). Using a series of fact. ** To whom correspondence should be addressed: 1808 7th Ave. S., peptides in which the hydrophobicity of the nonpolar face was DREB 640, Department of Medicine, UAB Medical Center, Birming- increased by substituting Ala, Leu, or Val residues with Phe ham, AL 35294. Tel.: 205-934-1494; E-mail: [email protected]. residues, we have shown that greater hydrophobicity per se is The abbreviations used are: apoA-I, human apolipoprotein A-I; not sufficient to increase the anti-inflammatory properties of a Ac, acetyl; DPH-PC, 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexade- canoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero- 3-phosphocholine; EPC, egg phosphatidylcholine; HDL, high density lipoprotein(s); HPLC, high pressure liquid chromatography; HPODE, 3-phosphocholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- hydroperoxyoctadecadienoic acid; LDL, low density lipoprotein(s); LUV, choline; RBC, red blood cells; REES, red edge excitation shift; MLV, large unilamellar vesicle; PAPC, 1-palmitoyl-2-arachidonyl-sn-glycero- multilamellar vesicle. This paper is available on line at http://www.jbc.org 26509 This is an Open Access article under the CC BY license. 26510 Anti-inflammatory Class A Amphipathic Peptides FIG.1. A, helical wheel representation of the parent molecule 2F, the antiatherogenic 4F, and the four 3F analogs. The wheel is projected along the axis of the helix from N to C terminus with the hydrophobic side facing downward. The primary structure is given above each wheel diagram. The amino acid composition of each of these peptides is the same, but the sequences are different. The plus and minus signs denote the charge on the amino acids at neutral pH. Red denotes an acidic residue, blue denotes a basic residue, and boldface black denotes aromatic residues. The line shows the interface between the hydrophobic face (lower part) and the hydrophilic face (upper half) as given by us earlier (20). Although the hydrophobic faces appear to be similar in each peptide, their abilities to allow water to penetrate into the lipid are different. B, space-filling models of the parent peptide, 2F, the antiatherogenic peptide, 4F, and the four 3F peptides. These models were created using silicon graphics; the lysine 3 14 residues were snorkeled (22), and the models were energy-minimized. The view down the long axis of the helix shows that 2F, 3F , and 3F are wedge shaped, whereas 4F, 3F-1, and 3F-2 are more cylindrical in shape. The blue color in the space-filling model denotes a nitrogen atom, whereas the red color denotes an oxygen atom. class A amphipathic helical peptide (17). The addition of one alogs of 3F. In one analog, with the amino acid sequence Ac- more Phe to the nonpolar face of Ac-18A-NH (2F) either at DKLKAFYDKVFEWAKEAF-NH (referred to as 3F-1), the al- 2 2 3 14 position 3 (3F )orat14(3F ) (Fig. 1) resulted in peptides that iphatic residues are at the center of the nonpolar face, and were no longer able to inhibit LDL-induced monocyte chemo- aromatic residues are near the interfacial Lys residues, tactic activity (17). However, a peptide analog containing four whereas in another 3F analog with the amino acid sequence Phe residues on the nonpolar face (4F) was the most active in Ac-DKWKAVYDKFAEAFKEFL-NH (referred to as 3F-2), the this series in inhibiting LDL-induced monocyte chemotactic -electron-containing aromatic residues are at the center of the activity, whereas the extensively studied 2F analog was not as nonpolar face (see Fig. 1A). The primary sequence and wheel 3 14 effective. Furthermore, 2F did not inhibit atherosclerosis in a projection of these peptides along with those of 2F, 3F ,3F , diet-induced mouse model of atherosclerosis (19), whereas 4F and 4F for comparison are shown in Fig. 1A. The space-filling was highly effective (18). The further addition of Phe residues models of these peptides with the Lys residues snorkeled (20, (5F, 6F, and 7F) did not enhance this property. We therefore 21) are shown in Fig. 1B. We have studied the physical-chem- hypothesized that a particular arrangement of aromatic resi- ical and anti-inflammatory properties of these two peptides 3 14 dues on the nonpolar face is more important in producing a and 4F in comparison with 3F and 3F . The results show that peptide with maximum anti-inflammatory properties than is although all of the peptides have similar abilities to form an the overall hydrophobicity of the peptide. amphipathic helix, the two peptides 3F-1 and 3F-2 are biolog- To investigate this, we have synthesized two additional an- ically active, similar to 4F. This effect appears to be related to Anti-inflammatory Class A Amphipathic Peptides 26511 method with a Protein Technologies PS-3 automatic peptide synthe- the position of aromatic residues with respect to the surface of sizer using the procedures described previously (12, 17). Peptides were lipoprotein particles, which affect molecular arrangements purified using a preparative HPLC system (Beckman Gold), and the near the interface and alter the lateral interactions within the purity of the peptides was determined by mass spectral analysis and particle. Changes in these interactions could promote the analytical HPLC. transfer of oxidized lipids (found on the surface of LDL parti- Circular Dichroism—CD spectra were recorded on an AVIV model cles) to HDL-like particles, thus making LDL less effective in 215 spectropolarimeter using a quartz cell with a 0.1-cm path length at 25 °C. Peptide solutions in phosphate-buffered saline were used at inducing monocyte chemotaxis, an important step in the initi- concentrations ranging from 10 to 400 M. The effect of lipid binding on ation of atherogenesis. the secondary structure of the peptide was studied using peptide-lipid complexes (1:10 mol/mol) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phos- EXPERIMENTAL PROCEDURES phocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine Materials—Tissue culture materials and other reagents were ob- (DMPC). Since the results were similar, we have reported results with tained from sources previously described (6, 7). Lipids used in these POPC in order to be able to compare with the other studies. These investigations were obtained from Avanti Polar Lipids (Alabaster, AL) complexes were prepared by adding the appropriate volume of peptide except for the fluorescent phospholipid 2-(3-(diphenylhexatrienyl)pro- solution to POPC multilamellar vesicles (MLVs). The MLVs were pre- panoyl)-1- hexadecanoyl-sn-glycero-3- phosphocholine (DPH-PC) that pared by dissolving a known amount of lipid in chloroform and slowly was obtained from Molecular Probes, Inc. (Eugene, OR). Acetonitrile, removing the solvent by evaporation under a thin stream of nitrogen. dimethylformamide, chloroform, methanol, ammonium formate, formic Residual solvent was removed by storing the lipid film under vacuum acid, and acetic acid, were all obtained from Fisher, and acetic anhy- overnight. An appropriate volume of buffer was added to hydrate the dride diisopropylethylamine and pipyridine were from Sigma. Amino thin lipid film, which was then vortexed. The lipid-peptide complexes acids and condensing reagent O-benzotriazole-N,N,N,N-tetramethy- were prepared by adding the required volume of peptide solutions to luronium hexafluorophosphate and rink amide resin were purchased give the desired lipid to peptide molar ratio. CD measurements were from Advanced Chem Tech (Louisville, KY) and Peptides International also done in the presence of 50% trifluoroethanol. The program CD (Louisville, KY). Spectra Deconvolution (CDNN), version 2.1, was used to calculate the Hemolysis Assay—The hemolysis assay was carried out as described secondary structure content (28). by Tytler et al. (22). Briefly, red blood cells (RBCs) were collected from Right Angle Light Scattering Measurements—Association of these EDTA-treated human blood by centrifugation. The cell pellet was peptides with POPC was determined by following the dissolution of washed three times with phosphate-buffered saline to remove plasma POPC MLVs by right angle light scattering using an SLM 8000C and the buffy coat. A suspension of 1% erythrocytes in phosphate- photon counting spectrofluorometer as described (29). POPC MLVs buffered saline with or without peptide was incubated at 37 °C for 10 were prepared by evaporating a solution of POPC (Avanti Polar) under min. The suspension was centrifuged at 16,000  g for 3 min. Hemolysis nitrogen and hydrating the lipid film with phosphate-buffered saline was measured as hemoglobin content (absorbance at 540 nm) of the (pH 7.4). The sample containing 105 M POPC and an equimolar supernatant. Base-line hemolysis was the hemolysis of RBCs incubated amount of peptide was maintained at 25 °C and continuously stirred. with phosphate-buffered saline. Hemoglobin released by 0.1% Triton Turbidity clarification was monitored at 400 nm for 30 min. Complete X-100 was taken to be 100% lysis. Inhibition of lysis by peptides was dissolution of POPC vesicles was achieved by the addition of Triton measured by incubating RBCs with a 10 M concentration of peptides X-100 to a final concentration of 1 mM. and adding 2 M 18L to the cell suspension. Hemolysis was expressed as Surface Pressure Measurements—Monolayer exclusion pressure meas- a percentage of the Triton X-100 lysis. urements give the affinity of the peptides for a lipid-water interface. The Monocyte Chemotaxis Assay—The procedure detailed by Navab et al. procedure of Phillips and Krebs (30, 31) was followed using egg phosphati- (6) was followed. Briefly, human aortic endothelial cells and smooth dylcholine (EPC) as per their procedure. An insoluble monolayer of EPC muscle cells were isolated. Human aortic smooth muscle cells were was spread at the air-water interface in a Teflon dish at room tempera- grown in microtiter well plates until confluence, and the human aortic ture to give an initial surface pressure ( ) in the range of 5– 45 microne- endothelial cells were added. Cocultures were treated with 20 gof wtons/m. A solution of peptides in phosphate-buffered saline containing 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1 1.5 M guanidinium chloride was carefully injected in to the subphase to g/ml of hydroperoxyeicosatetraenoic acid (HPODE) in the absence or give a final concentration of 50 g/dl. The guanidinium chloride was presence of HDL (50 g of cholesterol) or peptides (66 M)for8h.The diluted in the subphase to a final concentration of 1mM to allow the supernatants were collected and assayed for chemotactic activity. peptides to renature. The subphase was stirred continuously, and the Determination of Lipid Hydroperoxide Content—Lipid hydroperox- increase in EPC monolayer surface pressure () was recorded until a ides were measured colorimetrically by the FOX2 assay (23). The ability steady state value was obtained. The value of the initial surface pressure of the peptides to remove lipid hydroperoxides from lipoproteins was ( ) at which the peptides no longer penetrate the EPC monolayer (i.e. the assessed by incubating each of them with plasma from Watanabe rab- exclusion pressure ( )) was calculated by extrapolating the  versus e i bits. Plasma from these rabbits is known to possess lipid hydroperox- linear regression fit to   0 micronewtons/m. ides (24, 25). 1 ml of plasma was incubated with peptide at a final Intrinsic Tryptophan Fluorescence—Fluorescence spectra of both concentration of 44 M for1hat37 °C. The lipoproteins were then peptide and peptide-lipid complexes were recorded at room temperature separated on two Superose 6 (Amersham Biosciences) columns in tan- with excitation at 295 nm using a SPF-500 spectrofluorometer. These dem using the Bio-Rad BioLogic Duoflow system. 