Class I defensins (BraDef) from broccoli (Brassica oleracea var. italica) seeds and their antimicrobial activity.

The objective of this study was to determine whether seeds of Brassica oleracea var. italica (i.e. broccoli, an edible plant) produce defensins that inhibit phytopathogenic fungi and pathogenic bacteria of clinical significance. Crude extracts obtained from broccoli seeds were fractioned by molecular exclusion techniques and analyzed by liquid chromatography-high-resolution mass spectrometry. Two peptides were identified, BraDef1 (10.68 kDa) and BraDef2 (9.9 kDa), which were categorized as Class I defensins based on (a) their primary structure, (b) the presence of four putative cysteine disulfide bridges, and (c) molecular modeling predictions. BraDef1 and BraDef2 show identities of, respectively, 98 and 71%, and 67 and 85%, with defensins from Brassica napus and Arabidopsis thaliana. BraDef (BraDef1 + BraDef2) disrupted membranes of Colletotrichum gloeosporioides and Alternaria alternata and also reduced hyphal growth of C. gloeosporioides by ~ 56% after 120 h of incubation. Pathogenic bacteria (Bacillus cereus 183, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and Vibrio parahaemolitycus) were susceptible to BraDef, but probiotic bacteria such as Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus casei were not inhibited. To our knowledge, this is the first report of defensins present in seeds of B. oleracea var. italica (i.e. edible broccoli). Our findings suggest an applied value for BraDef1/BraDef2 in controlling phytopathogenic fungi and pathogenic bacteria of clinical significance.

Characterization of the mature form of a ß-defensin-like peptide, Hoa-D1, in the lobster Homarus americanus.

We report on the characterization of the native form of an American lobster, Homarus americanus, ß-defensin-like putative antimicrobial peptide, H. americanus defensin 1 (Hoa-D1), sequenced employing top-down and bottom-up peptidomic strategies using a sensitive, chip-based nanoLC-QTOF-MS/MS instrument. The sequence of Hoa-D1 was determined by mass spectrometry; it was found to contain three disulfide bonds and an amidated C-terminus. The sequence was further validated by searching publicly-accessible H. americanus expressed sequence tag (EST) and transcriptome shotgun assembly (TSA) datasets. Hoa-D1, SYVRScSSNGGDcVYRcYGNIINGAcSGSRVccRSGGGYamide (with c representing a cysteine participating in a disulfide bond), was shown to be related to ß-defensin-like peptides previously reported from Panulirus japonicas and Panulirus argus. We found Hoa-D1 in H. americanus hemolymph, hemocytes, the supraoesophageal ganglion (brain), eyestalk ganglia, and pericardial organ extracts, as well as in the plasma of some hemolymph samples. Using discontinuous density gradient separations, we fractionatated hemocytes and localized Hoa-D1 to hemocyte sub-populations. While Hoa-D1 was detected in semigranulocytes and granulocytes using conventional proteomic strategies for analysis, the direct analysis of cell lysates exposed evidence of Hoa-D1 processing, including truncation of the C-terminal tyrosine residue, in the granulocytes, but not semigranulocytes. These measurements demonstrate the insights regarding post-translational modifications and peptide processing that can be revealed through the MS analysis of intact peptides. The identification of Hoa-D1 as a widely-distributed peptide in the lobster suggests the possibility that it may be pleiotropic, with functions in addition to its proposed role as an antimicrobial molecule in the innate immune system.

Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant.

The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-ß motif which consists of an α helix and a triple-stranded ß-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.

An Independent Evaluation of a Novel Peptide Mimetic, Brilacidin (PMX30063), for Ocular Anti-infective.

