Lectin -Defensin, Is a Θ Retrocyclin, an Antiretroviral

Retrocyclin, an Antiretroviral θ-Defensin, Is a Lectin Wei Wang, Alexander M. Cole, Teresa Hong, Alan J. Waring and Robert I. Lehrer This information is current as of September 29, 2021. J Immunol 2003; 170:4708-4716; ; doi: 10.4049/jimmunol.170.9.4708 http://www.jimmunol.org/content/170/9/4708 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Retrocyclin, an Antiretroviral ␪-Defensin, Is a Lectin1

Wei Wang,* Alexander M. Cole,* Teresa Hong,* Alan J. Waring,*† and Robert I. Lehrer2*‡

␪-Defensins are circular octadecapeptides that contain an internal tridisulfide ladder. Because retrocyclin-1, an ancestral hominid ␪-defensin, can protect human cells in vitro from infection by T- and M-tropic strains of HIV-1, we used surface plasmon resonance techniques to study its binding to glycoproteins and glycolipids implicated in HIV-1 entry. Retrocyclin-1 bound with high affinity to gp120 (Kd, 35.4 nM), CD4 (Kd, 31 nM), and galactosylceramide (Kd, 24.1 nM). Neither a circular form of retrocyclin without its tridisulfide ladder nor its ␤-hairpin precursor with these disulfides intact bound gp120 or CD4 effectively. Retrocyclin also bound fetuin, an extensively glycosylated protein, with high affinity, but it did not bind nonglycosylated gp120 or BSA. However, retrocyclin did bind to a neoglycoprotein, BSA, with covalently attached sugar residues. Experiments with glycosidase- treated fetuin, gp120, and CD4 revealed that both O-linked and N-linked sugars were used as binding sites. In a panel of retrocyclin variants, binding to immobilized gp120 and CD4 were highly correlated to each other and to the peptide’s ability to protect human PBMCs from infection by HIV-1. Although small, cysteine-rich antimicrobial peptides with lectin-like properties Downloaded from exist in plants, ␪-defensins are the first such molecules to be identified in vertebrates. Retrocyclin’s ability to recognize and bind carbohydrate-containing surface molecules is integrally related to its ability to protect cells from HIV-1 infection. The Journal of Immunology, 2003, 170: 4708–4716.

efensins are small, cysteine-rich antimicrobial peptides lenced by a premature stop codon mutation (11). Our phylogenetic

whose roles in innate host defense include exerting di- studies indicate that the mutation that silenced Homo sapiens ␪-de- http://www.jimmunol.org/ D rect antibacterial, antifungal, and antiviral activity (1) fensin production occurred after the orangutan and hominid lin- and enhancing certain adaptive immune responses (2–4). The ␣- eages separated (T. Nguyen, A. Cole, R. I. Lehrer, unpublished and ␤-defensins, the two most widely distributed mammalian de- observations). fensin subfamilies, have been studied in detail by many investiga- These experiments, undertaken to examine how retrocyclin-1 tors (5). conferred protection against HIV-1, provided compelling evidence Recently, three representatives of a third defensin subfamily, that the molecules acted as lectins. Although antimicrobial pep- ␪-defensins, were purified from the leukocytes (6, 7) and bone tides are well known to “recognize” membranes populated by an- marrow (8) of the rhesus macaque (Macaca mulatta). ␪-Defensins ionic phospholipids, this is the first demonstration that antimicro- are circular, tetracyclic peptides with three cysteine disulfide bonds bial peptides from a vertebrate can recognize sugar molecules. by guest on September 29, 2021 connecting their antiparallel ␤-sheets (6, 9). Their 18 residues come from two “demidefensin” precursors, each of which contrib- Materials and Methods utes nine residues (including three cysteines) to the mature peptide Peptides (6). Exhaustive head-to-tail splicing renders the mature octade- Table I shows the peptides used in this study. Twelve peptides were iden- capeptide circular, and an internal tridisulfide ladder renders it tet- tical with retrocyclin-1 (RC-100), except for a single tyrosine substitution racyclic. Because nonapeptide elements from identical or different that replaced a noncysteine residue. RC-101 differed from RC-100 only by demidefensin precursors can be spliced, this imparts an element of containing a single arginine to lysine substitution that facilitates fluorescent structural diversity to ␪-defensins without necessitating commen- labeling of RC101. Enantio peptides contained D-amino acids exclusively. In retro peptides, the residues were synthesized in reverse order to their surate genome expansion (7, 8). normal counterparts. Synthesis, purification, disulfide formation, and cy- Evidence appeared recently that human ␣-defensins human neu- clization of all peptides were as previously described (8, 11). Reduction of trophil peptide-1, -2, and -3 are expressed by human CD8 T cells RC-100 by DTT and its subsequent alkylation with iodoacetimide (IAA)3 and may prevent the development of clinical AIDS (10). Not only were performed as previously described for protegrins (12). are ␪-defensin genes the evolutionary descendants of ␣-defensin Antiviral activity genes, but ␪-defensin peptides can also protect cells from in vitro infection by HIV-1. Human bone marrow expresses mRNA ho- Two HIV-1 strains were studied (11): JR-CSF, an M-tropic strain that uses the CCR5 chemokine coreceptor for entry, and IIIB, a T-tropic strain that ␪ ϩ mologous to the rhesus -defensins; however, its translation is si- uses CXCR4. Primary CD4 lymphocytes, freshly purified from HIV-1- seronegative donors, were stimulated with a CD3-CD8 bispecific mAb and were maintained in RPMI 1640 with 10% FCS, 2 mM glutamine, 100 U of Departments of *Medicine and †Pediatrics and ‡Molecular Biology Institute, Univer- penicillin/ml, 10 ␮g of streptomycin/ml, and 50 U of IL-2/ml. After 7 days, sity of California, Los Angeles, CA 90095 when 98% of the cells coexpressed CD3 and CD4, they were infected with Received for publication October 31, 2002. Accepted for publication February HIV-1. 24, 2003. HIV-1 stocks were prepared and handled as previously described (11) 6 ϩ The costs of publication of this article were defrayed in part by the payment of page and were thawed just before use. About 10 primary CD4 PBMCs/ml charges. This article must therefore be hereby marked advertisement in accordance were resuspended at 37°C in fresh RPMI 1640 medium containing 10% with 18 U.S.C. Section 1734 solely to indicate this fact. FCS and 50 U of IL-2. Test peptides (final concentration, 20 ␮g/ml) were 1 This work was supported by the National Institutes of Health (Grants AI22839 and AI37945 to R.I.L. and Grant AI52017 to A.M.C.). 3 Abbreviations used in this paper: IAA, iodoacetimide; SPR, surface plasmon reso- 2 Address correspondence and reprint requests to Dr. Robert I. Lehrer, Department of nance; PNGase F, peptide N-glycosidase F; POPC, palmitoyl-oleoyl-phosphatidyl Medicine, Center for Healtlh Science 37-062, 10833 LeConte Avenue, University of choline; BSA-galactosamide, BSA-2-amido-2-deoxy-D-galactose; OX, oxidized; California, Los Angeles, CA 90095-1690. E-mail address: [email protected] RTD-1, rhesus ␪-defensin-1.