0.5-ml fractions were studies were done using DMPC as the lipid of choice in order to compare collected, and absorbance was monitored at 280 nm. The peaks were them with earlier published results of other peptides (17). The relative analyzed for hydroperoxide content. Briefly, 900 l of the FOX reagent fluorescence intensity was measured in ratio mode that corrects for containing 300 M FeSO 7H O, 120 M xylenol orange, 25 mM H SO , time dependent fluctuations in lamp intensity. Quenching of trypto- 4 2 2 4 and 4.4 mM butylated hydroxytoluene in methanol was added to 100 l phan fluorescence by potassium iodide and acrylamide was determined of the sample and incubated in the dark for 30 min, and absorbance was by adding aliquots of stock solutions of potassium iodide (4 M) and measured at 560 nm. The values were compared with a standard of acrylamide (4 M) to a 2.7-ml solution of either peptide or peptide-lipid cumene hydroperoxide to quantitate the hydroperoxide content. complexes (32). Stock solutions of potassium iodide contained 1 mM Lipoproteins—Low density lipoprotein (d  1.019 g/ml) and high sodium thiosulfate (Na S O ) to prevent the formation of I . Inner filter 2 2 3 3 density lipoproteins (d  1.063–1.21 g/ml) were isolated using the corrections were applied for acrylamide quenching (32). Emission spec- procedure by Havel et al. (26) from normal volunteers under the proto- tra were taken after each addition, and emission intensity at  was max col approved by Human Research Subject Protection committees of the determined. Peak emission intensity was used for all calculations. The University of Alabama at Birmingham and UCLA. In some cases, quenching data were analyzed according to the Stern-Volmer equation, butylated hydroxytoluene (20 mM in ethanol) was added to freshly isolated plasma at a concentration of 20 M, and the lipoprotein was F /F  1  K Q (Eq. 1) 0 sv separated by fast protein liquid chromatography using the methods previously described (27). The LDL, HDL, and lipoprotein-deficient where F and F represent the fluorescence in the absence and presence serum had endotoxin levels below 20 pg/ml, which is well below the of the quencher, and [Q] is the concentration of the quencher. K is the SV threshold needed for induction of monocyte adhesion or chemotactic Stern-Volmer constant. activity. The concentrations of lipoproteins reported in this study are Large Unilamellar Vesicles (LUVs)—Lipid films were made by dis- based on protein content. solving appropriate amounts of lipid in a mixture of chloroform/meth- Peptide Synthesis—Peptides were synthesized by the solid phase anol (2:1 (v/v)) and dried in a test tube under nitrogen to deposit the 26512 Anti-inflammatory Class A Amphipathic Peptides lipid as a thin film on the wall of the tube. Final traces of solvent were removed in a vacuum chamber attached to a liquid nitrogen trap for 2–3 h. Dried films were kept under argon gas at 30 °C if not used imme- diately. Films were hydrated with buffer, vortexed extensively at room temperature, and then subjected to five cycles of freezing and thawing. The homogeneous lipid suspensions were then further processed by 10 passes through two stacked 0.1-m polycarbonate filters (Nucleopore Filtration Products, Pleasanton, CA) in a barrel extruder (Lipex Bi- omembranes, Vancouver, BC) at room temperature. LUVs were kept on ice and used within a few hours of preparation. Lipid phosphorus was determined by the method of Ames (33). Fluorescence Spectroscopy Studies with DPH-PC—Peptide solutions were prepared in buffer containing 10 mM Hepes, 0.14 M NaCl, 0.1 mM EDTA, pH 7.4. The peptide concentrations were determined by absorb- ance at 280 nm against buffer as reference. Films containing POPC/ DPH-PC (400:1) were prepared from a solution in chloroform/methanol (2:1). The film was hydrated in buffer. 100-nm diameter LUVs were then made by extrusion as described previously. Fluorescence was measured in quartz cuvettes at 37 °C, in an SLM Aminco Bowman Series II spectrofluorometer, with an excitation of 355 nm and slits set to 4-nm bandwidth in both excitation and emission. The fluorescence emission of 2 ml of 50 M LUVs in the same buffer was recorded. Then small aliquots of a peptide solution, also in the corresponding buffer, were added successively, and the fluorescence emission was recorded after each addition. The emission maximum was found at 431 nm. Red Edge Excitation Shift (REES)—The emission spectra of trypto- phan were measured at 25 °C in an SLM Aminco Bowman Series II luminescence spectrofluorometer. The excitation wavelength was var- ied every 5 nm between 280 and 310 nm, and the emission spectra were recorded over 310 –350 nm three times and averaged. Measurements were done in quartz cuvettes containing 2 ml of buffer (10 mM NaH PO , 0.14 M NaF, 1 mM EDTA, pH 7.4). Aliquots of solutions of 2 4 peptides in buffer were added to the cuvette to a final concentration of 10 M. Measurements were repeated with the addition of 100 M EPC LUVs. To reduce scattered light intensity, excitation polarization was set to 90° and emission to 0°, with 4-nm bandwidth. Spectra were corrected for instrumental factors, and controls were subtracted. Peak emission wavelengths were recorded. Statistical Analysis—Statistical analysis was performed as previ- ously described (18) using one-way analysis of variance, and signifi- FIG.2. A, ability of peptides to scavenge lipid hydroperoxides from cance was defined as p  0.05. Post hoc Tukey tests were performed. LDL. The effect of the 3F peptides and 4F on lipid hydroperoxide Peptides were compared with base line and with each other. levels in LDL was determined. 1 ml of plasma from Watanabe rabbits (used as a source of oxidized LDL) was incubated with peptides at a RESULTS final concentration of 44 M, and the lipoproteins were separated on Superose 6 columns using fast protein liquid chromatography. The Removal of Hydroperoxides from LDL and Inhibition of hydroperoxide concentration in the LDL fraction was determined PAPC HPODE-induced Monocyte Chemotaxis—We previ- using cumene hydroperoxide as a standard. The data was analyzed ously reported that the anti-inflammatory properties of these using one-way analysis of variance, and the significance value is marked. Compared with the control samples, 4F, 3F-2, 3F-1, and 3F peptides correlate well with their ability to scavenge seeding reduced the concentration of lipid hydroperoxide significantly. The molecules from the LDL surface, inhibiting LDL-induced reduction seen by 4F, 3F-2, and 3F-1 appeared to be statistically more monocyte chemotaxis (6, 7). To determine their ability to scav- significant (**, p  0.001) than that seen by 3F (†, p  0.001). enge hydroperoxides from LDL, the peptides were incubated However, 3F is not significantly different from the control. B, inhi- with plasma from Watanabe rabbits that are known to have bition of oxidized lipid-induced monocyte chemotaxis. The effect of the 3F peptides on PAPC HPODE-induced monocyte chemotaxis oxidized LDL (24, 25). We used 4F as a positive control peptide was determined at 66 M concentration of peptide. 20 g of PAPC was to compare the present results with the previously published added together with 1 g/ml of HPODE to cocultures of human artery results (17). The lipoproteins were separated on Superose 6 wall cells as described previously (7, 8), and human HDL at 50 g/ml columns, and the fractions were tested for hydroperoxide con- cholesterol or no addition was made to the cocultures (No Addition), or the peptides were also added at 66 M.After8hofincubation, tent. As shown in Fig. 2A, 3F-2, 3F-1, and 4F significantly supernatants were collected and assayed for monocyte chemotactic reduced hydroperoxides by about 75% from about 190 M to activity using standard neuroprobe chambers. The data are mean 3 14 about 50 M, whereas 3F and 3F had relatively small effects S.D. of the number of migrated monocytes in nine fields for triplicate on the hydroperoxide concentrations in LDL. samples. *, p  0.05 compared with PAPC HPODE alone. LDL-induced monocyte chemotaxis is due to the oxidation of LDL phospholipids containing arachidonic acid. This generates effects. This parallels their ability to scavenge lipid hydroper- specific biologically active phospholipids, which in turn cause oxide molecules from LDL. There was no statistical difference the artery wall cells to produce the potent monocyte chemoat- between the activities of 3F-2 and 3F-1. However, the peptide tractant MCP-1, which in turn induces monocyte chemotaxis 4F was the most effective and significantly different from 3F-1 (6, 7). We compared the ability of these peptides to inhibit the and 3F-2. monocyte chemotactic activity resulting from the incubation of Inhibition of 18L-induced Red Cell Lysis by 3F Peptide An- human artery wall cells with the arachidonic acid containing alogs—Our earlier results showed that apoA-I and apoA-I mi- phospholipid, PAPC, and the fatty acid hydroperoxide, metic peptides are not themselves lytic at low peptide/lipid HPODE. The effect of these peptides on PAPC-HPODE-in- ratios, and furthermore they stabilize membranes from lysis duced monocyte chemotaxis (Fig. 2B) showed that, whereas induced by the lytic peptide 18L (22). We tested the 3F peptides both 3F-2 and 3F-1 effectively inhibited PAPC-HPODE-in- for lytic activity using RBCs and compared them to 2F and 4F 3 14 duced monocyte chemotaxis, 3F and 3F showed only small (control peptides). The lysis caused by 0.1% Triton X-100 was Anti-inflammatory Class A Amphipathic Peptides 26513 TABLE I Comparison of some physical properties of peptides a b c Peptide HPLC retention time Exclusion pressure t min micronewtons/m s 3F-1 20.8 37 59 3F-2 20.0 33 65 3d 3F 21.0 38 44 14d 3F 21.2 39 34 4F 22.0 40 65 HPLC retention time is the time taken for the peptide to elute from a C-18 Vydac column using a gradient of 25–58% acetonitrile in water containing 0.1% trifluoroacetic acid. Exclusion pressure from an EPC monolayer. The reproducibility of these values is 1 micronewton/m. t is the time taken for reducing the light scattering intensity by 50%, calculated from light scattering studies presented in Fig. 4. Data from Ref. 17. peptide 3F-2 has the shortest retention time on a C-18 reversed phase HPLC column, and 3F has the highest retention time. Exclusion pressure from an EPC monolayer was also the least for the peptide 3F-2, and highest for 3F (Table I). The results suggest that among the four 3F analogs, the presence of aro- matic amino acids at the center of the nonpolar face decreases the lipid-associating ability (binding to reversed phase HPLC column containing C-18 acyl chain) and EPC monolayer exclu- sion pressure. The addition of another Phe to produce 4F mar- ginally increases the retention time and exclusion pressure 3 14 compared with 3F and 3F . Tryptophan Fluorescence and Quenching Studies—In the peptide 3F-2, Trp is at the center of the -helix nonpolar face, whereas it is at the polar-nonpolar interface in the other three peptide analogs. All of the peptides show a significant shift in fluorescence maximum ( ) with DMPC, indicating that all max of the four peptides associate similarly with lipid (Table II). FIG.3. A, effect of the peptides on 18L-induced hemolysis. The effect The shift in  from solution state to lipid-associated state for of the peptides on 18L-induced RBC lysis was studied by coincubating max 3 14 RBCs with 18L and the control peptide, 4F, and the four 3F analogs. 3F and 3F are 14 and 13 nm, respectively, whereas the other Hemolysis was measured as hemoglobin content in the lysate (absorb- two peptides shifted less (11 nm). Peptide 3F-2 shows the ance of the lysate at 540 nm). Hemolysis by 0.1% Triton X-100 was largest increase in fluorescence intensity on binding to DMPC. taken as 100% lysis. The data are expressed as a percentage of Triton This appears to be due to the position of Trp at the center of the X-100 lysis. The data were analyzed by one-way analysis of variance. All of the peptides protect the RBCs from 18L-induced lysis. However, nonpolar face. From aqueous phase to lipid-associated form, the protection by 3F is not statistically significant. All of the others this Trp residue undergoes the greatest change in environ- were significantly protective. **, p  0.001. B, correlation between RBC ment. Quenching studies by both iodide and acrylamide (Table lysis and lipid hydroperoxide scavenging by peptides. The percentage of II) indicate that the Trp residues become less exposed to the lysis obtained with the different peptides (Fig. 3A) and the lipid hy- droperoxide content in the LDL fraction of Watanabe plasma after quenchers (both KI and acrylamide) in the presence of lipid. incubation with the peptides (Fig. 2A) are plotted for each peptide. 