OBJECTIVE: Brilacidin (BRI), a novel defensin mimetic, was evaluated as an ocular anti-infective. METHODS: In vitro: Potency based on MIC90s was compared for 50 Staphylococcus aureus (SA), 50 Staphylococcus epidermidis (SE), and 25 each of Streptococcus pneumonia (SP), Streptococcus viridans (SV), Moraxella (MS), Haemophilus influenzae (HI), Pseudomonas aeruginosa (PA), and Serratia marcescens (SM). In vivo: Using established methods, ocular toxicity was graded with Draize testing. For efficacy testing, both corneas of 24 rabbits were infected with methicillin-resistant S. aureus (MRSA), whereas the corneal epithelium was removed in the left eye. After 4 h, 21 topical drops over 5 h were administered to 4 groups: BRI 0.5%, vancomycin (VAN) 5%, saline, and no treatment. The eyes were clinically graded and the corneas were harvested for colony counts. RESULTS: In vitro: Both SA and SE had the lowest minimum inhibitory concentrations among the bacterial groups. The MIC90s to BRI for SP, SV, MS, HI, PA, and SM were 4, 32, 256, 32, 16, and 128-fold higher, respectively, than SA and SE. In vivo: Draize testing determined BRI 0.5% to be minimally irritating. For abraded corneas, BRI was not statistically different from VAN for reducing MRSA. BRI was bactericidal. For intact corneas, VAN reduced more CFU than BRI. BRI reduced CFU in abraded corneas more than intact corneas suggesting poor corneal penetration. CONCLUSIONS: BRI has Gram-positive in vitro activity; topical BRI 0.5% was minimally irritating; and BRI 0.5% was equally efficacious as VAN in a MRSA keratitis model when the corneal epithelium was removed.

Isolation of a Hemagglutinin with Potent Antiproliferative Activity and a Large Antifungal Defensin from Phaseolus vulgaris cv. Hokkaido Large Pinto Beans.

Lectins (hemagglutinins) are defined as sugar-binding proteins or glycoproteins with various biological activities. A 60 kDa dimeric hemagglutinin with a blocked N-terminus was isolated in large yield (190 mg/60 g) from the common edible bean Phaseolus vulgaris cv. Hokkaido large pinto bean. Its hemagglutinating, antifungal, and antitumor activities as well as the effects of carbohydrate and metal ions on its hemagglutinating activity were examined. It inhibited the proliferation of nasopharyngeal carcinoma (CNE2), human breast cancer (MCF7), and hepatoma (HepG2) cells. The IC50 values toward HepG2, MCF7, and CNE2 cells after treatment for 48 h were 8.1, 6.07, and 7.49 µM, respectively, which were relatively low among lectins of different P. vulgaris cultivars. From the pinto beans, a 10888 Da antifungal peptide with similarity to plant defensins as revealed by mass spectroscopic analysis was also isolated with a yield of 3.2 mg of proteins from 60 g of beans. The large defensin was capable of inhibiting mycelial growth in Mycosphaerella arachidicola, Setosphaeria turcica, Bipolaris maydis, and Fusarium oxysporum but not in Valsa mali.

Isolation, purification, and characterization of a stable defensin-like antifungal peptide from Trigonella foenum-graecum (fenugreek) seeds.

A novel defensin-like antifungal peptide (Tf-AFP) with molecular mass of 10.3 kDa was isolated from seeds of Trigonella foenum-graecum (fenugreek) by ammonium sulfate precipitation, cation-exchange, gel-filtration, hydrophobic chromatography, and RP-HPLC. Mass spectroscopic analysis revealed the intact mass of the purified antifungal peptide as 10321.5 Da and high similarity to plant defensins and other antifungal proteins in database search. 2D-PAGE showed pI value to be 8.8 and absence of isoforms. Isolated Tf-AFP inhibited growth of fungal species such as Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani. The antifungal activity was inhibited in the presence of 50 mM NaCl. Circular dichroism analysis demonstrated that the protein is rich in ß-sheet structure and highly stable over a wide range of temperatures. Surprisingly, reduction of disulfide bridges and chemical denaturation did not produce large changes in secondary structure as judged by circular dichroism as well as by fluorescence spectroscopy.

An insect defensin-derived ß-hairpin peptide with enhanced antibacterial activity.

