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The Journal of Immunology, 2003, 170: 4708-4716.
Copyright © 2003 by The American Association of Immunologists

Retrocyclin, an Antiretroviral {theta}-Defensin, Is a Lectin1

Wei Wang*, Alexander M. Cole*, Teresa Hong*, Alan J. Waring*,{dagger} and Robert I. Lehrer2,*,{ddagger}

Departments of * Medicine and {dagger} Pediatrics and {ddagger} Molecular Biology Institute, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{theta}-Defensins are circular octadecapeptides that contain an internal tridisulfide ladder. Because retrocyclin-1, an ancestral hominid {theta}-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 {beta}-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 exist in plants, {theta}-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Defensins are small, cysteine-rich antimicrobial peptides whose roles in innate host defense include exerting direct antibacterial, antifungal, and antiviral activity (1) and enhancing certain adaptive immune responses (2, 3, 4). The {alpha}- and {beta}-defensins, the two most widely distributed mammalian defensin subfamilies, have been studied in detail by many investigators (5).

Recently, three representatives of a third defensin subfamily, {theta}-defensins, were purified from the leukocytes (6, 7) and bone marrow (8) of the rhesus macaque (Macaca mulatta). {theta}-Defensins are circular, tetracyclic peptides with three cysteine disulfide bonds connecting their antiparallel {beta}-sheets (6, 9). Their 18 residues come from two "demidefensin" precursors, each of which contributes nine residues (including three cysteines) to the mature peptide (6). Exhaustive head-to-tail splicing renders the mature octadecapeptide circular, and an internal tridisulfide ladder renders it tetracyclic. Because nonapeptide elements from identical or different demidefensin precursors can be spliced, this imparts an element of structural diversity to {theta}-defensins without necessitating commensurate genome expansion (7, 8).

Evidence appeared recently that human {alpha}-defensins human neutrophil peptide-1, -2, and -3 are expressed by human CD8 T cells and may prevent the development of clinical AIDS (10). Not only are {theta}-defensin genes the evolutionary descendants of {alpha}-defensin genes, but {theta}-defensin peptides can also protect cells from in vitro infection by HIV-1. Human bone marrow expresses mRNA homologous to the rhesus {theta}-defensins; however, its translation is silenced by a premature stop codon mutation (11). Our phylogenetic studies indicate that the mutation that silenced Homo sapiens {theta}-defensin production occurred after the orangutan and hominid lineages separated (T. Nguyen, A. Cole, R. I. Lehrer, unpublished observations).

These experiments, undertaken to examine how retrocyclin-1 conferred protection against HIV-1, provided compelling evidence that the molecules acted as lectins. Although antimicrobial peptides are well known to "recognize" membranes populated by anionic phospholipids, this is the first demonstration that antimicrobial peptides from a vertebrate can recognize sugar molecules.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides

Table I shows the peptides used in this study. Twelve peptides were identical with retrocyclin-1 (RC-100), except for a single tyrosine substitution that replaced a noncysteine residue. RC-101 differed from RC-100 only by containing a single arginine to lysine substitution that facilitates fluorescent labeling of RC101. Enantio peptides contained D-amino acids exclusively. In retro peptides, the residues were synthesized in reverse order to their normal counterparts. Synthesis, purification, disulfide formation, and cyclization of all peptides were as previously described (8, 11). Reduction of RC-100 by DTT and its subsequent alkylation with iodoacetimide (IAA)3 were performed as previously described for protegrins (12).


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  		Table I. Peptides used in this studya

 
Antiviral activity

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 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 penicillin/ml, 10 µg of streptomycin/ml, and 50 U of IL-2/ml. After 7 days, when 98% of the cells coexpressed CD3 and CD4, they were infected with HIV-1.

HIV-1 stocks were prepared and handled as previously described (11) and were thawed just before use. About 106 primary CD4+ PBMCs/ml were resuspended at 37°C in fresh RPMI 1640 medium containing 10% FCS and 50 U of IL-2. Test peptides (final concentration, 20 µg/ml) were added, and HIV-1 was introduced 3 h later at a multiplicity of infection of 0.01 tissue culture-infective dose at 50% per cell. After three additional hours, these cells were washed twice and transferred to 24-well plates. Each well contained 5 x 105 cells in 2 ml of medium ± 20 µg/ml of a retrocyclin. Every 3 days, we removed 1 ml of supernatant for HIV-1 p24 Ag quantitation by ELISA (DuPont Pharmaceuticals, Wilmington, DE) and replaced it with 1 ml of fresh medium containing the retrocyclin peptide. Only the results from day 9 supernatants are presented in this report.