3F-1 The K values suggest that the Trp environment is similar in SV 3 14 (), 3F-2 (E), 3F (ƒ), 3F (‚), 4F (). The regression coefficient was all the peptides in aqueous and lipid-bound states. not calculated, since data from separate experiments are combined. Right Angled Light Scattering Studies—Turbidity clarifica- tion (Fig. 4) studies indicate that all of the peptides clear considered to be 100%. The 3F peptides and 4F by themselves multilamellar vesicles of POPC. The peptide 3F-2 appears to did not cause any lysis, even at a concentration of 10 M, clear turbidity due to POPC multilamellar vesicles as rapidly 3 14 whereas 2 M 18L was able to almost completely lyse the cells as the 3F and 3F peptides. However, the peptide 3F-1 ap- (98% of the level of Triton X-100). We also tested these peptides pears to clarify the least, suggesting that this peptide forms for their ability to inhibit 18L-mediated lysis (Fig. 3A). There is larger complexes than the other peptides. Calculation of t , the a range of potencies exhibited by these peptides in inhibiting time taken for reducing the light scattering intensity by 50%, 18L-mediated hemolysis. The results indicate that 3F was from Fig. 4 indicates that 3F-1 and 3F-2 have the fastest rates not as effective in inhibiting 18L-mediated red cell lysis, of clarification (Table I), in agreement with these two peptides whereas 2F and 3F caused about 40% inhibition of 18L lysis. having increased surface pressure and increased HPLC reten- The peptides that were most effective in inhibiting 18L-induced tion times. cell lysis were 3F-1, 3F-2, and 4F. Both 3F-1 and 4F inhibited Circular Dichroism Studies—Ellipticity at 222 nm was de- lysis by 65%, whereas 3F-2 was the most potent among the four pendent on the concentration of the peptide for all of the 3F peptides in inhibiting 18L-induced hemolysis (90% inhibition). analogs. Fig. 5 shows that there is an increase in ellipticity due A plot of reduction of lipid hydroperoxides by peptides (Fig. 2A) to self-association, up to 300 M for all four peptides, beyond versus 18L-induced percent RBC lysis (Fig. 3A) showed a re- which it appears to plateau. The concentration for attaining markable correlation (Fig. 3B). 50% of maximal helicity varies for these peptides and indicates HPLC Retention Times and Surface Pressure—Table I lists the differences in self-association of peptides. However, at 400 the HPLC retention time on a C-18 column and exclusion M, all four peptides seem to have similar helicities (40 – 60%). pressure from an EPC monolayer. Among the four 3F analogs, The arrangement of hydrophobic residues on the nonpolar face 26514 Anti-inflammatory Class A Amphipathic Peptides TABLE II Fluorescence data for 3F peptides The values given are an average of three experiments, 10 M peptides in each case. Peptide/DMPC ratio was 1:5 (mol/mol) in each case. AU, arbitrary units; K , the Stern-Volmer quenching constant. SV I K (KI) K max max sv sv(acrylamide) Peptide PBS DMPC PBS DMPC PBS DMPC PBS DMPC 1 1 nm AU M M 3F-1 351 340 1880 1809 7.05 5.18 27.12 14.72 3F-2 349 338 1739 5331 9.74 5.19 36.64 18.64 3F 351 337 1237 1849 7.14 4.72 32.01 16.39 3F 349 336 1727 2102 12.4 5.65 35.81 16.06 bic environment. However, if the DPH-PC in the PC bilayer is exposed to water, the fluorescence is quenched. This lipid can therefore be used to determine the extent of water penetration into the membrane. We determined the extent to which the ad- dition of the peptides increased the quenching of the DPH-PC probe embedded in LUVs of POPC. We compared the properties of the four 3F peptides with that of 4F. Peptide 4F caused the greatest quenching of the fluorescence of DPH-PC (Fig. 6). At low peptide concentrations, the peptides 3F-2 and 3F-1 were 3 14 the next most potent. The peptides 3F and 3F were not able to efficiently quench the DPH fluorescence. The presence of the aromatic groups in clusters could change the packing of the lipid acyl chains to allow for a greater penetration of water, which would cause quenching of the probe. Although one would not expect many water molecules to penetrate into the mem- brane, water molecules may gain access transiently, or the access into the membrane could occur as a result of water FIG.4. Peptides solubilize POPC vesicles. 50 M POPC was used. binding to the peptides, as has been observed in other systems Peptides were added at 1:1 (mol/mol lipid/peptide) concentrations, and (35). Alternatively, the peptides themselves may cause the clarification was followed for 30 min at 25 °C. POPC, no peptide added 3 14 quenching of the DPH-PC. (solid line), 3F-1 (), 3F-2 (E), 3F (ƒ), 3F (‚), 4F (). The results described in the legend to Fig. 6 were obtained by adding peptide to LUVs, maintaining a lipid/peptide ratio of 5 or greater. We have also measured the relative fluorescence intensity of DPH-PC in lipid particles that were solubilized by peptide (results not shown). This was done by incubating MLVs of POPC/DPH-PC (400:1) with a solution of one of the peptides at a lipid/peptide molar ratio of either 1:1 or 2:1. These mix- tures were maintained at 25 °C for up to 10 h. The resulting fluorescence of the solubilized lipid was compared with that of an identical mixture without peptide. Unlike the case described above, of peptide added to LUVs, these solubilized lipid micelles exhibited fluorescence intensi- ties that were similar to but slightly greater than that of the lipid alone. There was little difference in the fluorescence emis- sion from the mixtures containing the different peptides. These results suggest that in the case of the LUVs, the lipid was not converted into a micellar form in the time and peptide concen- trations used. We interpret the small effect of the peptides on the DPH-PC fluorescence in the solubilized micelles as being a FIG.5. Concentration-dependent helicity of peptides. Concen- 3 consequence of the peptide interacting with only a small frac- trations ranging from 10 to 400 M were used. , 3F-1; E, 3F-2; ƒ,3F ; tion of the total lipid that is present at the edge of the discoidal ‚,3F ; , 4F. The vertical lines correspond to the concentrations at which the half-maximal helix content is attained. a, 3F-2; b, 4F; c and micelles. The biological effects presented in the present work, 3 14 d, 3F-1 and 3F ; e,3F . of inhibition of hemolysis (Fig. 2) and of monocyte chemotaxis (Fig. 3), do not involve the use of complexes in the form of appears to determine helicity in solution. All of the peptides discoidal micelles. possess similar helicity values (50 10%) at two different We have also analyzed the quenching of DPH-PC in LUVs at concentrations (100 and 400 M) in the presence of POPC or low peptide concentrations, where solubilization of the lipid 50% trifluoroethanol (results not shown). These results indi- would be minimal. We have calculated the dependence of cate that the potential to form an amphipathic helical structure quenching on peptide concentration for each of the peptides in for all of these sequences is similar, and the secondary struc- the range between 0 and 3.5 M peptide (Fig. 6). The slopes of ture in the presence of lipid is also similar. these plots are given in Table III. The peptide 4F is clearly the Fluorescence Studies with DPH-PC—DPH-PC has been pre- most potent in promoting quenching of the DPH-PC fluores- viously used to study the penetration of water into phospholip- cence, followed by 3F-2. 3F-1 has a somewhat higher slope than 3 14 ids bilayers (34). This lipid, when incorporated into POPC 3F or 3F , but the difference is not statistically significant vesicles, produces increased fluorescence due to the hydropho- because of the markedly nonlinear dependence of quenching on Anti-inflammatory Class A Amphipathic Peptides 26515 FIG.6. Quenching of DPH-PC fluorescence by peptides. , 3 14 3F-1; E, 3F-2; ƒ,3F ; ‚,3F ; , 4F. Data shown are the average of duplicates. Relative intensities have an error of 0.005. The experi- ment was repeated with independently prepared liposomes and peptide solutions and exhibited the same relative order among the different peptides but with somewhat greater error between experiments than the precision within an experiment. An analysis is given in Table III. TABLE III Analysis of the DPH-PC quenching data Slope (relative fluorescence intensity/ Regression Peptide M peptide) coefficient (r ) 3F-1 0.017 0.002 0.96 3F-2 0.0221 0.0003 0.99 3F 0.015 0.003 0.96 3F 0.015 0.001 0.99 4F 0.0247 0.0003 0.99 the concentration of 3F-1, resulting in an increase in the error of the estimation of the slope at this range of very low peptide concentrations. FIG.7. The red edge excitation shift. Shown is the effect of exci- REES—REES is the shift in the wavelength of maximum tation on the emission spectra of intrinsic fluorescence of Trp of the fluorescence emission toward higher wavelengths, caused by a 3 14 peptides 3F-1 (), 3F-2 (E), 3F (ƒ), 3F (‚), and 4F () and peptide- 3 14 change in the excitation wavelength toward the red edge of the EPC complexes 3F-1 (f), 3F-2 (), 3F (), 3F (Œ), and 4F (). A, 3F-1 3 14 and 3F-2 in buffer and EPC complexes; B,3F and 3F ; C, 4F. Repro- absorption band. This phenomenon occurs with fluorophores in ducible results were obtained in a separate experiment. a motionally restricted environment in which the solvent dipole reorientation is slower than the lifetime of the excited state. Despite all of these similarities, there is a significant difference REES has been used as a measure of the rigidity of the envi- in the biological potency of these peptides with respect to inhi- ronment of Trp in proteins/peptides (36). There can also be bition of 18L-induced hemolysis (Fig. 3A) and anti-inflamma- other factors such as proton exchange in the excited state or tory action (Fig. 2, A and B). These facts suggest that some other electrostatic effects that influence REES (37). However, relatively subtle differences among these peptides in their in- for a series of peptides with similar amino acid compositions, teraction with membranes account for the larger differences in conformation, and Trp emission wavelength, as with most pro- the observed anti-inflammatory properties. teins (36), the magnitude of REES is dependent on local mo- It is clear that the arrangement of aliphatic and aromatic tion. Despite the fact that the Trp environment is similar in all amino acids on the hydrophobic face of the helix has significant of the peptides studied here, there is a clear difference between effects on biological properties of Class A amphipathic helical the more biologically active analogs 3F-2, 3F-1, or 4F that do peptides. In the present study, we introduce two new 3F ana- not exhibit REES either in lipid or buffer (Fig. 7, A and C) and 3 14 logs, 3F-1 and 3F-2, with the aromatic groups being either at the less active analogs 3F and 3F that exhibit a significant the hydrophobic/hydrophilic interface of the amphipathic helix effect. In the presence of LUVs of egg PC, the change in emis- 14 3 in 3F-1 or at the center of the hydrophobic face in 3F-2. This sion  at  is about 20 nm for 3F and 14 nm for 3F . max ex arrangement of aromatic residues changes the angle of the These results suggest that the Trp is motionally restricted in 3 14 nonpolar face. In both 3F-1 and 3F-2, the angle is 180°, 3F and 3F , whereas it is not in 4F, 3F-1, and 3F-2. whereas it is 160° in the other peptides (Fig. 1A). Thus, the DISCUSSION surface area of the nonpolar face is much greater in these two The series of peptides studied here have many structural new analogs. CD results in 50% trifluoroethanol and in the similarities. All of the 3F peptides have identical amino acid presence of POPC indicate that all of the sequences have sim- compositions. In the presence of lipid, all of these peptides have ilar potential to form amphipathic helical structures (results similar secondary structures, and they all have similar hydro- not shown). Concentration-dependent CD studies in aqueous phobicities. They insert in a similar fashion into monolayers of