Table I. Peptides used in this studya

Sequence

b Number Name Comments Massav 13568101113151618 RC-100 Retrocyclin-1 Net charge ϩ4 1918.43 GICRC ICGRG ICRCI CGR 3 RC-101 K9-retrocyclin-1 R K 1890.42 GICRC ICGKG ICRCI CGR 3 RC-102 Y6-retrocyclin-1 I Y 1968.45 GICRC YCGRG ICRCI CGR 3 RC-103 Y15-retrocyclin-1 I Y 1968.45 GICRC ICGRG ICRCY CGR 3 RC-104 Y2-retrocyclin-1 I Y 1968.45 GYCRC ICGRG ICRCI CGR 3 RC-105 Y11-retrocyclin-1 I Y 1968.45 GICRC ICGRG YCRCI CGR 3 RC-106 Y4-retrocyclin-1 R Y 1882.39 GICYC ICGRG ICRCI CGR 3 RC-107 Y9-retrocyclin-1 R Y 1882.39 GICIC ICGYG ICRCI CGR 3 RC-108 Y13-retrocyclin-1 R Y 1882.39 GICIC ICGRG ICYCI CGR 3 RC-109 Y18-retrocyclin-1 R Y 1882.39 GICIC ICGRG ICRCI CGY RC-111 Retro-retrocyclin-1 All L 18 3 1 1918.43 RGCIC RCIGR GCICR CIG RC-112 Enantio-retrocyclin-1 All D 1 3 18 1918.43 GICRC ICGRG ICRCI CGR RC-113 Enantio-RC-101 All D 1 3 18 1890.42 GICRC ICGKG ICRCI CGR 3 3 RC-114 K9,Y15-retrocyclin-1 R K, I Y 1940.43 GICRC ICGKG ICRCY CGR 3 RC-115 Y10-retrocyclin-1 G Y 2024.56 GICRC ICGRY ICRCI CGR 3 RC-116 Y2-retrocyclin-1 I Y 1968.45 RYICR CICGR GICRC ICG 3 Downloaded from RC-117 Y8-retrocyclin-1 G Y 2024.56 GICRC ICYRG ICRCI CGR 3 RC-118 Y17-retrocyclin-1 G Y 2024.56 GICRC ICGRG ICRCI CYR a The sequence of each synthetic 18-residue precursor, starting from its N terminus, is shown. Any residues that differ from their counterpart in retrocyclin-1 (RC-100) are in boldface. The enantio peptides contained D-amino acids exclusively. In the retro peptides, the order of the amino acids was reversed, relative to that of their L-amino acid counterparts. All peptides were oxidized and rendered cyclic prior to testing. b ϭ Massav average mass. http://www.jimmunol.org/ added, and HIV-1 was introduced 3 h later at a multiplicity of infection of a rate of 10 ␮l/min. Each binding study was done in triplicate, and the chip 0.01 tissue culture-infective dose at 50% per cell. After three additional was regenerated between trials by washing it with 0.1 M HCl and 0.1 hours, these cells were washed twice and transferred to 24-well plates. M NaOH. Each well contained 5 ϫ 105 cells in 2 ml of medium Ϯ 20 ␮g/ml of a retrocyclin. Every 3 days, we removed 1 ml of supernatant for HIV-1 p24 Special reagents Ag quantitation by ELISA (DuPont Pharmaceuticals, Wilmington, DE) and Recombinant human soluble CD4 (full-length 45-kDa glycosylated ectodo- replaced it with 1 ml of fresh medium containing the retrocyclin peptide. main) was produced by ExpresSFϩ (Protein Sciences, Meriden, CT) Spo- Only the results from day 9 supernatants are presented in this report. doptera frugiperda cells in serum-free medium and was purchased from Protein Sciences. Glycosylated HIV-1 gp120 (LAV) envelope glycoprotein

