Defensin Domain Research Articles

Defensins of the stable fly (Stomoxys Calcitrans) have developmental-specific regulation and evolve at different rates.

Organisms produce antimicrobial peptides (AMPs) either in response to infection (induced) or continuously (constitutively) to combat microbes encountered during normal trophic activities and/or through pathogenic infections. The expression of AMPs is tightly regulated often with specificity to particular tissues or developmental stages. As a result, AMPs face varying selective pressures based on the microbes the organism's tissue or developmental stage encounters. Here, we analyzed the evolution and developmental-specific expression of Defensins, which are ancient AMPs in insects, in the stable fly (Stomoxys calcitrans). Stable fly larvae inhibit microbe-rich environments, whereas adult flies, as blood-feeders, experience comparatively fewer encounters with diverse microbial communities. Using existing RNA-seq datasets, we identified six Defensins that were only expressed in larvae (larval Defensins) and five that were not expressed in larvae (non-larval Defensins). Each of the non-larval Defensins was expressed in at least one adult tissue sample. Half of the larval Defensins were induced by mating or feeding in adults, and all three of the induced Defensins were located downstream of canonical binding sites for an Imd transcription factor involved in the highly conserved NF-κB signaling that regulates induction of AMPs. The larval and non-larval Defensins were located in distinct genomic regions, and the amino acid sequences of the larval Defensins formed a monophyletic clade. There were more amino acid substitutions across non-larval Defensins, with multiple genes losing a highly conserved furin cleavage site thought to be required for the removal of the amino terminus from the mature Defensin domain. However, larval Defensins had a higher proportion of radical amino acid substitutions, altering amino acid size and polarity. Our results reveal insights into the developmental stage-specific regulation of AMPs, and they suggest different regulatory regimes impose unique selection pressures on AMPs, possibly as a result of variation in exposure to microbial communities across development.

Legume NCRs and nodule-specific defensins of actinorhizal plants—Do they share a common origin?

The actinorhizal plant Datisca glomerata (Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbiotic Frankia clade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed in D. glomerata nodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodule-specific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels, DgDef1, was characterized. Promoter-GUS studies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity. In vitro studies with E. coli and Sinorhizobium meliloti 1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality for S. meliloti 1021 at 20.8 μM. Analysis of the S. meliloti 1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages.

Isolation and In Silico Characterization of Defensin Genes from Some Indigenous Plant Species of Pakistan

Aims: Damage of crop plants due to pathogenic attacks, postharvest crops spoilage and lethal effects of chemical pesticides has enforced scientists to find some potential natural alternative. Plants produce different types of antimicrobial peptides including defensins in response to biotic and abiotic stresses. Defensins are small cysteine rich, cationic peptide with 40-45 amino acid residues with a variety of biological activities. Hence defensins have pharmaceutical and agricultural significance especially wide range of antifungal activities.
 Methodology: Nine plant species viz Brassica napus, Brassica nigra, Conyza bonariensis, Alhagi marorum, Sonchus arvensis, Brassica compestris, Eruca sativa, Cirsium arvensis and Brassica juncea were selected for the study. Four set of primers were applied on extracted genomic DNAs and four amplified genes were isolated from different plants species by PCR. The amplified genes were cloned via pTZ57R/T in E.coli. The cloned fragments were sequenced and characterized by different bioinformatics tool such as coding length and peptide sequence, site of cell aggregation, peptide physico-chemical properties, antimicrobial properties and gene expression pattern.
 Result: The amplified products from B. napus, B. nigra, B. compestris, E. sativa and B. juncea were about 330 bp in length and showed upto 85% nucleotide homology to the reported defensins. Deduced amino acid sequence from Bn-Def, Es-Def Bj-Def and Bc- Def showed the conserved defensin domain. These sequences were also characterized for different characteristic like charged amino acids, pI value, shelflife and stability. Sonchus arvensis didn’t give any specific product by prescribed set of primers. However the products from Alhagi marorum was about 770 bp and B. napus was of 270 bp but both give homology below 30%.
 Conclusion: This study showed that in silico characterization of deduced antimicrobial peptides from different plant species has depicted these as an effective alternative to communal therapeutics. However, further work is needed to verify defensins specific activity at protein level.

Seed-derived defensins from Scots pine: structural and functional features.