Insect defensins are a class of small, cysteine-rich antimicrobial peptides primarily active on Gram-positive bacteria. Their roles in maggot therapy for treating chronic wound infection have been reported recently. However, a relatively narrow antibacterial spectrum together with the lack of a cost-effective means of commercial-scale production has limited their application. To further exploit the therapeutic potential of these molecules, we engineered the carboxyl-terminal ß-sheet of navidefensin2-2, an insect defensin from Nasonia vitripennis, based on its structural similarity to naturally occurring microbicidal ß-hairpin peptides. The designed peptide of 14 residues, referred to as NvBH, spans the ß-sheet region of the defensin with two amino acids substituted for assembly of a disulfide-bonded amphipathic ß-hairpin structure. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with circular dichroism (CD) analysis shows that the oxidized NvBH (oNvBH), produced from the synthetic peptide by air oxidization in an alkaline environment, folds into a typical ß-hairpin structure linked by two disulfide bridges (Cys1-Cys4; Cys2-Cys3). However, such a structure appears not to be functionally necessary as synthetic NvBH with a spontaneously oxidized disulfide bridge (Cys2-Cys3) (termed poNvBH) displayed similar antibacterial potency to oNvBH. In comparison with oNvBH, poNvBH exhibited higher serum stability and more resistance on tryptic digestion. These two forms of peptides are capable of killing an array of Gram-positive (including antibiotic-resistant strains of Staphylococcus) and Gram-negative bacterial pathogens at low micromolar concentrations through a membrane disruptive mode of action. Our work indicates that the ß-sheet region of insect defensins is a promising subdomain of proteins in anti-infective drug discovery.

Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata.

Defensins are a large family of small, cysteine-rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C-terminal pro-peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt-thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC50 ) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5-10 times less effective at killing necrotrophs, but only two-fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat plants prior to the plants being inoculated with crown rust, they reduced the number of pustules per leaf area, as well as the amount of chlorosis induced by infection. Similar to observations in vitro, NaD1 was more effective as an antifungal control agent than NaD2. Further investigation revealed that both NaD1 and NaD2 permeabilized the plasma membranes of Puccinia spp. This study provides evidence that both secreted (NaD2) and nonsecreted (NaD1) defensins may be useful for broad-spectrum resistance to pathogens.

Antibacterial activity of defensin PaDef from avocado fruit (Persea americana var. drymifolia) expressed in endothelial cells against Escherichia coli and Staphylococcus aureus.

Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP) represent attractive control agents. Mexican avocado (Persea americana var. drymifolia) is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp) encoding a protein (78 aa) homologous with plant defensins (>80%). We expressed the defensin PaDef cDNA (pBME3) in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 µg/mL of total protein from clones (>55%). Also, S. aureus viability was inhibited from 50 µg/mL total protein (27-38%) but was more evident at 100 µg/mL (52-65%). This inhibition was higher than the effect showed by polyclonal population (~23%). Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

Identification and mechanism of action of the plant defensin NaD1 as a new member of the antifungal drug arsenal against Candida albicans.

In recent decades, pathogenic fungi have become a serious threat to human health, leading to major efforts aimed at characterizing new agents for improved treatments. Promising in this context are antimicrobial peptides produced by animals and plants as part of innate immune systems. Here, we describe an antifungal defensin, NaD1, with activity against the major human pathogen Candida albicans, characterize the mechanism of killing, and identify protection strategies used by the fungus to survive defensin treatment. The mechanism involves interaction between NaD1 and the fungal cell surface followed by membrane permeabilization, entry into the cytoplasm, hyperproduction of reactive oxygen species, and killing induced by oxidative damage. By screening C. albicans mutant libraries, we identified that the high-osmolarity glycerol (HOG) pathway has a unique role in protection against NaD1, while several other stress-responsive pathways are dispensable. The involvement of the HOG pathway is consistent with induction of oxidative stress by NaD1. The HOG pathway has been reported to have a major role in protection of fungi against osmotic stress, but our data indicate that osmotic stress does not contribute significantly to the adverse effects of NaD1 on C. albicans. Our data, together with previous studies with human beta-defensins and salivary histatin 5, indicate that inhibition of the HOG pathway holds promise as a broad strategy for increasing the activity of antimicrobial peptides against C. albicans.

Defensin like peptide from Panulirus argus relates structurally with beta defensin from vertebrates.

Naturally occurring antimicrobial peptides take place in the first line of host defense against pathogen as part of the humoral innate immune response. ß-defensins are among the most abundant antimicrobial peptides in mammals, and thought to be solely found in vertebrates until a recent report describing the cloning and sequencing of defensin like peptides in the spiny lobster Panulirus japonicus. In the current study, we cloned and sequenced two genes from the hemocytes of the spiny lobster Panulirus argus encoding for two isoforms of defensin-like peptides, thus confirming the presence of this protein in the Panulirus genus. The 44 amino acids mature peptides showed the conservation of cysteine pattern characterizing the ß-defensins, as well as known amino acids residues critical to exert their antimicrobial activity. They are also amphipathics, hydrophobics, and display an overall positive charge (+1) located at the C-terminus. The tertiary structure obtained by homology modeling indicated that likely conformations of lobster peptides are highly similar to ß-defensins from vertebrates. The phylogenetic study carried out by probabilistic methods confirmed the relation with ancestral ß-defensin from vertebrates. The finding of a putative defensin-like peptide in the expressed sequence tag (EST) of the lobster Homarus americanus with high homology with those of P. argus described in this study, would indicate the presence of this peptides in Palinuridae family. Taking into account all similarities between these peptides with ß-defensins from vertebrates, it is conceivable to further support the finding of a new family of ß-defensins in invertebrate.