Surface plasmon resonance (SPR) experiments by guest on September 29, 2021 (catalog number 2003-LAV) was also purchased from Protein Sciences. SPR experiments were performed using CM5 sensor chips on a BiaCore Recombinant HIV-1 glycosylated HXB2 gp41 viral protein, produced in 2000 system (BiaCore, Piscataway, NJ). HBS-EP running buffer (pH 7.4) Pichia pastoris, contained amino acids 546–682 of the gp41 ectodomain contained 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% and was from Advanced Biotechnologies (Columbia, MD). HIV-1 SF2 polysorbate 20. The proteins (gp120-LAV, gp41, CD4, nonglycosylated gp120 (Env 2-3) was from the AIDS Research and Reference Reagent gp120, fetuin, and BSA) were dissolved at 20 ␮g/ml in 10 mM sodium Program (National Institutes of Health, Division of AIDS, Rockville, MD). acetate (pH 5.0) and were immobilized on a CM5 sensor chip using the It represented amino acids 28–509 of gp120, had a mass of 54,000, and amine coupling method. The chip was activated by mixing 400 mM N-eth- was produced in yeast by Chiron Corporation (Emeryville, CA) (13, 14). yl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 100 mM Three mouse mAbs were also obtained from the National Institutes of N-hydroxysuccinimide. An immobilization level of ϳ6000 response units Health AIDS Research and Reference Reagent Program: HIV-1 gp120 was attained for each bound protein. Residual reactive groups on the chip mAb (697-30D), LAI/BRU V3 mAb (4G10), and HIV-1 IIIB gp120 mAb surface were blocked using 1.0 M ethanolamine/HCl (pH 8.5). (902). Ab 902 was raised in mice immunized with a vaccinia virus-gp160 The flow cell-1 chip, which served as a control, lacked immobilized construct, binds the immunodominant loop of gp120, and recognizes protein but was treated with N-ethyl-N-(3-dimethylaminopropyl)-carbodi- strains LAV and IIIB (15, 16). Ab 4G10 was raised against hybrid HbcAg/ imide hydrochloride, N-hydroxysuccinimide, and ethanolamine/HCl. Bind- HIV-1 gp120 particles (aa 303–327) and recognizes LAI gp160 and the V3 ing signals were corrected for nonspecific binding by subtracting the flow peptide (aa 308–322) in ELISA (17). Ab 697-30D recognizes an epitope cell-1 signal. To regenerate chip surfaces, bound ligands were removed common to 75% of clade B primary isolates and is specific for the V2 with 10 mM HCl. Data were analyzed with BIAevaluation 3.1 software region of gp120 (18). (Biacore, Piscataway, NJ), and curve fitting was done with the assumption BSA, fetuin, and monosaccharides were purchased from Sigma-Aldrich. of one-to-one binding. High-mannose oligosaccharides were from GlyKo (Novato, CA) and gly- In some experiments, fetuin that had been immobilized on a CM5 chip cosylated ceramides were from Matreya (State College, PA). Fetuin and was deglycosylated in the BiaCore by perfusing the chambers for3hat the following glycosidases were purchased from ProZyme in a GlycoPro 37°C with PNGase F (5 U), Sialidase A (0.005 U), and/or endo-O-glyco- deglycosylation kit: GE41 PNGase F, GE23 Sialidase A, and GE43 endo- sidase (0.00125 U) (ProZyme, San Leandro, CA). These enzymes were O-glycosidase. The two neoglycoproteins used in this study were purchased delivered in 450 ␮l of 50 mM sodium phosphate buffer (pH 7.0). from Sigma-Aldrich. BSA-p-aminophenyl-␣-D-mannopyranoside contained 27 mol of monosaccharide/mol BSA, and BSA-2-amido-2-deoxy-D-galactose Interactions with glycolipids (BSA-galactosamide) contained 23 mol of galactosamine/mol BSA. Liposomes containing palmitoyl-oleoyl-phosphatidyl choline (POPC) Ϯ Results 25% ceramide or galactosylceramide were extruded through a 100-nM pore size filter. BiaCore L1 chips were precleaned and washed twice with Because RC-100 was active against HIV-1 strains using either the 40 mM n-octyl ␤-D-glucopyranoside (Sigma-Aldrich, St. Louis, MO). Li- CXCR4 or CCR5 coreceptors (11), we suspected that it might bind posomes (1 mg/ml in 150 mM NaCl and10 mM HEPES buffer (pH 7.4)) either to gp120 or to CD4. To test this, we immobilized ϳ6000 were attached by passaging 30 ␮l of the preparation across the chip’s response units of glycosylated gp120, CD4, or BSA (a control) on surface over a period of 15 min. Any unbound liposomes were washed away with 0.1 M HCl and 0.1 M NaOH. BSA (0.1 mg/ml) was used to a BiaCore CM5 chip. Fig. 1a shows that immobilized gp120 bound detect nonspecific binding on the liposome-coated surfaces. To assess their RC-100 and CD4, but not BSA. Fig. 1b shows that immobilized binding to the chip surface, retrocyclin peptides were injected for 3 min at CD4 bound RC-100 but not gp120. Evidently, RC-100 and gp120 4710 BINDING PROPERTIES OF RETROCYCLIN