The recombinant PsDef5.1 defensin inhibits the growth of phytopathogenic fungi, Gram-positive and Gram-negative bacteria, and human pathogen Candida albicans. Expression of seed-derived Scots pine defensins is tissue-specific and developmentally regulated. Plant defensins are ubiquitous antimicrobial peptides that possess a broad spectrum of activities and multi-functionality. The genes for these antimicrobial proteins form a multigenic family in the plant genome and are expressed in every organ. Most of the known defensins have been isolated from seeds of various monocot and dicot species, but seed-derived defensins have not yet been characterized in gymnosperms. This study presents the isolation of two new 249bp cDNA sequences from Scots pine seeds with 97.9% nucleotide homology named PsDef5.1 and PsDef5.2. Their deduced amino acid sequences have typical plant defensin features, including an endoplasmic reticulum signal sequence of 31 amino acids (aa), followed by a characteristic defensin domain of 51 aa. To elucidate the functional activity of new defensins, we expressed the mature form of PsDef5.1 in a prokaryotic system. The purified recombinant peptide exhibited activity against the phytopathogenic fungi and Gram-negative and Gram-positive bacteria with the IC50 of 5-18µM. Moreover, it inhibited the growth of the human pathogen Candida albicans with the IC50 of 6.0µM. Expression analysis showed that transcripts of PsDef5.1-2 genes were present in immature and mature pine seeds and different parts of seedlings at the early stage of germination. In addition, unlike the PsDef5.2, the PsDef5.1 gene was expressed in the reproductive organs. Our findings indicate that novel defensins are promising candidates for transgenic application and the development of new antimicrobial drugs.

Peptides Derived From the α-Core and γ-Core Regions of a Putative Silybum marianum Flower Defensin Show Antifungal Activity Against Fusarium graminearum.

Fusarium graminearum is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved β-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant Silybum marianum. The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAPα1-21 and SmAPα10-21) and two from the γ-core region (SmAPγ27-44 and SmAPγ29-35). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAPα1-21, SmAPα10-21, and SmAPγ27-44 inhibited the growth of the phytopathogen F. graminearum at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells.

In silico identification and expression analyses of Defensin genes in the mealworm beetle Tenebrio molitor

AbstractDefensins are a major family of antimicrobial peptides that serve as the innate immune defense of both vertebrates and invertebrates. Due to their antimicrobial, chemotactic, and regulatory activities, Defensins have been exploited for their therapeutic potential. Insect Defensins are cysteine‐rich and contain an N‐terminal loop, α‐helix, and antiparallel β‐sheet, forming a “cysteine‐stabilized alpha beta (CSαβ)” or “loop–helix‐sheet” structure. In this study, we identified the full‐length open reading frame (ORF) sequences of Defensin (TmDef) and Defensin‐like (TmDef‐like) genes from the mealworm beetle Tenebrio molitor using in silico methods. TmDef and TmDef‐like genes encode the peptides of 72 and 71 amino acid residues, respectively. TmDefensin is comprised of a Defensin domain and the TmDefensin‐like is comprised of a signal peptide of 21 amino acid residues. Phylogenetic analysis revealed close similarities of TmDefensin with the Defensin of Acalolepta luxuriosa of the longhorn beetle family. The expression of TmDef mRNA was found to be greater than that of TmDef‐like mRNA and was mostly expressed in the pupal and adult stages. Tissue distribution showed high expression of TmDef‐like mRNA in larval hemocytes, gut, integument, and fat body, while in adults, the expression was high in gut and hemocytes. Following bacterial and fungal stimulation in vivo, TmDef was upregulated at 24 h post‐infection in whole body, fat body, and hemocytes of the larvae. Even TmDef‐like mRNA was upregulated in the gut and hemocytes at 12 and 9 h post‐infection respectively. These results suggest that TmDef and TmDef‐like genes play important roles in protecting T. molitor from microbial contact.

Molecular Evolutionary Analysis of a-Defensin Peptides in Vertebrates

?-Defensin is a group of polypeptides with antimicrobial activity found in the host defense system and it is widely distributed in, but not limited to mammalian epithelial cells and phagocytes. These molecules protect the organism from a diverse spectrum of bacteria, viruses, fungi, and protozoan parasites. Different studies have revealed widesequence variation within ?-defensin sequences, but the underlying evolutionary cause is not well-studied. In this study, the ?-defensin gene from 25 vertebrate species has been comprehensively collected and computationally analyzed. NCBI gene and nucleotide databases were accessed to extract meta-information about ?-defensin gene's defensin domain and leader propeptide sequences. Full coding sequences downloaded from nucleotide database by splitting out intron sequence. MEGA software used to construct phylogenetic tree using Neighbor-Joining method, which indicates that ?-defensin gene evolution does not matches with species evolution. Selection analysis was carried out using Data Monkey web-server's FEL, SLAC, IFEL, MEME, TOGGLE and REL program on both propeptide and defensin super-family codon-aligned sequences to test different hypothesises. Positively selected sites were found on both propeptide and defensin domain, but the effect of negative selection pressure was higher on leader sequences. It was found that mutations in the positively selected sites of defensin domain had stabilizing effect on protein. Phyre2 web-server was used for homology modeling of selected ?-defensin genes. Structural variation is observed on ?-defensinproteins which may indicate heterogeneous structure-function relationship between species that reflects its interaction with diverse pathogens. This study provides a new perspective on the relationships among ?-defensin gene repertoires which will help to infer its evolution.