Lucifensin, a novel insect defensin of medicinal maggots: synthesis and structural study.

Recently, we identified a new insect defensin, named lucifensin that is secreted/excreted by the blowfly Lucilia sericata larvae into a wound as a disinfectant during the medicinal process known as maggot therapy. Here, we report the total chemical synthesis of this peptide of 40 amino acid residues and three intramolecular disulfide bridges by using three different protocols. Oxidative folding of linear peptide yielded a peptide with a pattern of disulfide bridges identical to that of native lucifensin. The synthetic lucifensin was active against Gram-positive bacteria and was not hemolytic. We synthesized three lucifensin analogues that are cyclized through one native disulfide bridge in different positions and having the remaining four cysteines substituted by alanine. Only the analogue cyclized through a Cys16-Cys36 disulfide bridge showed weak antimicrobial activity. Truncating lucifensin at the N-terminal by ten amino acid residues resulted in a drop in antimicrobial activity. Linear lucifensin having all six cysteine residues alkylated was inactive. Circular dichroism spectra measured in the presence of α-helix-promoting compounds showed different patterns for lucifensin and its analogues. Transmission electron microscopy revealed that Bacillus subtilis treatment with lucifensin induced significant changes in its envelope.

Lucifensin, the long-sought antimicrobial factor of medicinal maggots of the blowfly Lucilia sericata.

A novel homologue of insect defensin designated lucifensin (Lucilia defensin) was purified from the extracts of various tissues (gut, salivary glands, fat body, haemolymph) of green bottle fly (Lucilia sericata) larvae and from their excretions/secretions. The primary sequence of this peptide of 40 residues and three intramolecular disulfide bridges was determined by ESI-QTOF mass spectrometry and Edman degradation and is very similar to that of sapecin and other dipteran defensins. We assume that lucifensin is the key antimicrobial component that protects the maggots when they are exposed to the highly infectious environment of a wound during the medicinal process known as maggot therapy. We also believe that lucifensin is that long-sought larger molecular weight antimicrobial factor of the Lucilia sericata excretions/secretions believed to be effective against pathogenic elements of the wound microbial flora.

Synthesis of kalasinamide, a putative plant defense phototoxin.

The first total synthesis of the azaanthracene kalasinamide (1) is described, and the discrepancy in the reported (13)C NMR data and melting points for the natural product from two different sources is resolved. Kalasinamide is prone to autosensitized photooxidation, in solution and in the solid state, to give the corresponding quinone, marcanine A (8). This transformation may be representative of a novel and more general step in the biosynthesis of (aza)anthraquinones. Through its ability to generate toxic singlet oxygen, kalasinamide may serve a protective role, defending the plant against predation and the invasion of microbial pathogens, following mechanical insult.

Aurelin, a novel antimicrobial peptide from jellyfish Aurelia aurita with structural features of defensins and channel-blocking toxins.

A novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against Gram-positive and Gram-negative bacteria, was purified from the mesoglea of a scyphoid jellyfish Aurelia aurita by preparative gel electrophoresis and RP-HPLC. Molecular mass (4296.95 Da) and complete amino acid sequence of aurelin (AACSDRAHGHICESFKSFCKDSGRNGVKLRANCKKTCGLC) were determined. Aurelin has six cysteines forming three disulfide bonds. The total RNA was isolated from the jellyfish mesoglea, RT-PCR and cloning were performed, and cDNA was sequenced. A 84-residue preproaurelin contains a putative signal peptide (22 amino acids) and a propiece of the same size (22 amino acids). Aurelin has no structural homology with any previously identified antimicrobial peptides but reveals partial similarity both with defensins and K+ channel-blocking toxins of sea anemones and belongs to ShKT domain family.