FIGURE 1. Binding of retrocyclin (RC-100) to glycosylated gp120 and CD4. a, Binding of RC-100 (solid line), CD4 (dotted line), and BSA (dashed line) to immobilized gp120. b, Binding of RC-100 (solid line), gp120 (dotted line), and BSA (dashed line) to immobilized CD4. c, Binding of RC-100 (solid line), gp120 (dotted line), and CD4 (dashed line) to immobilized BSA. The concentration of RC-100 was 1 ␮g/ml. Downloaded from bind CD4 at different sites. Immobilized BSA did not bind RC- nor oligomannose-8-D1D2 inhibited the binding of retrocyclin to 100, gp120, or CD4 (Fig. 1c). gp120. Fig. 2, a and b, shows binding of several retrocyclin analogs to From the isotherms in Fig. 4, we calculated retrocyclin’s bind-

CD4 or gp120. Whereas RC-100 (retrocyclin-1) and RC-101, its ing afﬁnity (KD) to glycosylated gp120 (35.4 nM) and CD4 (31.0

3 http://www.jimmunol.org/ arg9 lys9 variant, bound CD4 and gp120 extensively, several nM). Because certain glycolipids provide cell surface receptors for variants did not bind at all, despite differing from RC-100 only by HIV-1, we also studied binding to some immobilized (glyco)lipids: the replacement of arg6 (RC-106), arg9 (RC-107), or arg13 (RC- galactosylceramide, ceramide, and POPC (Table II). Galactosyl- 108) with a tyrosine. RC-111, the retro form of RC-100, showed ceramide was bound with a KD of 24.1 nM by RC-100 and with a little binding to CD4 and gp120, and RC-112, its enantio form, KD of 25.5 nM by RC-101. Their KD values for ceramide binding bound slightly to both molecules (data not shown). The binding of were 3- to 4-fold higher (RC-100, 71.3 nM; RC-101, 107 nM), and modiﬁed retrocyclins to gp120 and to CD4 were highly correlated these values were higher still for binding POPC (RC-100, 116.3 (r2 ϭ 0.978) and were an indication that these glycoproteins dis- nM; RC-101, 213.3 nM). Binding of retrocyclin analogs to played a similar binding site(s), despite their different sequences galactosylceramide generally paralleled their binding to gp120 and structures (Fig. 2c). or CD4 (Fig. 5), except for RC-113 (enantio-RC-101) and by guest on September 29, 2021

Suspecting that these retrocyclin-binding sites might contain RC-103 (Y15-RC-100), which bound galactosylceramide some- carbohydrates, we tested this hypothesis by comparing the binding what better than either glycoprotein did. The correlations between of RC-100 to glycosylated and nonglycosylated gp120. As shown binding of peptides in this panel to galactosylceramide and their in Fig. 3a, the peptide bound only to glycosylated gp120. We also binding to CD4 (r2 ϭ 0.508) or between their binding to galacto- tested the ability of high concentrations (1 mg/ml) of various car- sylceramide and gp120 (r2 ϭ 0.434) were less marked than was the bohydrates to inhibit retrocyclin’s binding to gp120 or CD4. None correlation of their binding to CD4 and gp120 (r2 ϭ 0.978; Fig. 2). of the following was inhibitory: 1) D and L enantiomers of glucose, We performed the experiments summarized in Fig. 6 to examine galactose, and mannose, or 2) N-acetyl-neuraminic acid, D-glu- the relationship between binding and protecting cells from infec- cosamine, and methyl-␣-D-mannose. In addition, neither oligom- tion by HIV-1 strains JR-CSF and IIIB. At least three experiments, annose-8 D1D3 (cyanovirin’s high-afﬁnity binding site on gp120) each with PBMCs from a different normal donor, were done with