solution indicate that all of the peptides self-associate, al- EPC as shown with surface pressure measurements and into though at different concentrations (Fig. 5). bilayers as evidenced by the fluorescence emission from Trp. ApoA-I has been shown to inhibit LDL-induced monocyte 26516 Anti-inflammatory Class A Amphipathic Peptides chemotaxis (6, 7). The seeding molecules in LDL, hydroper- scavenging lipid hydroperoxide from LDL. In addition, we have oxyeicosatetraenoic acid and HPODE, induce the formation of seen that an amphipathic helical peptide from apoJ sequence, specific oxidized phospholipids, which cause monocyte chemo- which does not possess either Tyr or Trp, is able to inhibit lipid taxis. Removal of these lipid hydroperoxides by HDL and hydroperoxide-induced monocyte chemotaxis. This suggests that in the peptides studied here, other factors such as the apoA-I inhibits this process (6, 7). In these studies, we have used HPODE-PAPC, which directly generates the oxidized environment of the nonpolar face may play a major role, al- though the ability of the Trp and Tyr residues to act as free phospholipids that induce monocyte chemotaxis. In this report, radical scavengers cannot be ignored presently. we show that HPODE-PAPC-induced monocyte chemotaxis is There are two physical properties that vary in the same way inhibited by both 3F-2 and 3F-1. These two peptides and 4F as the biological activity. These are the REES (Fig. 7) and the were better inhibitors than HDL, whereas 3F was relatively quenching of DPH-PC (Fig. 6). The REES suggests that the ineffective and 3F had no significant effect (Fig. 2B). This more active peptides 3F-1, 3F-2, and 4F are in a less confor- suggests that the arrangement of aromatic amino acids on the mationally restricted environment, and the increased quench- nonpolar face plays an important role in the complex process of ing of DPH-PC indicates that these active peptides, particu- inhibiting oxidized phospholipid-induced monocyte chemo- larly 4F (Fig. 6), allow a greater penetration of water molecules taxis. It is interesting to note that although 3F clarifies lipid into the hydrophobic milieu of the membrane. We are currently similarly to other peptides and possesses a relatively higher working on further defining the factors among these peptides HPLC retention time and a higher monolayer exclusion pres- that account for the differences in both these physical proper- sure than 3F-1 and 3F-2, it is not as effective in inhibiting ties as well as their different biological effects. However, one monocyte chemotaxis. The peptides that were effective in in- feature that strikes us is the difference in “cross-sectional hibiting oxidized phospholipid-induced monocyte chemotaxis shape” of these peptides when modeled as -helices with the also effectively removed lipid hydroperoxides from LDL (Fig. 3 14 Lys residues in a snorkel position (Fig. 1B). The peptide helices 2A). The peptides 3F and 3F were relatively ineffective in would lie near the lipoprotein (or peptide-lipid) interface with removing the LDL-lipid hydroperoxides from WHHL rabbit the hydrophobic face of the helix facing toward the lipid (bot- plasma and in inhibiting oxidized phospholipid-induced mono- tom of the molecular models shown in Fig. 1, A and B). The cyte chemotaxis. Preliminary studies suggest that oral admin- molecular models show that the less active peptides (2F, 3F , istration of D-4F to LDL receptor null and apoE null mice and 3F ) have a relatively small cross-sectional area of the causes the rapid formation of small HDL-like particles contain- helix nonpolar face so that they are wedge-shaped. In contrast, ing peptide, cholesterol, apoA-I, and paraoxonase (15). These peptides 3F-1, 3F-2, and 4F have a more cylindrical shape, results coupled with the previous observations that class A because the nonpolar face of the helix is larger (Fig. 1B). We amphipathic helical peptide associates with HDL (16) suggest suggest that these peptides affect the lateral pressure profile that the peptides exhibit anti-inflammatory properties in lipid- and molecular packing of the lipoprotein particle surface in associated form. different ways. We have earlier used inhibition of 18L-induced red cell he- The concept of lateral pressure profile was developed by molysis (22) to screen the properties of class A peptides. The Cantor (42) and used initially to explain the action of general inhibition of superoxide production and neutrophil degranula- anesthetics but has been shown to have applicability to may tion by class A peptides reported by us earlier (38) and 18L- aspects of membrane structure and function (43). In this de- induced lysis presented here suggest that the mechanism of scription, the lateral interactions in the phospholipid-water inhibition of hemolysis and inhibition of HPODE-induced interface vary with the depth of penetration of a molecule such monocyte chemotaxis may all be related to stabilization of as a peptide into the phospholipid milieu. Presently, the rela- membrane by class A peptide analogs. We had shown in a tive depths of penetration of wedge- and cylinder-shaped pep- dose-response study that 50 g of 18A was able to inhibit tides are not known. However, relative to the wedge-shaped 18L-induced hemolysis by 50% (22), a result similar to the peptides, the cylinder- shaped peptides seem to perturb the present observations with 2F. In the present group of peptides, lipid acyl chain packing more because of their larger helix we have compared the relative potency of class A peptides to nonpolar face. This concept of looser acyl chain packing with inhibit 18L-induced hemolysis. Analogs 4F, 3F-2, and 3F-1 the active peptides is supported by the results of red edge were significantly effective in inhibiting 18L-induced hemoly- excitation shift experiments (Fig. 7) and fluorescence studies 3 14 sis, whereas 2F, 3F , and 3F were not as effective (Fig. 3A). It with DPH-PC, which show the quenching of DPH-PC fluores- is interesting to note that there is a close relationship between cence by the presence of water molecules in the hydrophobic the ability of the model peptides to inhibit 18L-induced RBC milieu (Fig. 6). In contrast, the effect of wedge-shaped peptide lysis and their ability to lower LDL lipid hydroperoxides (Fig. molecules appears to be mainly on the interfacial region, since 3B). Molecular modeling studies (Fig. 1B) show that whereas they have a small nonpolar face (Fig. 1B), thus causing mini- the three less effective peptides show a similar wedge-shaped mal effects on the packing of the lipid acyl chains. cross-section, the active peptides possess hydrophobic residues In summary, our studies show that factors other than overall that would give a cylindrical shape to the peptides. hydrophobicity are important for the anti-inflammatory prop- Tryptophan and tyrosine residues, when in close proximity erties of class A peptides. Furthermore, for the first time, these to a lipid interface, are known to engage in electron transfer studies differentiate the role of aromatic hydrophobic residues reactions that enable them to function as free radical scaven- and aliphatic residues in altering the biological activity of a gers capable of quenching hydroperoxide-initiated lipid peroxi- class A amphipathic helical peptide. The effects of interaction dation (39 – 41). Since these peptides possess Tyr and Trp res- of class A amphipathic helical peptides on lipid packing depend idues, they may play a role as scavengers of lipid upon the nature of hydrophobic residues on the nonpolar face. hydroperoxides. The presence of multiple phenylalanine resi- The presence of water in the milieu of the hydrophobic region of dues in these peptides may further enhance the stability of the the peptide-lipid complex could determine the extent of anti- tyrosyl radical (39). In the active 3F-2 analog, Tyr and Trp are one turn apart on the nonpolar face. However, Trp and Tyr are not in close proximity either in the helical wheel representation 2 M. Navab, G. M. Anantharamaiah, and A. M. Fogelman, unpub- or in the linear sequence of 4F, yet this peptide is effective in lished results. Anti-inflammatory Class A Amphipathic Peptides 26517 17. Datta, G., Chaddha, M., Hama, S., Navab, M., Fogelman, A. M., Garber, D. W., atherogenicity of class A amphipathic helical peptides. The Mishra, V. K., Epand, R. M., Epand, R. F., Lund-Katz, S., Phillips, M. C., presence of water molecules in turn could allow for the transfer Segrest, J. P., and Anantharamaiah, G. M. (2001) J. Lipid Res. 42, 1096 –1104 of oxidized lipids from the LDL surface to peptide-containing 18. Anantharamaiah, G. M., Datta, G., and Garber, D. W. (2001) Current Sci. 81, particles, thereby rendering LDL less effective in inducing 53– 65 monocyte chemotaxis, an important step in the initiation of 19. Garber, D. W., Handattu, S., Aslan, I., Datta, G., Chaddha, M., Anantharamaiah, G. M. (2003) Atherosclerosis 168, 229 –237 atherogenesis. 20. Anantharamaiah, G. M., Jones, J. L., Brouillette, C. G., Schmidt, B-H. C., Hughes, T. A., Bhown, A. S., Segrest, J. P. (1985) J. Biol. Chem. 260, Acknowledgment—We thank Martin Jones for help with molecular 10248 –10255 modeling. 21. Epand, R. M., Shai, Y., Segrest, J. P., Anantharamaiah, G. M. 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Aromatic Residue Position on the Nonpolar Face of Class A Amphipathic Helical Peptides Determines Biological Activity

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 24, Issue of June 18, pp. 26509 –26517, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Aromatic Residue Position on the Nonpolar Face of Class A Amphipathic Helical Peptides Determines Biological Activity* Received for publication, December 30, 2003, and in revised form, April 8, 2004 Published, JBC Papers in Press, April 8, 2004, DOI 10.1074/jbc.M314276200 Geeta Datta‡, Raquel F. Epand§, Richard M. Epand§, Manjula Chaddha‡, Matthew A. Kirksey‡, ¶ ¶ David W. Garber‡, Sissel Lund-Katz , Michael C. Phillips , Susan Hama , Mohamad Navab , Alan M. Fogelman , Mayakonda N. Palgunachari‡, Jere P. Segrest‡, and G. M. Anantharamaiah‡** From the ‡Departments of Medicine, Biochemistry and Molecular Genetics and the Atherosclerosis Research Unit, University of Alabama at Birmingham, Birmingham, Alabama 35294, the §Department of Biochemistry, McMaster University Health Sciences Centre, Hamilton, Ontario L8N 3Z5, Canada, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, and the Department of Medicine and the Atherosclerosis Research Unit, UCLA Cardiology, UCLA, Los Angeles, California 90095 of intense investigation in many laboratories because of its The apolipoprotein A-I mimetic peptide 4F (Ac-DW- FKAFYDKVAEKFKEAF-NH ), with four Phe residues on strong antiatherogenic properties (1–7). Two major theories on the nonpolar face of the amphipathic -helix, is strongly the role of HDL in the development of atherosclerosis are as anti-inflammatory, whereas two 3F analogs (3F and follows: (i) reverse cholesterol transport (HDL transfers choles- 3F ) are not. To understand how changes in helix non- terol from peripheral tissues to the liver for excretion) (5), and polar face structure affect function, two additional 3F (ii) the lipid oxidation theory (it has been shown that HDL analogs, Ac-DKLKAFYDKVFEWAKEAF-NH (3F-1) and 2 traps oxidized lipids that are responsible for the production of Ac-DKWKAVYDKFAEAFKEFL-NH (3F-2), were de- cytokines) (7). Several enzymes (notably platelet-activating signed using the same amino acid composition as 3F factor acetylhydrolase and paraoxonase) that are present on and 3F . The aromatic residues in 3F-1 and 3F-2 are the surface of HDL are responsible for destroying the biological near the polar-nonpolar interface and at the center of activity of oxidized lipids (6). Different strategies have been the nonpolar face of the helix, respectively. Like 4F, but used by several laboratories to support both theories (8, 9). 