Surface plasmon resonance (SPR) experiments

SPR experiments were performed using CM5 sensor chips on a BiaCore 2000 system (BiaCore, Piscataway, NJ). HBS-EP running buffer (pH 7.4) contained 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% polysorbate 20. The proteins (gp120-LAV, gp41, CD4, nonglycosylated gp120, fetuin, and BSA) were dissolved at 20 µg/ml in 10 mM sodium acetate (pH 5.0) and were immobilized on a CM5 sensor chip using the amine coupling method. The chip was activated by mixing 400 mM N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 100 mM N-hydroxysuccinimide. An immobilization level of ~6000 response units was attained for each bound protein. Residual reactive groups on the chip surface were blocked using 1.0 M ethanolamine/HCl (pH 8.5).

The flow cell-1 chip, which served as a control, lacked immobilized protein but was treated with N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride, N-hydroxysuccinimide, and ethanolamine/HCl. Binding signals were corrected for nonspecific binding by subtracting the flow cell-1 signal. To regenerate chip surfaces, bound ligands were removed with 10 mM HCl. Data were analyzed with BIAevaluation 3.1 software (Biacore, Piscataway, NJ), and curve fitting was done with the assumption of one-to-one binding.

In some experiments, fetuin that had been immobilized on a CM5 chip was deglycosylated in the BiaCore by perfusing the chambers for 3 h at 37°C with PNGase F (5 U), Sialidase A (0.005 U), and/or endo-O-glycosidase (0.00125 U) (ProZyme, San Leandro, CA). These enzymes were delivered in 450 µl of 50 mM sodium phosphate buffer (pH 7.0).

Interactions with glycolipids

Liposomes containing palmitoyl-oleoyl-phosphatidyl choline (POPC) ± 25% ceramide or galactosylceramide were extruded through a 100-nM pore size filter. BiaCore L1 chips were precleaned and washed twice with 40 mM n-octyl {beta}-D-glucopyranoside (Sigma-Aldrich, St. Louis, MO). Liposomes (1 mg/ml in 150 mM NaCl and10 mM HEPES buffer (pH 7.4)) were attached by passaging 30 µl of the preparation across the chip’s 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 detect nonspecific binding on the liposome-coated surfaces. To assess their binding to the chip surface, retrocyclin peptides were injected for 3 min at a rate of 10 µl/min. Each binding study was done in triplicate, and the chip was regenerated between trials by washing it with 0.1 M HCl and 0.1 M NaOH.

Special reagents

Recombinant human soluble CD4 (full-length 45-kDa glycosylated ectodomain) was produced by ExpresSF+ (Protein Sciences, Meriden, CT) Spodoptera frugiperda cells in serum-free medium and was purchased from Protein Sciences. Glycosylated HIV-1 gp120 (LAV) envelope glycoprotein (catalog number 2003-LAV) was also purchased from Protein Sciences. Recombinant HIV-1 glycosylated HXB2 gp41 viral protein, produced in Pichia pastoris, contained amino acids 546–682 of the gp41 ectodomain and was from Advanced Biotechnologies (Columbia, MD). HIV-1 SF2 gp120 (Env 2-3) was from the AIDS Research and Reference Reagent Program (National Institutes of Health, Division of AIDS, Rockville, MD). It represented amino acids 28–509 of gp120, had a mass of 54,000, and was produced in yeast by Chiron Corporation (Emeryville, CA) (13, 14). Three mouse mAbs were also obtained from the National Institutes of Health AIDS Research and Reference Reagent Program: HIV-1 gp120 mAb (697-30D), LAI/BRU V3 mAb (4G10), and HIV-1 IIIB gp120 mAb (902). Ab 902 was raised in mice immunized with a vaccinia virus-gp160 construct, binds the immunodominant loop of gp120, and recognizes strains LAV and IIIB (15, 16). Ab 4G10 was raised against hybrid HbcAg/HIV-1 gp120 particles (aa 303–327) and recognizes LAI gp160 and the V3 peptide (aa 308–322) in ELISA (17). Ab 697-30D recognizes an epitope common to 75% of clade B primary isolates and is specific for the V2 region of gp120 (18).