FIGURE 2. Correlation of binding to gp120 and CD4. Binding isotherms for RC-100 and its analogs are shown for immobilized gp120 in a and for immobilized CD4 in b. Each retrocyclin is identiﬁed by a number close to its isotherm. The isotherms for RC-104, 105, 112, and 113 (data not shown) fell between those for RC-103 and RC-111. c, Binding of the peptides to gp120 and CD4 was very highly correlated (r2 ϭ 0.978). The Journal of Immunology 4711

FIGURE 4. Binding afﬁnity by kinetic analysis. The binding constants of retrocyclin for glycosylated gp120 and CD4 shown in this ﬁgure were

calculated from the on and off rates in the experiments. Downloaded from

strain. The discontinuous relationship for the IIIB stain suggests that an additional factor, perhaps involving interaction(s) with FIGURE 3. Effect of carbohydrates on binding. a compares binding of CXCR4, remains to be identified. retrocyclin (RC-100) with immobilized gp120, nonglycosylated gp120, Two of the peptides listed in Table I bound gp120 with higher and gp41. b, Contains symbols identifying the carbohydrates in c and d. c, http://www.jimmunol.org/ Effects of selected carbohydrates on the binding of 1 ␮g/ml retrocyclin affinity than did retrocyclin: RC-115 (Kd, 21.8 nM) and RC-116 (RC-100) to gp120. d, Effects of these carbohydrates on the binding of 1 (Kd, 19.8 nM). Both of them also bound CD4 with higher affinity ␮g/ml RC-100 to CD4. (RC-115, Kd, 17.8 nM; RC-116, Kd, 20.1 nM). Because both peptides contain a tyrosine residue that can be radiolabeled, they could prove useful for phamacokinetic and binding studies. each peptide. Protective activity was assessed by calculating the If retrocyclins bind gp120 and CD4 by acting as lectins, we ratio of day 9 supernatant p24 Ag concentrations (untreated cul- reasoned that they would also bind glycoproteins. We selected tures/peptide-treated cultures). Because the ordinate shows this ra- bovine fetuin, a glycoprotein that comprises up to 45% of the ϳ tio on a log10 scale, a value of 4 (i.e., a 4 log10 reduction) is protein in FCS, to test this. Fully glycosylated fetuin contains 51 by guest on September 29, 2021 equivalent to a 10,000-fold (or 99.99%) reduction in supernatant mols of sugar/mol of protein and is ϳ20% carbohydrate by mass. p24 Ag concentration, relative to control. Fetuin’s three N-linked sites contain bi- (19) and triantennary For simplicity, only data from cultures treated with 20 ␮gof structures (20, 21), and its three O-linked sites bear sialylated di- peptide/ml are shown. Open circles represent RC-100, RC-101, and tetrasaccharides (22). We also used fetuin along with gp120 to RC-115, and RC-116, the only retrocyclins highly active against examine the contribution of RC-100’s circular structure and its both HIV-1 strains. The open triangles represent RC-102, RC-103, internal disulfide ladder to binding. Intact RC-100 bound both im- and RC-104, peptides that were considerably more active against mobilized glycoproteins well (Fig. 7a). After reduction and alky- the JR-CSF strain than against strain IIIB. Although a correlation lation, the still circular peptide bound neither glycoprotein, even between binding and protective activity is evident for both HIV-1 though they remained circular (albeit without a disulfide ladder; strains, the relationship appears continuous only for the JR-CSF Fig. 7c), and completely lost its protective activity against HIV-1

Table II. Binding to (glyco)lipidsa

Association Rate ka Dissociation Rate Afﬁnity KD Immobilized Lipid “Receptor” Ligand (1/MS) Kd (1/S) (nM) POPC/galactosyl ceramide (3:1) RC-100 7.72 ϫ 104 1.86 ϫ 10Ϫ3 24.1 RC-101 6.80 ϫ 104 1.74 ϫ 10Ϫ3 25.5 RC-103 2.90 ϫ 104 5.14 ϫ 10Ϫ3 177.0 gp120 6.87 ϫ 104 5.96 ϫ 10Ϫ4 8.7

POPC/ceramide (3:1) RC-100 2.44 ϫ 104 1.74 ϫ 10Ϫ3 71.3 RC-101 2.21 ϫ 104 2.36 ϫ 10Ϫ3 107.0 RC-103 2.60 ϫ 104 6.74 ϫ 10Ϫ3 259.2 gp120 5.21 ϫ 104 1.23 ϫ 10Ϫ3 23.6