3 14 in contrast to 3F and 3F , these peptides effectively However, recently, it has been suggested that the two mecha- inhibited lytic peptide-induced hemolysis, oxidized nisms may be two sides of the same coin (10). phospholipid-induced monocyte chemotaxis, and scav- We have used apoA-I mimetic class A amphipathic helical enged lipid hydroperoxides from low density lipopro- peptides to understand the functions of apoA-I that may be tein. High pressure liquid chromatography retention playing a primary role in inhibiting atherosclerosis (10, 11). times and monolayer exclusion pressures indicated that With this approach, we have shown that either intraperitonial there is no direct correlation of peptide function with administration of the class A amphipathic helical peptide 5F or lipid affinity. Fluorescence studies suggested that, al- the oral administration of peptide 4F synthesized from all-D- though the peptides bind phospholipids similarly, the amino acids inhibit atherosclerosis in dyslipidemic mouse mod- Trp residue in 4F, 3F-1, and 3F-2 is less motionally re- 3 14 stricted than in 3F and 3F . Based on these results and els without altering plasma cholesterol levels (12, 13). Further- molecular modeling studies, we propose that the ar- more, investigations using apoA-I peptides support the concept rangement of aromatic residues in class A amphipathic that their ability to inhibit atherosclerosis is related to their helical molecules regulates entry of reactive oxygen spe- ability to remove “seeding molecules” (reactive lipid hydroper- cies into peptide-phospholipid complexes, thereby re- oxides formed by the oxidation of phospholipids containing ducing the extent of monocyte chemotaxis, an important arachidonic acid) from the low density lipoprotein (LDL) sur- step in atherosclerosis. face, thereby inhibiting free radical oxidation chain reactions and the subsequent propagation and release of cytokines (14). Preliminary studies suggest that oral administration of D-4F to Human apolipoprotein A-I (apoA-I), the major protein com- LDL receptor null and apoE null mice causes the rapid forma- ponent of high density lipoproteins (HDL), has been the subject tion and clearance of small HDL-like particles containing pep- tide, cholesterol, and apoA-I and paraoxonase, capable of con- * This work was supported in part by National Institutes of Health verting proinflammatory HDL into anti-inflammatory HDL Grants PO1 HL34343, 30568, and 22633 and Canadian Institutes of (15). These results, coupled with the previous observations that Health Research Grant MT-7654. M. N., S. H., A. M. F., and G. M. A. are principals in Bruin Pharma, a start-up biotech company. The costs class A amphipathic helical peptide associates with HDL (16), of publication of this article were defrayed in part by the payment of suggest that the peptide is active in a lipid-associated form. page charges. This article must therefore be hereby marked “advertise- However, not all class A amphipathic helical peptides are ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this anti-inflammatory to the same extent (17, 18). Using a series of fact. ** To whom correspondence should be addressed: 1808 7th Ave. S., peptides in which the hydrophobicity of the nonpolar face was DREB 640, Department of Medicine, UAB Medical Center, Birming- increased by substituting Ala, Leu, or Val residues with Phe ham, AL 35294. Tel.: 205-934-1494; E-mail: [email protected]. residues, we have shown that greater hydrophobicity per se is The abbreviations used are: apoA-I, human apolipoprotein A-I; not sufficient to increase the anti-inflammatory properties of a Ac, acetyl; DPH-PC, 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexade- canoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero- 3-phosphocholine; EPC, egg phosphatidylcholine; HDL, high density lipoprotein(s); HPLC, high pressure liquid chromatography; HPODE, 3-phosphocholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- hydroperoxyoctadecadienoic acid; LDL, low density lipoprotein(s); LUV, choline; RBC, red blood cells; REES, red edge excitation shift; MLV, large unilamellar vesicle; PAPC, 1-palmitoyl-2-arachidonyl-sn-glycero- multilamellar vesicle. This paper is available on line at http://www.jbc.org 26509 This is an Open Access article under the CC BY license. 26510 Anti-inflammatory Class A Amphipathic Peptides FIG.1. A, helical wheel representation of the parent molecule 2F, the antiatherogenic 4F, and the four 3F analogs. The wheel is projected along the axis of the helix from N to C terminus with the hydrophobic side facing downward. The primary structure is given above each wheel diagram. The amino acid composition of each of these peptides is the same, but the sequences are different. The plus and minus signs denote the charge on the amino acids at neutral pH. Red denotes an acidic residue, blue denotes a basic residue, and boldface black denotes aromatic residues. The line shows the interface between the hydrophobic face (lower part) and the hydrophilic face (upper half) as given by us earlier (20). Although the hydrophobic faces appear to be similar in each peptide, their abilities to allow water to penetrate into the lipid are different. B, space-filling models of the parent peptide, 2F, the antiatherogenic peptide, 4F, and the four 3F peptides. These models were created using silicon graphics; the lysine 3 14 residues were snorkeled (22), and the models were energy-minimized. The view down the long axis of the helix shows that 2F, 3F , and 3F are wedge shaped, whereas 4F, 3F-1, and 3F-2 are more cylindrical in shape. The blue color in the space-filling model denotes a nitrogen atom, whereas the red color denotes an oxygen atom. class A amphipathic helical peptide (17). The addition of one alogs of 3F. In one analog, with the amino acid sequence Ac- more Phe to the nonpolar face of Ac-18A-NH (2F) either at DKLKAFYDKVFEWAKEAF-NH (referred to as 3F-1), the al- 2 2 3 14 position 3 (3F )orat14(3F ) (Fig. 1) resulted in peptides that iphatic residues are at the center of the nonpolar face, and were no longer able to inhibit LDL-induced monocyte chemo- aromatic residues are near the interfacial Lys residues, tactic activity (17). However, a peptide analog containing four whereas in another 3F analog with the amino acid sequence Phe residues on the nonpolar face (4F) was the most active in Ac-DKWKAVYDKFAEAFKEFL-NH (referred to as 3F-2), the this series in inhibiting LDL-induced monocyte chemotactic -electron-containing aromatic residues are at the center of the activity, whereas the extensively studied 2F analog was not as nonpolar face (see Fig. 1A). The primary sequence and wheel 3 14 effective. Furthermore, 2F did not inhibit atherosclerosis in a projection of these peptides along with those of 2F, 3F ,3F , diet-induced mouse model of atherosclerosis (19), whereas 4F and 4F for comparison are shown in Fig. 1A. The space-filling was highly effective (18). The further addition of Phe residues models of these peptides with the Lys residues snorkeled (20, (5F, 6F, and 7F) did not enhance this property. We therefore 21) are shown in Fig. 1B. We have studied the physical-chem- hypothesized that a particular arrangement of aromatic resi- ical and anti-inflammatory properties of these two peptides 3 14 dues on the nonpolar face is more important in producing a and 4F in comparison with 3F and 3F . The results show that peptide with maximum anti-inflammatory properties than is although all of the peptides have similar abilities to form an the overall hydrophobicity of the peptide. amphipathic helix, the two peptides 3F-1 and 3F-2 are biolog- To investigate this, we have synthesized two additional an- ically active, similar to 4F. This effect appears to be related to Anti-inflammatory Class A Amphipathic Peptides 26511 method with a Protein Technologies PS-3 automatic peptide synthe- the position of aromatic residues with respect to the surface of sizer using the procedures described previously (12, 17). Peptides were lipoprotein particles, which affect molecular arrangements purified using a preparative HPLC system (Beckman Gold), and the near the interface and alter the lateral interactions within the purity of the peptides was determined by mass spectral analysis and particle. Changes in these interactions could promote the analytical HPLC. transfer of oxidized lipids (found on the surface of LDL parti- Circular Dichroism—CD spectra were recorded on an AVIV model cles) to HDL-like particles, thus making LDL less effective in 215 spectropolarimeter using a quartz cell with a 0.1-cm path length at 25 °C. Peptide solutions in phosphate-buffered saline were used at inducing monocyte chemotaxis, an important step in the initi- concentrations ranging from 10 to 400 M. The effect of lipid binding on ation of atherogenesis. the secondary structure of the peptide was studied using peptide-lipid complexes (1:10 mol/mol) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phos- EXPERIMENTAL PROCEDURES phocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine Materials—Tissue culture materials and other reagents were ob- (DMPC). Since the results were similar, we have reported results with tained from sources previously described (6, 7). Lipids used in these POPC in order to be able to compare with the other studies. These investigations were obtained from Avanti Polar Lipids (Alabaster, AL) complexes were prepared by adding the appropriate volume of peptide except for the fluorescent phospholipid 2-(3-(diphenylhexatrienyl)pro- solution to POPC multilamellar vesicles (MLVs). The MLVs were pre- panoyl)-1- hexadecanoyl-sn-glycero-3- phosphocholine (DPH-PC) that pared by dissolving a known amount of lipid in chloroform and slowly was obtained from Molecular Probes, Inc. (Eugene, OR). Acetonitrile, removing the solvent by evaporation under a thin stream of nitrogen. dimethylformamide, chloroform, methanol, ammonium formate, formic Residual solvent was removed by storing the lipid film under vacuum acid, and acetic acid, were all obtained from Fisher, and acetic anhy- overnight. An appropriate volume of buffer was added to hydrate the dride diisopropylethylamine and pipyridine were from Sigma. Amino thin lipid film, which was then vortexed. The lipid-peptide complexes acids and condensing reagent O-benzotriazole-N,N,N,N-tetramethy- were prepared by adding the required volume of peptide solutions to luronium hexafluorophosphate and rink amide resin were purchased give the desired lipid to peptide molar ratio. CD measurements were from Advanced Chem Tech (Louisville, KY) and Peptides International also done in the presence of 50% trifluoroethanol. The program CD (Louisville, KY). Spectra Deconvolution (CDNN), version 2.1, was used to calculate the Hemolysis Assay—The hemolysis assay was carried out as described secondary structure content (28). by Tytler et al. (22). Briefly, red blood cells (RBCs) were collected from Right Angle Light Scattering Measurements—Association of these EDTA-treated human blood by centrifugation. The cell pellet was peptides with POPC was determined by following the dissolution of washed three times with phosphate-buffered saline to remove plasma POPC MLVs by right angle light scattering using an SLM 8000C and the buffy coat. A suspension of 1% erythrocytes in phosphate- photon counting spectrofluorometer as described (29). POPC MLVs buffered saline with or without peptide was incubated at 37 °C for 10 were prepared by evaporating a solution of POPC (Avanti Polar) under min. The suspension was centrifuged at 16,000  g for 3 min. Hemolysis nitrogen and hydrating the lipid film with phosphate-buffered saline was measured as hemoglobin content (absorbance at 540 nm) of the (pH 7.4). The sample containing 105 M POPC and an equimolar supernatant. Base-line hemolysis was the hemolysis of RBCs incubated amount of peptide was maintained at 25 °C and continuously stirred. with phosphate-buffered saline. Hemoglobin released by 0.1% Triton Turbidity clarification was monitored at 400 nm for 30 min. Complete X-100 was taken to be 100% lysis. Inhibition of lysis by peptides was dissolution of POPC vesicles was achieved by the addition of Triton measured by incubating RBCs with a 10 M concentration of peptides X-100 to a final concentration of 1 mM. and adding 2 M 18L to the cell suspension. Hemolysis was expressed as Surface Pressure Measurements—Monolayer exclusion pressure meas- a