A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance.

The metal tolerance of metal hyper-accumulating plants is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the Zn hyper-accumulating plant Arabidopsis halleri ssp. halleri, we carried out a functional screening of an A. halleri cDNA library in the yeast Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. The screening revealed four A. halleri defensin genes (AhPDFs), which induced Zn but not cadmium (Cd) tolerance in yeast. The expression of AhPDF1.1 under the control of the 35S promoter in A. thaliana made the transgenic plants more tolerant to Zn than wild-type plants, but did not change the tolerance to Cd, copper (Cu), cobalt (Co), iron (Fe) or sodium (Na). Thus, AhPDF1.1 is able to confer Zn tolerance both to yeast and plants. In A. halleri, defensins are constitutively accumulated at a higher level in shoots than in A. thaliana. A. halleri defensin pools are Zn-responsive, both at the mRNA and protein levels. In A. thaliana, some but not all defensin genes are induced by ZnCl2 treatment, and these genes are not induced by NaCl treatment. Defensins, found in a very large number of organisms, are known to be involved in the innate immune system but have never been found to play any role in metal physiology. Our results support the proposition that defensins could be involved in Zn tolerance in A. halleri, and that a role for plant defensins in metal physiology should be considered.

Antimicrobial peptide induction in the haemolymph of the Mexican scorpion Centruroides limpidus limpidus in response to septic injury.

Antimicrobial peptides (AMPs) are essential components of host defences against infectious microrganisms. In chelicerate organisms they have been implicated in three alternative defensive systems: one is defined by the immediate up-regulation of genes encoding AMPs, another is characterized by the inducible systemic release of AMPs from cellular reservoirs and the third alternative is the systemic constitutive production of AMPs. In this work we used a differential high-performance liquid chromatography and mass spectrometry approach to show that septic injury elicits an immune response in the haemolymph of the Mexican scorpion Centruroides limpidus limpidus. We isolated several haemolymph components, one of which was characterized extensively (amino acid sequence, disulphide pairing, cDNA and genomic clones) and demonstrated to be a novel member of the invertebrate defensin family and consequently named C. limpidus limpidus defensin-like peptide (Cll-dlp). This peptide accumulates in the haemolymph in response to septic injury, independently of transcriptional regulation.

Purification, characterization, and sequencing of a novel type of antimicrobial peptides, Fa-AMP1 and Fa-AMP2, from seeds of buckwheat (Fagopyrum esculentum Moench.).

Novel antimicrobial peptides (AMP), designated Fa-AMP1 and Fa-AMP2, were purified from the seeds of buckwheat (Fagopyrum esculentum Moench.) by gel filtration on Sephadex G75, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They were basic peptides having isoelectric points of over 10. Fa-AMP1 and Fa-AMP2 had molecular masses of 3,879 Da and 3,906 Da on MALDI-TOF MS analysis, and their extinction coefficients in 1% aqueous solutions at 280 nm were 42.8 and 38.9, respectively. Half of all amino acid residues of Fa-AMP1 and Fa-AMP2 were cysteine and glycine, and they had continuous sequences of cysteine and glycine. The concentrations of peptides required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, and Gram-positive and -negative bacteria were 11 to 36 microg/ml. The structural and antimicrobial characteristics of Fa-AMPs indicated that they are a novel type of antimicrobial peptides belonging to a plant defensin family.

Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain.

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.

Retrocyclin: a primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1.

Human bone marrow expresses a pseudogene that encodes an antimicrobial peptide homologous to rhesus monkey circular minidefensins (delta-defensins). We prepared the putative ancestral human peptide by solid-phase synthesis and named it "retrocyclin." Retrocyclin did not cause direct inactivation of HIV-1, and its modest antibacterial properties resembled those of its rhesus homologs. Nevertheless, retrocyclin had a remarkable ability to inhibit proviral DNA formation and to protect immortalized and primary human CD4(+) lymphocytes from in vitro infection by both T-tropic and M-tropic strains of HIV-1. Confocal fluorescent microscopy studies performed with BODIPY-FL-labeled RC-101, a close analog of retrocyclin, showed that the peptide formed patch-like aggregates on the surface of CD4(+) cells. These findings suggest that retrocyclin interferes with an early stage of HIV-1 infection and that retrocyclin-like agents might be useful topical agents to prevent sexually acquired HIV-1 infections.