POPC RC-100 1.22 ϫ 104 1.42 ϫ 10Ϫ3 116.3 RC-101 1.20 ϫ 104 2.56 ϫ 10Ϫ3 213.3 RC-103 1.01 ϫ 104 2.61 ϫ 10Ϫ3 258.4 gp120 1.70 ϫ 104 1.89 ϫ 10Ϫ3 111.2

a Binding of retrocyclin, two retrocyclin analogs, and gp120 to tethered liposomes composed of POPC Ϯ (galactosyl)ceramide was examined by SPR. The rate and afﬁnity constants shown in this table were calculated from kinetic experiments, like those illustrated in Fig. 7. 1/MS ϭ per molar per second; 1/S ϭ per second. 4712 BINDING PROPERTIES OF RETROCYCLIN

FIGURE 5. Binding correlation. The ordinate of this X-Y plot represents the binding of a 1-␮g/ml solution of the peptide to immobilized galactosyl ceramide, and the abscissa shows its binding to CD4. Peptides are identified by adjacent numbers that correspond to those used in Table I. A similar result with a slightly lower correlation coefficient (r2 ϭ 0.434) FIGURE 7. Contribution of disulfides and circularity to binding. The was obtained when we plotted binding of galactosyl ceramide against bind- left panel shows binding to immobilized gp120, and the right panel shows ing of glycosylated gp120. binding to immobilized fetuin. In both panels, a identifies intact RC-100, b represents the ␤-hairpin noncircular form of RC-100, and c represents circular RC-100 with reduced and alkylated disulfides. Downloaded from (data not shown). The noncyclic, ␤-hairpin form of RC-100 showed reduced binding to gp120 and fetuin (Fig. 7b) and reduced protective activity (11). fides. RC-100 (oxidized (OX)) is the not-yet-cyclized, ␤-hairpin Because the nonglycosylated gp120 used in Fig. 3a may well form of retrocyclin with three intramolecular disulfides. RC- have been misfolded, we immobilized glycosylated fetuin, gp120, 100(IAA) is a cyclic form of RC-100 whose disulfides had been and CD4 on the sensor chips and exposed them to deglycosylated reduced and alkylated with IAA. http://www.jimmunol.org/ enzymes that selectively removed O-linked or N-linked sugar res- Removing either O-linked or N-linked sugars reduced retrocy- idues. These results are shown in Fig. 8. These studies were per- clin’s binding to fetuin, gp120, and CD4 substantially. Removing formed with three forms of retrocyclin. RC-100, the “normal” both the O-linked and N-linked sugars virtually abolished binding form, is a cyclic octadecapeptide with three intramolecular disul- to all three proteins. The ␤-hairpin form of retrocyclin (RC- 100(OX)) bound very poorly to CD4 or gp120, but retained con- siderable affinity for native fetuin. Removing either the N-linked or O-linked sugars from fetuin decreased binding by RC-100(OX). After both O-linked and N-linked sugars were removed from fetuin, binding of RC-100(OX) was virtually nil. The cyclic but di- by guest on September 29, 2021 sulfide-free form of retrocyclin, RC-100(IAA), did not bind any of the ligands. It was shown in Fig. 1c that retrocyclin does not bind BSA. We took advantage of this finding to test several BSA-based neoglycoproteins that were formed by covalently attaching sugar deriv- atives to BSA. Fig. 9a shows that retrocyclin (RC-100) can bind to BSA derivatized with galactosamide, and Fig. 9c shows that it can bind BSA derivatized with mannopyranose. Retrocyclin bound to

BSA-galactosamide (Kd, 242 nM) and BSA-mannopyranose (Kd, 1050 nM) with lower afﬁnity than it bound gp120 (Kd, 35.4 nM), CD4 (Kd, 31.0 nM), and fetuin (Kd, 41.6 nM). We also used these neoglycoproteins to test binding by RTD-1, a ␪-defensin of the rhesus macaque (Fig. 9, b and d). RTD-1 bound to BSA-galactos-

amide with a Kd of 106 nM, and it bound BSA-mannopyranose with a Kd of 551 nM. Fig. 10 provides additional confirmation that the ability of retrocyclin to bind gp120 is mediated, in large part, by its affinity for N-linked oligosaccharides. Whereas removal of these sugars had a FIGURE 6. Correlation between binding and protective activity. Each minimal effect on the binding of CD4 or of two murine mAbs to data point represents a different peptide from the panel listed in Table I. gp120, the binding of retrocyclin-1 was diminished by 60%. A Retrocyclin, RC-101, RC-115, and RC-116 are represented by open circles. third mAb, HIV gp120 mAb 670-30D, behaved similarly to the RC-102, RC-103, and RC-104 are represented by open triangles. All of the two shown in the figure (data not shown). other peptides are represented by a filled circle. Protective activity is represented on the ordinate as the log decrease in supernatant p24 Ag con- 10 Discussion centrations, relative to controls, on day 9 after introducing HIV-1 into the ␪ Ն Certain -defensins, including retrocyclin-1 and RTD-1 of rhesus culture. A reduction 4 log10 (i.e., 99.99%) is indistinguishable from com- plete protection, given the sensitivity of the p24 assay (ϳ10 pg/ml). The macaque, can protect susceptible human cells from in vitro infec- top row (a–c) compares binding of the various retrocyclin peptides to im- tion by T- and M-tropic strains of HIV-1 (11). Hoping to gain mobilized gp120, CD4, and galactosylceramide with their activity against insight into their mechanism of action, we used SPR to examine strain JR-CSF. The bottom row (d–f) compares binding to these receptors binding of retrocyclins to molecules implicated in the cellular up- with their activity against strain IIIB. take of HIV-1 (11, 23). Retrocyclin-1 bound with high affinity to The Journal of Immunology 4713