percentage of the Triton X-100 lysis. urements give the affinity of the peptides for a lipid-water interface. The Monocyte Chemotaxis Assay—The procedure detailed by Navab et al. procedure of Phillips and Krebs (30, 31) was followed using egg phosphati- (6) was followed. Briefly, human aortic endothelial cells and smooth dylcholine (EPC) as per their procedure. An insoluble monolayer of EPC muscle cells were isolated. Human aortic smooth muscle cells were was spread at the air-water interface in a Teflon dish at room tempera- grown in microtiter well plates until confluence, and the human aortic ture to give an initial surface pressure ( ) in the range of 5– 45 microne- endothelial cells were added. Cocultures were treated with 20 gof wtons/m. A solution of peptides in phosphate-buffered saline containing 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1 1.5 M guanidinium chloride was carefully injected in to the subphase to g/ml of hydroperoxyeicosatetraenoic acid (HPODE) in the absence or give a final concentration of 50 g/dl. The guanidinium chloride was presence of HDL (50 g of cholesterol) or peptides (66 M)for8h.The diluted in the subphase to a final concentration of 1mM to allow the supernatants were collected and assayed for chemotactic activity. peptides to renature. The subphase was stirred continuously, and the Determination of Lipid Hydroperoxide Content—Lipid hydroperox- increase in EPC monolayer surface pressure () was recorded until a ides were measured colorimetrically by the FOX2 assay (23). The ability steady state value was obtained. The value of the initial surface pressure of the peptides to remove lipid hydroperoxides from lipoproteins was ( ) at which the peptides no longer penetrate the EPC monolayer (i.e. the assessed by incubating each of them with plasma from Watanabe rab- exclusion pressure ( )) was calculated by extrapolating the  versus e i bits. Plasma from these rabbits is known to possess lipid hydroperox- linear regression fit to   0 micronewtons/m. ides (24, 25). 1 ml of plasma was incubated with peptide at a final Intrinsic Tryptophan Fluorescence—Fluorescence spectra of both concentration of 44 M for1hat37 °C. The lipoproteins were then peptide and peptide-lipid complexes were recorded at room temperature separated on two Superose 6 (Amersham Biosciences) columns in tan- with excitation at 295 nm using a SPF-500 spectrofluorometer. These dem using the Bio-Rad BioLogic Duoflow system. 0.5-ml fractions were studies were done using DMPC as the lipid of choice in order to compare collected, and absorbance was monitored at 280 nm. The peaks were them with earlier published results of other peptides (17). The relative analyzed for hydroperoxide content. Briefly, 900 l of the FOX reagent fluorescence intensity was measured in ratio mode that corrects for containing 300 M FeSO 7H O, 120 M xylenol orange, 25 mM H SO , time dependent fluctuations in lamp intensity. Quenching of trypto- 4 2 2 4 and 4.4 mM butylated hydroxytoluene in methanol was added to 100 l phan fluorescence by potassium iodide and acrylamide was determined of the sample and incubated in the dark for 30 min, and absorbance was by adding aliquots of stock solutions of potassium iodide (4 M) and measured at 560 nm. The values were compared with a standard of acrylamide (4 M) to a 2.7-ml solution of either peptide or peptide-lipid cumene hydroperoxide to quantitate the hydroperoxide content. complexes (32). Stock solutions of potassium iodide contained 1 mM Lipoproteins—Low density lipoprotein (d  1.019 g/ml) and high sodium thiosulfate (Na S O ) to prevent the formation of I . Inner filter 2 2 3 3 density lipoproteins (d  1.063–1.21 g/ml) were isolated using the corrections were applied for acrylamide quenching (32). Emission spec- procedure by Havel et al. (26) from normal volunteers under the proto- tra were taken after each addition, and emission intensity at  was max col approved by Human Research Subject Protection committees of the determined. Peak emission intensity was used for all calculations. The University of Alabama at Birmingham and UCLA. In some cases, quenching data were analyzed according to the Stern-Volmer equation, butylated hydroxytoluene (20 mM in ethanol) was added to freshly isolated plasma at a concentration of 20 M, and the lipoprotein was F /F  1  K Q (Eq. 1) 0 sv separated by fast protein liquid chromatography using the methods previously described (27). The LDL, HDL, and lipoprotein-deficient where F and F represent the fluorescence in the absence and presence serum had endotoxin levels below 20 pg/ml, which is well below the of the quencher, and [Q] is the concentration of the quencher. K is the SV threshold needed for induction of monocyte adhesion or chemotactic Stern-Volmer constant. activity. The concentrations of lipoproteins reported in this study are Large Unilamellar Vesicles (LUVs)—Lipid films were made by dis- based on protein content. solving appropriate amounts of lipid in a mixture of chloroform/meth- Peptide Synthesis—Peptides were synthesized by the solid phase anol (2:1 (v/v)) and dried in a test tube under nitrogen to deposit the 26512 Anti-inflammatory Class A Amphipathic Peptides lipid as a thin film on the wall of the tube. Final traces of solvent were removed in a vacuum chamber attached to a liquid nitrogen trap for 2–3 h. Dried films were kept under argon gas at 30 °C if not used imme- diately. Films were hydrated with buffer, vortexed extensively at room temperature, and then subjected to five cycles of freezing and thawing. The homogeneous lipid suspensions were then further processed by 10 passes through two stacked 0.1-m polycarbonate filters (Nucleopore Filtration Products, Pleasanton, CA) in a barrel extruder (Lipex Bi- omembranes, Vancouver, BC) at room temperature. LUVs were kept on ice and used within a few hours of preparation. Lipid phosphorus was determined by the method of Ames (33). Fluorescence Spectroscopy Studies with DPH-PC—Peptide solutions were prepared in buffer containing 10 mM Hepes, 0.14 M NaCl, 0.1 mM EDTA, pH 7.4. The peptide concentrations were determined by absorb- ance at 280 nm against buffer as reference. Films containing POPC/ DPH-PC (400:1) were prepared from a solution in chloroform/methanol (2:1). The film was hydrated in buffer. 100-nm diameter LUVs were then made by extrusion as described previously. Fluorescence was measured in quartz cuvettes at 37 °C, in an SLM Aminco Bowman Series II spectrofluorometer, with an excitation of 355 nm and slits set to 4-nm bandwidth in both excitation and emission. The fluorescence emission of 2 ml of 50 M LUVs in the same buffer was recorded. Then small aliquots of a peptide solution, also in the corresponding buffer, were added successively, and the fluorescence emission was recorded after each addition. The emission maximum was found at 431 nm. Red Edge Excitation Shift (REES)—The emission spectra of trypto- phan were measured at 25 °C in an SLM Aminco Bowman Series II luminescence spectrofluorometer. The excitation wavelength was var- ied every 5 nm between 280 and 310 nm, and the emission spectra were recorded over 310 –350 nm three times and averaged. Measurements were done in quartz cuvettes containing 2 ml of buffer (10 mM NaH PO , 0.14 M NaF, 1 mM EDTA, pH 7.4). Aliquots of solutions of 2 4 peptides in buffer were added to the cuvette to a final concentration of 10 M. Measurements were repeated with the addition of 100 M EPC LUVs. To reduce scattered light intensity, excitation polarization was set to 90° and emission to 0°, with 4-nm bandwidth. Spectra were corrected for instrumental factors, and controls were subtracted. Peak emission wavelengths were recorded. Statistical Analysis—Statistical analysis was performed as previ- ously described (18) using one-way analysis of variance, and signifi- FIG.2. A, ability of peptides to scavenge lipid hydroperoxides from cance was defined as p  0.05. Post hoc Tukey tests were performed. LDL. The effect of the 3F peptides and 4F on lipid hydroperoxide Peptides were compared with base line and with each other. levels in LDL was determined. 1 ml of plasma from Watanabe rabbits (used as a source of oxidized LDL) was incubated with peptides at a RESULTS final concentration of 44 M, and the lipoproteins were separated on Superose 6 columns using fast protein liquid chromatography. The Removal of Hydroperoxides from LDL and Inhibition of hydroperoxide concentration in the LDL fraction was determined PAPC HPODE-induced Monocyte Chemotaxis—We previ- using cumene hydroperoxide as a standard. The data was analyzed ously reported that the anti-inflammatory properties of these using one-way analysis of variance, and the significance value is marked. Compared with the control samples, 4F, 3F-2, 3F-1, and 3F peptides correlate well with their ability to scavenge seeding reduced the concentration of lipid hydroperoxide significantly. The molecules from the LDL surface, inhibiting LDL-induced reduction seen by 4F, 3F-2, and 3F-1 appeared to be statistically more monocyte chemotaxis (6, 7). To determine their ability to scav- significant (**, p  0.001) than that seen by 3F (†, p  0.001). enge hydroperoxides from LDL, the peptides were incubated However, 3F is not significantly different from the control. B, inhi- with plasma from Watanabe rabbits that are known to have bition of oxidized lipid-induced monocyte chemotaxis. The effect of the 3F peptides on PAPC HPODE-induced monocyte chemotaxis oxidized LDL (24, 25). We used 4F as a positive control peptide was determined at 66 M concentration of peptide. 20 g of PAPC was to compare the present results with the previously published added together with 1 g/ml of HPODE to cocultures of human artery results (17). The lipoproteins were separated on Superose 6 wall cells as described previously (7, 8), and human HDL at 50 g/ml columns, and the fractions were tested for hydroperoxide con- cholesterol or no addition was made to the cocultures (No Addition), or the peptides were also added at 66 M.After8hofincubation, tent. As shown in Fig. 2A, 3F-2, 3F-1, and 4F significantly supernatants were collected and assayed for monocyte chemotactic reduced hydroperoxides by about 75% from about 190 M to activity using standard neuroprobe chambers. The data are mean 3 14 about 50 M, whereas 3F and 3F had relatively small effects S.D. of the number of migrated monocytes in nine fields for triplicate on the hydroperoxide concentrations in LDL. samples. *, p  0.05 compared with PAPC HPODE alone. LDL-induced monocyte chemotaxis is due to the oxidation of LDL phospholipids containing arachidonic acid. This generates effects. This parallels their ability to scavenge lipid hydroper- specific biologically active phospholipids, which in turn cause oxide molecules from LDL. There was no statistical difference the artery wall cells to produce the potent monocyte chemoat- between the activities of 3F-2 and 3F-1. However, the peptide tractant MCP-1, which in turn induces monocyte chemotaxis 4F was the most effective and significantly different from 3F-1 (6, 7). We compared the ability of these peptides to inhibit the and 3F-2. monocyte chemotactic activity resulting from the incubation of Inhibition of 18L-induced Red Cell Lysis by 3F Peptide An- human artery wall cells with the arachidonic acid containing alogs—Our earlier results showed that apoA-I and apoA-I mi- phospholipid, PAPC, and the fatty acid hydroperoxide, metic peptides are not themselves lytic at low peptide/lipid HPODE. The effect of these peptides on PAPC-HPODE-in- ratios, and furthermore they stabilize membranes from lysis duced monocyte chemotaxis (Fig. 2B) showed that, whereas induced by the lytic peptide 18L (22). We tested the 3F peptides both 3F-2 and 3F-1 effectively inhibited PAPC-HPODE-in- for lytic activity using RBCs and compared them to 2F and 4F 3 14 duced monocyte chemotaxis, 3F and 3F showed only small (control peptides). The lysis caused by 0.1% Triton X-100 was Anti-inflammatory Class A Amphipathic Peptides 26513 TABLE I Comparison of some physical properties of peptides a b c Peptide HPLC retention time Exclusion pressure t min micronewtons/m s 3F-1 20.8 37 59 3F-2 20.0 33 65 3d 3F 21.0 38 44 14d 3F 21.2 39 34 4F 22.0 40 65 HPLC retention time is the time taken for the peptide to elute from a C-18 Vydac column using a gradient of 25–58% acetonitrile in water containing 0.1% trifluoroacetic acid. Exclusion pressure from an EPC monolayer. The reproducibility of these values is 1 micronewton/m. t is the time taken for reducing the light scattering intensity by 50%, calculated from light scattering studies presented in Fig. 4. Data from Ref. 17. peptide 3F-2 has the shortest retention time on a C-18 reversed phase HPLC column, and 3F has the highest retention time. Exclusion pressure from an EPC monolayer was also the least for the peptide 3F-2, and highest for 3F (Table I). The results suggest that among the four 3F analogs, the presence of aro- matic amino acids at the center of the nonpolar face decreases the lipid-associating ability (binding to reversed phase HPLC column containing C-18 acyl chain) and EPC monolayer exclu- sion pressure. The addition of another Phe to produce 4F mar- ginally increases the retention time and exclusion pressure 3 14 compared with 3F and 3F . Tryptophan Fluorescence and Quenching Studies—In the peptide 3F-2, Trp is at the center of the -helix nonpolar face, whereas it is at the polar-nonpolar interface in the other three peptide analogs. All of the peptides show a significant shift in fluorescence maximum ( ) with DMPC, indicating that all max of the four peptides associate similarly with lipid (Table II). FIG.3. A, effect of the peptides on 18L-induced hemolysis. The effect The shift in  from solution state to lipid-associated state for of the peptides on 18L-induced RBC lysis was studied by coincubating max 3 14 RBCs with 18L and the control peptide, 4F, and the four 3F analogs. 