BSA, fetuin, and monosaccharides were purchased from Sigma-Aldrich. High-mannose oligosaccharides were from GlyKo (Novato, CA) and glycosylated ceramides were from Matreya (State College, PA). Fetuin and the following glycosidases were purchased from ProZyme in a GlycoPro deglycosylation kit: GE41 PNGase F, GE23 Sialidase A, and GE43 endo-O-glycosidase. The two neoglycoproteins used in this study were purchased from Sigma-Aldrich. BSA-p-aminophenyl-{alpha}-D-mannopyranoside contained 27 mol of monosaccharide/mol BSA, and BSA-2-amido-2-deoxy-D-galactose (BSA-galactosamide) contained 23 mol of galactosamine/mol BSA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Because RC-100 was active against HIV-1 strains using either the CXCR4 or CCR5 coreceptors (11), we suspected that it might bind either to gp120 or to CD4. To test this, we immobilized ~6000 response units of glycosylated gp120, CD4, or BSA (a control) on a BiaCore CM5 chip. Fig. 1a shows that immobilized gp120 bound RC-100 and CD4, but not BSA. Fig. 1b shows that immobilized CD4 bound RC-100 but not gp120. Evidently, RC-100 and gp120 bind CD4 at different sites. Immobilized BSA did not bind RC-100, gp120, or CD4 (Fig. 1c).



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  		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 µg/ml.

 
Fig. 2, a and b, shows binding of several retrocyclin analogs to CD4 or gp120. Whereas RC-100 (retrocyclin-1) and RC-101, its arg9->lys9 variant, bound CD4 and gp120 extensively, several variants did not bind at all, despite differing from RC-100 only by the replacement of arg6 (RC-106), arg9 (RC-107), or arg13 (RC-108) with a tyrosine. RC-111, the retro form of RC-100, showed little binding to CD4 and gp120, and RC-112, its enantio form, bound slightly to both molecules (data not shown). The binding of modified retrocyclins to gp120 and to CD4 were highly correlated (r2 = 0.978) and were an indication that these glycoproteins displayed a similar binding site(s), despite their different sequences and structures (Fig. 2c).



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  		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 = 0.978).

 
Suspecting that these retrocyclin-binding sites might contain carbohydrates, we tested this hypothesis by comparing the binding of RC-100 to glycosylated and nonglycosylated gp120. As shown in Fig. 3a, the peptide bound only to glycosylated gp120. We also tested the ability of high concentrations (1 mg/ml) of various carbohydrates to inhibit retrocyclin’s binding to gp120 or CD4. None of the following was inhibitory: 1) D and L enantiomers of glucose, galactose, and mannose, or 2) N-acetyl-neuraminic acid, D-glucosamine, and methyl-{alpha}-D-mannose. In addition, neither oligomannose-8 D1D3 (cyanovirin’s high-affinity binding site on gp120) nor oligomannose-8-D1D2 inhibited the binding of retrocyclin to gp120.



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  		FIGURE 3. Effect of carbohydrates on binding. a compares binding of retrocyclin (RC-100) with immobilized gp120, nonglycosylated gp120, and gp41. b, Contains symbols identifying the carbohydrates in c and d. c, Effects of selected carbohydrates on the binding of 1 µg/ml retrocyclin (RC-100) to gp120. d, Effects of these carbohydrates on the binding of 1 µg/ml RC-100 to CD4.

 
From the isotherms in Fig. 4, we calculated retrocyclin’s binding affinity (KD) to glycosylated gp120 (35.4 nM) and CD4 (31.0 nM). Because certain glycolipids provide cell surface receptors for HIV-1, we also studied binding to some immobilized (glyco)lipids: galactosylceramide, ceramide, and POPC (Table II). Galactosylceramide was bound with a KD of 24.1 nM by RC-100 and with a KD of 25.5 nM by RC-101. Their KD values for ceramide binding were 3- to 4-fold higher (RC-100, 71.3 nM; RC-101, 107 nM), and these values were higher still for binding POPC (RC-100, 116.3 nM; RC-101, 213.3 nM). Binding of retrocyclin analogs to galactosylceramide generally paralleled their binding to gp120 or CD4 (Fig. 5), except for RC-113 (enantio-RC-101) and RC-103 (Y15-RC-100), which bound galactosylceramide somewhat better than either glycoprotein did. The correlations between binding of peptides in this panel to galactosylceramide and their binding to CD4 (r2 = 0.508) or between their binding to galactosylceramide and gp120 (r2 = 0.434) were less marked than was the correlation of their binding to CD4 and gp120 (r2 = 0.978; Fig. 2).



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  		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.