FIGURE 8. Retrocyclin binds to O- linked and N-linked sugars. All three panels show binding of retrocyclin to immobilized fetuin that was unmodified (solid line), treated with PNGase F to remove its N-linked sugars (dotted line), treated with Sialidase A and endo- O-glycosidase to remove its O-linked Downloaded from sugars (dashed line), or treated with all three glycosidases to remove O-linked and N-linked sugars (dash and dots). The left panel shows binding by unmodified retrocyclin. The center panel shows binding by a ␤-hairpin synthetic precursor of retrocyclin. This precursor http://www.jimmunol.org/ contained three intramolecular disulfides but had not been cyclized. The right panel shows binding by cyclic retrocyclin, whose disulfides had been reduced with DTT and then alkylated with IAA. by guest on September 29, 2021

CD4 (Kd, 31 nM), the primary host cell receptor for HIV-1; to replaced with a tyrosine. Ten of the 12 tyrosine-containing variants galactosylceramide (Kd, 24.1), an alternative cell surface receptor afforded much reduced protection, especially against the IIIB for HIV-1 in CD4-negative cells (24, 25); and to gp120 (Kd, 34.1 strain of HIV-1. The two that retained potent antiviral activity, nM), the envelope glycoprotein of HIV-1. RC-115 and RC-116, had their tyrosine substitutions in a ␤-turn. Removing the O-linked or N-linked sugars from immobilized, gly- Neither retro-RC-100 nor enantio-RC-100 showed potent protec- cosylated CD4 or gp120 reduced retrocyclin’s binding considerably, tive or binding activity, despite a general similarity to RC-100. and removing both O-linked and N-linked sugars almost abolished Overall, these studies emphasize the importance of the ␤-sheet retrocyclin’s binding. We also performed this experiment in reverse. residues for effective binding and antiviral protection. Whereas neither retrocyclin nor RTD-1 (a ␪-defensin of rhesus ma- Overall, binding of retrocyclin analogs to CD4, gp120, and caques) bound to native BSA, BSA with covalently attached galac- galactosylceramide correlated well with their protective activity tosamine or mannopyranose residues bound both ␪-defensins. The word lectin derives from the past participle of legere (Latin for “to against the JR-CSF strain of HIV-1 (Fig. 6). Of the seven peptides select”) and describes proteins with carbohydrate-binding properties. most active against the JR-CSF strain, four (RC-100, RC-101, RC- ␪-Defensins are the smallest lectins yet described, as well as the only 115, and RC-116) also showed excellent activity against the IIIB known circular molecules of animal origin. strain. Although these four were effective in binding CD4, gp120, To see whether retrocyclin’s binding and protective properties and galactosylceramide, the correlation between binding and pro- were related, we tested a panel of peptides that included retro and tection in the entire peptide panel was less distinct. Binding of the enantio versions of retrocyclin-1 (RC-100) and twelve peptides peptides to CXCR-4, which has two potential N-glycosylation sites wherein a single (noncysteine) residue of this peptide had been and is used by HIV-1 strain IIIB, has not yet been studied. 4714 BINDING PROPERTIES OF RETROCYCLIN

has a conserved cystine-disulfide core (27). The smallest of these, Ac-AMP2 from Amaranthus caudatus, has 30 residues including six cysteines. Ac-AMP2 exerts antifungal and antibacterial activity (29), binds chitin and chitotriose, and structurally resembles portions of the hevein domain. Certain plant-derived lectins can inhibit HIV-1 uptake (30, 31) or protect against herpes simplex, influenza, CMV, and respiratory syncytial virus (30). Cyanovi- rin-N, a 101-residue (11-kDa) protein from the cyanobacterium Nostoc ellipsosporum, is an extremely potent HIV-1 entry inhibitor that binds selectively, and with high affinity, to high-mannose oligosaccharides on gp120 (32). Concentrations of cyanovirin-N that prevent infection do not block attachment of HIV-1 virions to T cells, suggesting that it interferes with a postbinding event, possi- bly fusion (33). Like rhesus RTD-1, retrocyclin’s structure consists of antiparallel ␤-sheets bridged by three cysteine disulfides and connected by turns. These disulfide bridges were essential for gp120 binding and protective activity, and circularity enhanced both actions (Fig. Downloaded from 7). Structurally, ␪-defensins resemble tachyplesins (34) and protegrins (35–37). Tachyplesins such as T22 bind CXCR4 with high affinity and protect against HIV-1 strains that enter via this coreceptor (38). Both T22 (39) and ␣-defensins (40) mimic portions of gp41. Unlike T22, retrocyclin-1 protected against both T-tropic FIGURE 9. Binding of retrocyclin and rhesus ␪-defensin 1 (RTD-1 to and M-tropic HIV-1 strains. In addition, retrocyclin-1 bound