3F and 3F are 14 and 13 nm, respectively, whereas the other Hemolysis was measured as hemoglobin content in the lysate (absorb- two peptides shifted less (11 nm). Peptide 3F-2 shows the ance of the lysate at 540 nm). Hemolysis by 0.1% Triton X-100 was largest increase in fluorescence intensity on binding to DMPC. taken as 100% lysis. The data are expressed as a percentage of Triton This appears to be due to the position of Trp at the center of the X-100 lysis. The data were analyzed by one-way analysis of variance. All of the peptides protect the RBCs from 18L-induced lysis. However, nonpolar face. From aqueous phase to lipid-associated form, the protection by 3F is not statistically significant. All of the others this Trp residue undergoes the greatest change in environ- were significantly protective. **, p  0.001. B, correlation between RBC ment. Quenching studies by both iodide and acrylamide (Table lysis and lipid hydroperoxide scavenging by peptides. The percentage of II) indicate that the Trp residues become less exposed to the lysis obtained with the different peptides (Fig. 3A) and the lipid hy- droperoxide content in the LDL fraction of Watanabe plasma after quenchers (both KI and acrylamide) in the presence of lipid. incubation with the peptides (Fig. 2A) are plotted for each peptide. 3F-1 The K values suggest that the Trp environment is similar in SV 3 14 (), 3F-2 (E), 3F (ƒ), 3F (‚), 4F (). The regression coefficient was all the peptides in aqueous and lipid-bound states. not calculated, since data from separate experiments are combined. Right Angled Light Scattering Studies—Turbidity clarifica- tion (Fig. 4) studies indicate that all of the peptides clear considered to be 100%. The 3F peptides and 4F by themselves multilamellar vesicles of POPC. The peptide 3F-2 appears to did not cause any lysis, even at a concentration of 10 M, clear turbidity due to POPC multilamellar vesicles as rapidly 3 14 whereas 2 M 18L was able to almost completely lyse the cells as the 3F and 3F peptides. However, the peptide 3F-1 ap- (98% of the level of Triton X-100). We also tested these peptides pears to clarify the least, suggesting that this peptide forms for their ability to inhibit 18L-mediated lysis (Fig. 3A). There is larger complexes than the other peptides. Calculation of t , the a range of potencies exhibited by these peptides in inhibiting time taken for reducing the light scattering intensity by 50%, 18L-mediated hemolysis. The results indicate that 3F was from Fig. 4 indicates that 3F-1 and 3F-2 have the fastest rates not as effective in inhibiting 18L-mediated red cell lysis, of clarification (Table I), in agreement with these two peptides whereas 2F and 3F caused about 40% inhibition of 18L lysis. having increased surface pressure and increased HPLC reten- The peptides that were most effective in inhibiting 18L-induced tion times. cell lysis were 3F-1, 3F-2, and 4F. Both 3F-1 and 4F inhibited Circular Dichroism Studies—Ellipticity at 222 nm was de- lysis by 65%, whereas 3F-2 was the most potent among the four pendent on the concentration of the peptide for all of the 3F peptides in inhibiting 18L-induced hemolysis (90% inhibition). analogs. Fig. 5 shows that there is an increase in ellipticity due A plot of reduction of lipid hydroperoxides by peptides (Fig. 2A) to self-association, up to 300 M for all four peptides, beyond versus 18L-induced percent RBC lysis (Fig. 3A) showed a re- which it appears to plateau. The concentration for attaining markable correlation (Fig. 3B). 50% of maximal helicity varies for these peptides and indicates HPLC Retention Times and Surface Pressure—Table I lists the differences in self-association of peptides. However, at 400 the HPLC retention time on a C-18 column and exclusion M, all four peptides seem to have similar helicities (40 – 60%). pressure from an EPC monolayer. Among the four 3F analogs, The arrangement of hydrophobic residues on the nonpolar face 26514 Anti-inflammatory Class A Amphipathic Peptides TABLE II Fluorescence data for 3F peptides The values given are an average of three experiments, 10 M peptides in each case. Peptide/DMPC ratio was 1:5 (mol/mol) in each case. AU, arbitrary units; K , the Stern-Volmer quenching constant. SV I K (KI) K max max sv sv(acrylamide) Peptide PBS DMPC PBS DMPC PBS DMPC PBS DMPC 1 1 nm AU M M 3F-1 351 340 1880 1809 7.05 5.18 27.12 14.72 3F-2 349 338 1739 5331 9.74 5.19 36.64 18.64 3F 351 337 1237 1849 7.14 4.72 32.01 16.39 3F 349 336 1727 2102 12.4 5.65 35.81 16.06 bic environment. However, if the DPH-PC in the PC bilayer is exposed to water, the fluorescence is quenched. This lipid can therefore be used to determine the extent of water penetration into the membrane. We determined the extent to which the ad- dition of the peptides increased the quenching of the DPH-PC probe embedded in LUVs of POPC. We compared the properties of the four 3F peptides with that of 4F. Peptide 4F caused the greatest quenching of the fluorescence of DPH-PC (Fig. 6). At low peptide concentrations, the peptides 3F-2 and 3F-1 were 3 14 the next most potent. The peptides 3F and 3F were not able to efficiently quench the DPH fluorescence. The presence of the aromatic groups in clusters could change the packing of the lipid acyl chains to allow for a greater penetration of water, which would cause quenching of the probe. Although one would not expect many water molecules to penetrate into the mem- brane, water molecules may gain access transiently, or the access into the membrane could occur as a result of water FIG.4. Peptides solubilize POPC vesicles. 50 M POPC was used. binding to the peptides, as has been observed in other systems Peptides were added at 1:1 (mol/mol lipid/peptide) concentrations, and (35). Alternatively, the peptides themselves may cause the clarification was followed for 30 min at 25 °C. POPC, no peptide added 3 14 quenching of the DPH-PC. (solid line), 3F-1 (), 3F-2 (E), 3F (ƒ), 3F (‚), 4F (). The results described in the legend to Fig. 6 were obtained by adding peptide to LUVs, maintaining a lipid/peptide ratio of 5 or greater. We have also measured the relative fluorescence intensity of DPH-PC in lipid particles that were solubilized by peptide (results not shown). This was done by incubating MLVs of POPC/DPH-PC (400:1) with a solution of one of the peptides at a lipid/peptide molar ratio of either 1:1 or 2:1. These mix- tures were maintained at 25 °C for up to 10 h. The resulting fluorescence of the solubilized lipid was compared with that of an identical mixture without peptide. Unlike the case described above, of peptide added to LUVs, these solubilized lipid micelles exhibited fluorescence intensi- ties that were similar to but slightly greater than that of the lipid alone. There was little difference in the fluorescence emis- sion from the mixtures containing the different peptides. These results suggest that in the case of the LUVs, the lipid was not converted into a micellar form in the time and peptide concen- trations used. We interpret the small effect of the peptides on the DPH-PC fluorescence in the solubilized micelles as being a FIG.5. Concentration-dependent helicity of peptides. Concen- 3 consequence of the peptide interacting with only a small frac- trations ranging from 10 to 400 M were used. , 3F-1; E, 3F-2; ƒ,3F ; tion of the total lipid that is present at the edge of the discoidal ‚,3F ; , 4F. The vertical lines correspond to the concentrations at which the half-maximal helix content is attained. a, 3F-2; b, 4F; c and micelles. The biological effects presented in the present work, 3 14 d, 3F-1 and 3F ; e,3F . of inhibition of hemolysis (Fig. 2) and of monocyte chemotaxis (Fig. 3), do not involve the use of complexes in the form of appears to determine helicity in solution. All of the peptides discoidal micelles. possess similar helicity values (50 10%) at two different We have also analyzed the quenching of DPH-PC in LUVs at concentrations (100 and 400 M) in the presence of POPC or low peptide concentrations, where solubilization of the lipid 50% trifluoroethanol (results not shown). These results indi- would be minimal. We have calculated the dependence of cate that the potential to form an amphipathic helical structure quenching on peptide concentration for each of the peptides in for all of these sequences is similar, and the secondary struc- the range between 0 and 3.5 M peptide (Fig. 6). The slopes of ture in the presence of lipid is also similar. these plots are given in Table III. The peptide 4F is clearly the Fluorescence Studies with DPH-PC—DPH-PC has been pre- most potent in promoting quenching of the DPH-PC fluores- viously used to study the penetration of water into phospholip- cence, followed by 3F-2. 