 

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  		Table II. Binding to (glyco)lipidsa

 


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  		FIGURE 5. Binding correlation. The ordinate of this X-Y plot represents the binding of a 1-µg/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 = 0.434) was obtained when we plotted binding of galactosyl ceramide against binding of glycosylated gp120.

 
We performed the experiments summarized in Fig. 6 to examine the relationship between binding and protecting cells from infection by HIV-1 strains JR-CSF and IIIB. At least three experiments, each with PBMCs from a different normal donor, were done with each peptide. Protective activity was assessed by calculating the ratio of day 9 supernatant p24 Ag concentrations (untreated cultures/peptide-treated cultures). Because the ordinate shows this ratio on a log10 scale, a value of 4 (i.e., a 4 log10 reduction) is equivalent to a 10,000-fold (or 99.99%) reduction in supernatant p24 Ag concentration, relative to control.



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  		FIGURE 6. Correlation between binding and protective activity. Each data point represents a different peptide from the panel listed in Table I. Retrocyclin, RC-101, RC-115, and RC-116 are represented by open circles. RC-102, RC-103, and RC-104 are represented by open triangles. All of the other peptides are represented by a filled circle. Protective activity is represented on the ordinate as the log10 decrease in supernatant p24 Ag concentrations, relative to controls, on day 9 after introducing HIV-1 into the culture. A reduction >=4 log10 (i.e., 99.99%) is indistinguishable from complete protection, given the sensitivity of the p24 assay (~10 pg/ml). The top row (a–c) compares binding of the various retrocyclin peptides to immobilized gp120, CD4, and galactosylceramide with their activity against strain JR-CSF. The bottom row (d–f) compares binding to these receptors with their activity against strain IIIB.

 
For simplicity, only data from cultures treated with 20 µg of peptide/ml are shown. Open circles represent RC-100, RC-101, RC-115, and RC-116, the only retrocyclins highly active against both HIV-1 strains. The open triangles represent RC-102, RC-103, and RC-104, peptides that were considerably more active against the JR-CSF strain than against strain IIIB. Although a correlation between binding and protective activity is evident for both HIV-1 strains, the relationship appears continuous only for the JR-CSF strain. The discontinuous relationship for the IIIB stain suggests that an additional factor, perhaps involving interaction(s) with CXCR4, remains to be identified.

Two of the peptides listed in Table I bound gp120 with higher affinity than did retrocyclin: RC-115 (Kd, 21.8 nM) and RC-116 (Kd, 19.8 nM). Both of them also bound CD4 with higher affinity (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.

If retrocyclins bind gp120 and CD4 by acting as lectins, we reasoned that they would also bind glycoproteins. We selected bovine fetuin, a glycoprotein that comprises up to 45% of the protein in FCS, to test this. Fully glycosylated fetuin contains ~51 mols of sugar/mol of protein and is ~20% carbohydrate by mass. Fetuin’s three N-linked sites contain bi- (19) and triantennary structures (20, 21), and its three O-linked sites bear sialylated di- and tetrasaccharides (22). We also used fetuin along with gp120 to examine the contribution of RC-100’s circular structure and its internal disulfide ladder to binding. Intact RC-100 bound both immobilized glycoproteins well (Fig. 7a). After reduction and alkylation, the still circular peptide bound neither glycoprotein, even though they remained circular (albeit without a disulfide ladder; Fig. 7c), and completely lost its protective activity against HIV-1 (data not shown). The noncyclic, {beta}-hairpin form of RC-100 showed reduced binding to gp120 and fetuin (Fig. 7b) and reduced protective activity (11).



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  		FIGURE 7. Contribution of disulfides and circularity to binding. The left panel shows binding to immobilized gp120, and the right panel shows binding to immobilized fetuin. In both panels, a identifies intact RC-100, b represents the {beta}-hairpin noncircular form of RC-100, and c represents circular RC-100 with reduced and alkylated disulfides.

 
Because the nonglycosylated gp120 used in Fig. 3a may well have been misfolded, we immobilized glycosylated fetuin, gp120, and CD4 on the sensor chips and exposed them to deglycosylated enzymes that selectively removed O-linked or N-linked sugar residues. These results are shown in Fig. 8. These studies were performed with three forms of retrocyclin. RC-100, the "normal" form, is a cyclic octadecapeptide with three intramolecular disulfides. RC-100 (oxidized (OX)) is the not-yet-cyclized, {beta}-hairpin form of retrocyclin with three intramolecular disulfides. RC-100(IAA) is a cyclic form of RC-100 whose disulfides had been reduced and alkylated with IAA.