neoglycoproteins). a, Binding of retrocyclin (RC-100) to BSA-galactos- http://www.jimmunol.org/ amide. b, Binding of RTD-1 to this neoglycoprotein. c, Binding of retro- gp120 and CD4 with three- to ﬁvefold higher afﬁnity than did T22,

cyclin to BSA-mannopyranose. d, Binding of RTD-1 to this neoglycopro- whose KD values for gp120 and CD4 were 167 nM and 101 nM, tein. Neither peptide bound unmodified BSA, even at concentrations of 10 respectively (41, 42). ␮g/ml (data not shown). The single most remarkable structural feature of ␪-defensins is their end-to-end circularization. Heretofore, naturally occurring Figs. 7–10 provide compelling evidence that retrocyclin is a circular peptides have been identified only in plants or microbes ␪ lectin. Twenty N-glycosylation sites exist in gp120 and carbohy- (reviewed in Ref. 9). -Defensins are not the smallest circular pep- drates account for ϳ55% of its mass (4). Although the binding of tides produced by a eukaryote. That distinction currently goes to retrocyclin-1 to CD4 or gp120 was not inhibited by competing sunflower trypsin inhibitor-1, a 14-residue serine protease inhibitor by guest on September 29, 2021 monosaccharides, this approach often fails, even when applied to from sunflower seeds (43). Cyclotides, the largest group of circular lectins of defined specificity (26). Perhaps we simply selected the peptides found in plants, have 28–37 aa residues, including six wrong sugar(s). Alternatively, high-affinity binding can result from cysteines that form a tri-disulfide knot. Circulins A–F (cyclotides incremental binding interactions involving multiple subsites of an ex- from Chassalia parvifolia) were discovered by screening natural tended receptor, as shown for hevein’s binding to chitin (27). Table II products for anti-HIV activity (44–46). Notwithstanding their suggests that retrocyclin’s high-affinity binding to galactosylceramide complexity, cyclotides can be synthesized (47). results from the combined effects of its binding to the acyl chains, to Attachment of gp120 to CD4 and chemokine coreceptors ini- sphingosine, and to galactose moieties of galactosylceramide. tiates membrane fusion by gp41 (48, 49). At 37°C, the binding In plants, many peptides that participate in host defense are lec- affinity between gp120 and CD4 was estimated as 5 Ϯ 3nMby tins that bind chitin, a linear polymer of ␤-1,4-linked N-acetyl-D- isothermal titration calorimetry and as 22 Ϯ 6 nM by surface plas- glucosamine present in fungal cell walls and insect exoskeletons. mon resonance assay (50). When we measured the interaction be- Many chitin-binding lectins have a structural motif called the tween gp120 and CD4 by SPR (at 25°C), the binding affinity was “hevein domain” (28), which is comprised of 40–45 residues and 10.4 nM (data not shown). Consequently, retrocyclin-1’saffinity

FIGURE 10. Binding to gp120 before and after selective deglycosylation. The binding of mouse mAbs (MAb1 and MAb2) and CD4 to immobilized gp120 was studied before and after the N-linked oligosaccharides of gp120 were removed with PNGase F. MAb1 (HIV-1 IIIB gp120 mAb #902) binds the immunodominant loop of gp120 (15, 16), and MAb2 (LAI/ BRU V3 mAb #4G10) recognizes LAI gp160 and aa 308–322 of the V3 peptide (17). The right panel shows binding of CD4 to gp120 before and after removal of its N-linked sugars, and the inset shows the much greater effect of this deglycosylation on binding by retrocyclin-1 (RC-100). The Journal of Immunology 4715

for gp120 and CD4 (KD,30–35 nM) is less strong than that be- 15. Pincus, S. H., K. Wehrly, and B. Chesebro. 1989. Treatment of HIV tissue culture tween gp120 and CD4. infection with monoclonal antibody-ricin A chain conjugates. J. Immunol. 142: 3070. We infer from our data that the ability of retrocyclin-1 to rec- 16. Chesebro, B., and K. Wehrly. 1988. Development of a sensitive quantitative focal ognize and bind oligosaccharide side chains on viral and/or cellu- assay for human immunodeﬁciency virus infectivity. J. Virol. 62:3779. lar glycoproteins allows it to interdict the operation of surface 17. von Brunn, A., M. Brand, C. Reichhuber, C. Morys-Wortmann, F. Deinhardt, and F. Schodel. 1993. Principal neutralizing domain of HIV-1 is highly immunogenic structures involved in HIV-1 entry. Although lectin domains are when expressed on the surface of hepatitis B core particles. Vaccine 11:817. found in many larger animal proteins, heretofore only one other 18. Israel, Z. R., M. K. 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