3F-1 has a somewhat higher slope than 3 14 ids bilayers (34). This lipid, when incorporated into POPC 3F or 3F , but the difference is not statistically significant vesicles, produces increased fluorescence due to the hydropho- because of the markedly nonlinear dependence of quenching on Anti-inflammatory Class A Amphipathic Peptides 26515 FIG.6. Quenching of DPH-PC fluorescence by peptides. , 3 14 3F-1; E, 3F-2; ƒ,3F ; ‚,3F ; , 4F. Data shown are the average of duplicates. Relative intensities have an error of 0.005. The experi- ment was repeated with independently prepared liposomes and peptide solutions and exhibited the same relative order among the different peptides but with somewhat greater error between experiments than the precision within an experiment. An analysis is given in Table III. TABLE III Analysis of the DPH-PC quenching data Slope (relative fluorescence intensity/ Regression Peptide M peptide) coefficient (r ) 3F-1 0.017 0.002 0.96 3F-2 0.0221 0.0003 0.99 3F 0.015 0.003 0.96 3F 0.015 0.001 0.99 4F 0.0247 0.0003 0.99 the concentration of 3F-1, resulting in an increase in the error of the estimation of the slope at this range of very low peptide concentrations. FIG.7. The red edge excitation shift. Shown is the effect of exci- REES—REES is the shift in the wavelength of maximum tation on the emission spectra of intrinsic fluorescence of Trp of the fluorescence emission toward higher wavelengths, caused by a 3 14 peptides 3F-1 (), 3F-2 (E), 3F (ƒ), 3F (‚), and 4F () and peptide- 3 14 change in the excitation wavelength toward the red edge of the EPC complexes 3F-1 (f), 3F-2 (), 3F (), 3F (Œ), and 4F (). A, 3F-1 3 14 and 3F-2 in buffer and EPC complexes; B,3F and 3F ; C, 4F. Repro- absorption band. This phenomenon occurs with fluorophores in ducible results were obtained in a separate experiment. a motionally restricted environment in which the solvent dipole reorientation is slower than the lifetime of the excited state. Despite all of these similarities, there is a significant difference REES has been used as a measure of the rigidity of the envi- in the biological potency of these peptides with respect to inhi- ronment of Trp in proteins/peptides (36). There can also be bition of 18L-induced hemolysis (Fig. 3A) and anti-inflamma- other factors such as proton exchange in the excited state or tory action (Fig. 2, A and B). These facts suggest that some other electrostatic effects that influence REES (37). However, relatively subtle differences among these peptides in their in- for a series of peptides with similar amino acid compositions, teraction with membranes account for the larger differences in conformation, and Trp emission wavelength, as with most pro- the observed anti-inflammatory properties. teins (36), the magnitude of REES is dependent on local mo- It is clear that the arrangement of aliphatic and aromatic tion. Despite the fact that the Trp environment is similar in all amino acids on the hydrophobic face of the helix has significant of the peptides studied here, there is a clear difference between effects on biological properties of Class A amphipathic helical the more biologically active analogs 3F-2, 3F-1, or 4F that do peptides. In the present study, we introduce two new 3F ana- not exhibit REES either in lipid or buffer (Fig. 7, A and C) and 3 14 logs, 3F-1 and 3F-2, with the aromatic groups being either at the less active analogs 3F and 3F that exhibit a significant the hydrophobic/hydrophilic interface of the amphipathic helix effect. In the presence of LUVs of egg PC, the change in emis- 14 3 in 3F-1 or at the center of the hydrophobic face in 3F-2. This sion  at  is about 20 nm for 3F and 14 nm for 3F . max ex arrangement of aromatic residues changes the angle of the These results suggest that the Trp is motionally restricted in 3 14 nonpolar face. In both 3F-1 and 3F-2, the angle is 180°, 3F and 3F , whereas it is not in 4F, 3F-1, and 3F-2. whereas it is 160° in the other peptides (Fig. 1A). Thus, the DISCUSSION surface area of the nonpolar face is much greater in these two The series of peptides studied here have many structural new analogs. CD results in 50% trifluoroethanol and in the similarities. All of the 3F peptides have identical amino acid presence of POPC indicate that all of the sequences have sim- compositions. In the presence of lipid, all of these peptides have ilar potential to form amphipathic helical structures (results similar secondary structures, and they all have similar hydro- not shown). Concentration-dependent CD studies in aqueous phobicities. They insert in a similar fashion into monolayers of solution indicate that all of the peptides self-associate, al- EPC as shown with surface pressure measurements and into though at different concentrations (Fig. 5). bilayers as evidenced by the fluorescence emission from Trp. ApoA-I has been shown to inhibit LDL-induced monocyte 26516 Anti-inflammatory Class A Amphipathic Peptides chemotaxis (6, 7). The seeding molecules in LDL, hydroper- scavenging lipid hydroperoxide from LDL. In addition, we have oxyeicosatetraenoic acid and HPODE, induce the formation of seen that an amphipathic helical peptide from apoJ sequence, specific oxidized phospholipids, which cause monocyte chemo- which does not possess either Tyr or Trp, is able to inhibit lipid taxis. Removal of these lipid hydroperoxides by HDL and hydroperoxide-induced monocyte chemotaxis. This suggests that in the peptides studied here, other factors such as the apoA-I inhibits this process (6, 7). In these studies, we have used HPODE-PAPC, which directly generates the oxidized environment of the nonpolar face may play a major role, al- though the ability of the Trp and Tyr residues to act as free phospholipids that induce monocyte chemotaxis. In this report, radical scavengers cannot be ignored presently. we show that HPODE-PAPC-induced monocyte chemotaxis is There are two physical properties that vary in the same way inhibited by both 3F-2 and 3F-1. These two peptides and 4F as the biological activity. These are the REES (Fig. 7) and the were better inhibitors than HDL, whereas 3F was relatively quenching of DPH-PC (Fig. 6). The REES suggests that the ineffective and 3F had no significant effect (Fig. 2B). This more active peptides 3F-1, 3F-2, and 4F are in a less confor- suggests that the arrangement of aromatic amino acids on the mationally restricted environment, and the increased quench- nonpolar face plays an important role in the complex process of ing of DPH-PC indicates that these active peptides, particu- inhibiting oxidized phospholipid-induced monocyte chemo- larly 4F (Fig. 6), allow a greater penetration of water molecules taxis. It is interesting to note that although 3F clarifies lipid into the hydrophobic milieu of the membrane. We are currently similarly to other peptides and possesses a relatively higher working on further defining the factors among these peptides HPLC retention time and a higher monolayer exclusion pres- that account for the differences in both these physical proper- sure than 3F-1 and 3F-2, it is not as effective in inhibiting ties as well as their different biological effects. However, one monocyte chemotaxis. The peptides that were effective in in- feature that strikes us is the difference in “cross-sectional hibiting oxidized phospholipid-induced monocyte chemotaxis shape” of these peptides when modeled as -helices with the also effectively removed lipid hydroperoxides from LDL (Fig. 3 14 Lys residues in a snorkel position (Fig. 1B). The peptide helices 2A). The peptides 3F and 3F were relatively ineffective in would lie near the lipoprotein (or peptide-lipid) interface with removing the LDL-lipid hydroperoxides from WHHL rabbit the hydrophobic face of the helix facing toward the lipid (bot- plasma and in inhibiting oxidized phospholipid-induced mono- tom of the molecular models shown in Fig. 1, A and B). The cyte chemotaxis. Preliminary studies suggest that oral admin- molecular models show that the less active peptides (2F, 3F , istration of D-4F to LDL receptor null and apoE null mice and 3F ) have a relatively small cross-sectional area of the causes the rapid formation of small HDL-like particles contain- helix nonpolar face so that they are wedge-shaped. In contrast, ing peptide, cholesterol, apoA-I, and paraoxonase (15). These peptides 3F-1, 3F-2, and 4F have a more cylindrical shape, results coupled with the previous observations that class A because the nonpolar face of the helix is larger (Fig. 1B). We amphipathic helical peptide associates with HDL (16) suggest suggest that these peptides affect the lateral pressure profile that the peptides exhibit anti-inflammatory properties in lipid- and molecular packing of the lipoprotein particle surface in associated form. different ways. We have earlier used inhibition of 18L-induced red cell he- The concept of lateral pressure profile was developed by molysis (22) to screen the properties of class A peptides. The Cantor (42) and used initially to explain the action of general inhibition of superoxide production and neutrophil degranula- anesthetics but has been shown to have applicability to may tion by class A peptides reported by us earlier (38) and 18L- aspects of membrane structure and function (43). In this de- induced lysis presented here suggest that the mechanism of scription, the lateral interactions in the phospholipid-water inhibition of hemolysis and inhibition of HPODE-induced interface vary with the depth of penetration of a molecule such monocyte chemotaxis may all be related to stabilization of as a peptide into the phospholipid milieu. Presently, the rela- membrane by class A peptide analogs. We had shown in a tive depths of penetration of wedge- and cylinder-shaped pep- dose-response study that 50 g of 18A was able to inhibit tides are not known. However, relative to the wedge-shaped 18L-induced hemolysis by 50% (22), a result similar to the peptides, the cylinder- shaped peptides seem to perturb the present observations with 2F. In the present group of peptides, lipid acyl chain packing more because of their larger helix we have compared the relative potency of class A peptides to nonpolar face. This concept of looser acyl chain packing with inhibit 18L-induced hemolysis. Analogs 4F, 3F-2, and 3F-1 the active peptides is supported by the results of red edge were significantly effective in inhibiting 18L-induced hemoly- excitation shift experiments (Fig. 7) and fluorescence studies 3 14 sis, whereas 2F, 3F , and 3F were not as effective (Fig. 3A). It with DPH-PC, which show the quenching of DPH-PC fluores- is interesting to note that there is a close relationship between cence by the presence of water molecules in the hydrophobic the ability of the model peptides to inhibit 18L-induced RBC milieu (Fig. 6). In contrast, the effect of wedge-shaped peptide lysis and their ability to lower LDL lipid hydroperoxides (Fig. molecules appears to be mainly on the interfacial region, since 3B). Molecular modeling studies (Fig. 1B) show that whereas they have a small nonpolar face (Fig. 1B), thus causing mini- the three less effective peptides show a similar wedge-shaped mal effects on the packing of the lipid acyl chains. cross-section, the active peptides possess hydrophobic residues In summary, our studies show that factors other than overall that would give a cylindrical shape to the peptides. hydrophobicity are important for the anti-inflammatory prop- Tryptophan and tyrosine residues, when in close proximity erties of class A peptides. Furthermore, for the first time, these to a lipid interface, are known to engage in electron transfer studies differentiate the role of aromatic hydrophobic residues reactions that enable them to function as free radical scaven- and aliphatic residues in altering the biological activity of a gers capable of quenching hydroperoxide-initiated lipid peroxi- class A amphipathic helical peptide. The effects of interaction dation (39 – 41). Since these peptides possess Tyr and Trp res- of class A amphipathic helical peptides on lipid packing depend idues, they may play a role as scavengers of lipid upon the nature of hydrophobic residues on the nonpolar face. hydroperoxides. The presence of multiple phenylalanine resi- The presence of water in the milieu of the hydrophobic region of dues in these peptides may further enhance the stability of the the peptide-lipid complex could determine the extent of anti- tyrosyl radical (39). In the active 3F-2 analog, Tyr and Trp are one turn apart on the nonpolar face. However, Trp and Tyr are not in close proximity either in the helical wheel representation 2 M. Navab, G. M. Anantharamaiah, and A. M. Fogelman, unpub- or in the linear sequence of 4F, yet this peptide is effective in lished results. Anti-inflammatory Class A Amphipathic Peptides 26517 17. Datta, G., Chaddha, M., Hama, S., Navab, M., Fogelman, A. M., Garber, D. W., atherogenicity of class A amphipathic helical peptides. The Mishra, V. K., Epand, R. M., Epand, R. F., Lund-Katz, S., Phillips, M. C., presence of water molecules in turn could allow for the transfer Segrest, J. P., and Anantharamaiah, G. M. (2001) J. Lipid Res. 42, 1096 –1104 of oxidized lipids from the LDL surface to peptide-containing 18. Anantharamaiah, G. M., Datta, G., and Garber, D. W. (2001) Current Sci. 81, particles, thereby rendering LDL less effective in inducing 53– 65 monocyte chemotaxis, an important step in the initiation of 19. Garber, D. W., Handattu, S., Aslan, I., Datta, G., Chaddha, M., Anantharamaiah, G. M. (2003) Atherosclerosis 168, 229 –237 atherogenesis. 20. Anantharamaiah, G. M., Jones, J. L., Brouillette, C. G., Schmidt, B-H. C., Hughes, T. A., Bhown, A. S., Segrest, J. P. (1985) J. Biol. Chem. 260, Acknowledgment—We thank Martin Jones for help with molecular 10248 –10255 modeling. 21. Epand, R. M., Shai, Y., Segrest, J. P., Anantharamaiah, G. M. 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