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  		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 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 {beta}-hairpin synthetic precursor of retrocyclin. This precursor 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.

 
Removing either O-linked or N-linked sugars reduced retrocyclin’s binding to fetuin, gp120, and CD4 substantially. Removing both the O-linked and N-linked sugars virtually abolished binding to all three proteins. The {beta}-hairpin form of retrocyclin (RC-100(OX)) bound very poorly to CD4 or gp120, but retained considerable 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 disulfide-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 derivatives 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 {theta}-defensin of the rhesus macaque (Fig. 9, b and d). RTD-1 bound to BSA-galactosamide with a Kd of 106 nM, and it bound BSA-mannopyranose with a Kd of 551 nM.



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  		FIGURE 9. Binding of retrocyclin and rhesus {theta}-defensin 1 (RTD-1 to neoglycoproteins). a, Binding of retrocyclin (RC-100) to BSA-galactosamide. b, Binding of RTD-1 to this neoglycoprotein. c, Binding of retrocyclin to BSA-mannopyranose. d, Binding of RTD-1 to this neoglycoprotein. Neither peptide bound unmodified BSA, even at concentrations of 10 µg/ml (data not shown).

 
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 minimal effect on the binding of CD4 or of two murine mAbs to gp120, the binding of retrocyclin-1 was diminished by 60%. A third mAb, HIV gp120 mAb 670-30D, behaved similarly to the two shown in the figure (data not shown).



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  		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).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Certain {theta}-defensins, including retrocyclin-1 and RTD-1 of rhesus macaque, can protect susceptible human cells from in vitro infection by T- and M-tropic strains of HIV-1 (11). Hoping to gain insight into their mechanism of action, we used SPR to examine binding of retrocyclins to molecules implicated in the cellular uptake of HIV-1 (11, 23). Retrocyclin-1 bound with high affinity to CD4 (Kd, 31 nM), the primary host cell receptor for HIV-1; to galactosylceramide (Kd, 24.1), an alternative cell surface receptor for HIV-1 in CD4-negative cells (24, 25); and to gp120 (Kd, 34.1 nM), the envelope glycoprotein of HIV-1.

Removing the O-linked or N-linked sugars from immobilized, glycosylated CD4 or gp120 reduced retrocyclin’s binding considerably, and removing both O-linked and N-linked sugars almost abolished retrocyclin’s binding. We also performed this experiment in reverse. Whereas neither retrocyclin nor RTD-1 (a {theta}-defensin of rhesus macaques) bound to native BSA, BSA with covalently attached galactosamine or mannopyranose residues bound both {theta}-defensins. The word lectin derives from the past participle of legere (Latin for "to select") and describes proteins with carbohydrate-binding properties. {theta}-Defensins are the smallest lectins yet described, as well as the only known circular molecules of animal origin.

To see whether retrocyclin’s binding and protective properties were related, we tested a panel of peptides that included retro and enantio versions of retrocyclin-1 (RC-100) and twelve peptides wherein a single (noncysteine) residue of this peptide had been replaced with a tyrosine. Ten of the 12 tyrosine-containing variants afforded much reduced protection, especially against the IIIB strain of HIV-1. The two that retained potent antiviral activity, RC-115 and RC-116, had their tyrosine substitutions in a {beta}-turn. Neither retro-RC-100 nor enantio-RC-100 showed potent protective or binding activity, despite a general similarity to RC-100. Overall, these studies emphasize the importance of the {beta}-sheet residues for effective binding and antiviral protection.

Overall, binding of retrocyclin analogs to CD4, gp120, and galactosylceramide correlated well with their protective activity against the JR-CSF strain of HIV-1 (Fig. 6). Of the seven peptides most active against the JR-CSF strain, four (RC-100, RC-101, RC-115, and RC-116) also showed excellent activity against the IIIB strain. Although these four were effective in binding CD4, gp120, and galactosylceramide, the correlation between binding and protection in the entire peptide panel was less distinct. Binding of the peptides to CXCR-4, which has two potential N-glycosylation sites and is used by HIV-1 strain IIIB, has not yet been studied.

Figs. 7–10 provide compelling evidence that retrocyclin is a lectin. Twenty N-glycosylation sites exist in gp120 and carbohydrates account for ~55% of its mass (4). Although the binding of retrocyclin-1 to CD4 or gp120 was not inhibited by competing monosaccharides, this approach often fails, even when applied to lectins of defined specificity (26). Perhaps we simply selected the wrong sugar(s). Alternatively, high-affinity binding can result from incremental binding interactions involving multiple subsites of an extended receptor, as shown for hevein’s binding to chitin (27). Table II suggests that retrocyclin’s high-affinity binding to galactosylceramide results from the combined effects of its binding to the acyl chains, to sphingosine, and to galactose moieties of galactosylceramide.

In plants, many peptides that participate in host defense are lectins that bind chitin, a linear polymer of {beta}-1,4-linked N-acetyl-D-glucosamine present in fungal cell walls and insect exoskeletons. Many chitin-binding lectins have a structural motif called the "hevein domain" (28), which is comprised of 40–45 residues and 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). Cyanovirin-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, possibly fusion (33).

Like rhesus RTD-1, retrocyclin’s structure consists of antiparallel {beta}-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. 7). Structurally, {theta}-defensins resemble tachyplesins (34) and protegrins (35, 36, 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 {alpha}-defensins (40) mimic portions of gp41. Unlike T22, retrocyclin-1 protected against both T-tropic and M-tropic HIV-1 strains. In addition, retrocyclin-1 bound gp120 and CD4 with three- to fivefold higher affinity than did T22, whose KD values for gp120 and CD4 were 167 nM and 101 nM, respectively (41, 42).

The single most remarkable structural feature of {theta}-defensins is their end-to-end circularization. Heretofore, naturally occurring circular peptides have been identified only in plants or microbes (reviewed in Ref. 9). {theta}-Defensins are not the smallest circular peptides produced by a eukaryote. That distinction currently goes to sunflower trypsin inhibitor-1, a 14-residue serine protease inhibitor from sunflower seeds (43). Cyclotides, the largest group of circular peptides found in plants, have 28–37 aa residues, including six cysteines that form a tri-disulfide knot. Circulins A–F (cyclotides from Chassalia parvifolia) were discovered by screening natural products for anti-HIV activity (44, 45, 46). Notwithstanding their complexity, cyclotides can be synthesized (47).

Attachment of gp120 to CD4 and chemokine coreceptors initiates membrane fusion by gp41 (48, 49). At 37°C, the binding affinity between gp120 and CD4 was estimated as 5 ± 3 nM by isothermal titration calorimetry and as 22 ± 6 nM by surface plasmon resonance assay (50). When we measured the interaction between gp120 and CD4 by SPR (at 25°C), the binding affinity was 10.4 nM (data not shown). Consequently, retrocyclin-1’s affinity for gp120 and CD4 (KD, 30–35 nM) is less strong than that between gp120 and CD4.

We infer from our data that the ability of retrocyclin-1 to recognize and bind oligosaccharide side chains on viral and/or cellular glycoproteins allows it to interdict the operation of surface structures involved in HIV-1 entry. Although lectin domains are found in many larger animal proteins, heretofore only one other small lectin peptide has been described: Selenocosmia huwena lectin-1, a hemagglutinating peptide in the venom of the Chinese bird spider Selenocosmia huwena. This peptide has 32 residues (including six cysteines) and a triple-stranded antiparallel {beta}-sheet structure that resembles the N terminus of hevein (51).

Sir Peter Medawar, the late British Nobelist, once described viruses as "a bit of bad news in a protein coat." For infections caused by viruses whose infectivity depends on coat proteins that contain sugars, {theta}-defensins (and other lectins) have potential in prevention and treatment.


   Acknowledgments
 
We thank Professor Ron Kaback for generously allowing us to use his BiaCore 2000 instrument. Several reagents were obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health), as described in Materials and Methods. mAb 4G10 from Dr. Albrecht von Brunn was also courtesy of the Medical Research Council AIDS Directed Program (U.K.).


   Footnotes
 
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.). Back

2 Address correspondence and reprint requests to Dr. Robert I. Lehrer, Department of Medicine, Center for Healtlh Science 37-062, 10833 LeConte Avenue, University of California, Los Angeles, CA 90095-1690. E-mail address: rlehrer{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: IAA, iodoacetimide; SPR, surface plasmon resonance; PNGase F, peptide N-glycosidase F; POPC, palmitoyl-oleoyl-phosphatidyl choline; BSA-galactosamide, BSA-2-amido-2-deoxy-D-galactose; OX, oxidized; RTD-1, rhesus {theta}-defensin-1. Back

Received for publication October 31, 2002. Accepted for publication February 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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