Evolution of primate α and θ defensins revealed by analysis of genomes

Defensins are endogenous peptides with cysteine-rich antimicrobial ability that contribute to host defence against bacterial, fungal and viral infections. There are three subfamilies of defensins in primates: α, β and θ-defensins. α-defensins are most present in neutrophils and Paneth cells; β-defensins are involved in protecting the skin and the mucous membranes of the respiratory, genitourinary and gastrointestinal tracts; and θ-defensins are physically distinguished as the only known fully-cyclic peptides of animal origin, which are first isolated from rhesus macaques. All three kinds of defensins have six conserved cysteines, three intramolecular disulfide bonds, a net positive charge, and β-sheet regions. α and θ-defensins are closely related, comparative amino acid sequences showed that the difference between them is that θ-defensins have an additional stop codon limits the initial defensin domain peptides to 12 residues. Humans, chimpanzees and gorillas do not produce θ-defensin peptides due to a premature stop codon present in the signal sequence of all θ-defensin pseudogenes. By using comprehensive computational searches, here we report the discovery of complete repertoires of the α and θ-defensin gene family in ten primate species. Consistent with previous studies, our phylogenetic analyses showed all primate θ-defensins evident formed one distinct clusters evolved from α-defensins. β-defensins are ancestors of both α and θ-defensins. Human has two copies of DEFA1 and DEFT1P, and two extra DEFA3 and DEFA10P genes compared with gorilla. As different primates inhabit in quite different ecological niches, the production of species-specific α and θ-defensins and these highly evolved θ-defensins in old world monkeys would presumably allow them to better respond to the specific microbial challenges that they face.

Molecular characterization and expression of a novel big defensin (Sb-BDef1) from ark shell, Scapharca broughtonii

Big defensins, endogenous cysteine-rich antimicrobial peptides (AMPs) with antimicrobial activity and immunomodulatory property, play crucial roles in host defense against various microbial pathogens. A novel big defensin (Sb-BDef1) of ark shell Scapharca broughtonii was identified by expressed sequence tag (EST) and RACE techniques. The Sb-BDef1 cDNA contained an open reading frame (ORF) of 336-bp encoding a polypeptide of 111 amino acids with a putative signal peptide of 21 amino acid residues, followed by a putative propeptide of 11 residues and a putative mature peptide of 79 residues. The mature peptide shared the common features of big defensins, including a high hydrophobic residues region (59%) in the N-terminus, a defensin domain in the C-terminus, which perfectly corresponds to the six conserved disulfide-bonded cysteine residues involved in the formation of the internal disulfide bridges (C1–C5, C2–C4 and C3–C6) in all big defensins from mollusk, horseshoe crab and amphioxus. Quantitative real-time PCR analysis revealed that the expression of Sb-BDef1 transcript was detected in all the tissues examined from normal ark shells, and the temporal expression of Sb-BDef1 mRNA was remarkably up-regulated at 8, 16 h in hemocytes, and at 16, 24 h in hepatopancreas after Vibrio anguillarum-challenge, respectively. These results suggested that Sb-BDef1 was a constitutive and inducible acute-phase protein and should be involved in immune response of Gram-negative microbial infection in ark shell S. broughtonii.

Recombinant expression, affinity purification and functional characterization of Scots pine defensin 1

Plants produce a variety of molecules to defend themselves from fungal pathogens. Defensins belong to the family of antimicrobial peptides that play a central role in innate immunity in all species of plants. We have previously reported the purification of antimicrobial peptides from Scots pine seedlings and the identification of some of them, including defensin, by mass spectrometry. In this study, we extend our original study on molecular cloning of Pinus sylvestris defensin 1 (PsDef1) by presenting the expression and affinity purification of recombinant defensin 1 (rPsDef1). The full-length coding sequence of PsDef1 has an open reading frame capable to encode a protein of 83 amino residues, including a signal peptide of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5,601.6Da. We have employed pET system to express mature form of PsDef1 fussed to GST. As GST-PsDef1 fusion protein was not biologically active, we removed GST moiety from the mature defensin 1 peptide by proteolytic cleavage with Factor Xa. The resulting rPsDef1 protein exhibited strong antifungal activity against a panel of pathogenic fungi which is comparable to that of endogenous Scots pine defensin 1. In addition, rPsDef1 was used to produce specific polyclonal antibodies. Using generated antibodies, we found that the level of PsDef1 is significantly increased in Scots pine seedlings during germination and in their response to pathogenic infection with Heterobasidion annosum.

A New Allergen from Ragweed (Ambrosia artemisiifolia) with Homology to Art v 1 from Mugwort

Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29-31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more alpha-arabinofuranosyl residues with some beta-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked beta-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.

Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa ( p1) and 5.8 kDa ( p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin 1 cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins.

Expression of BrD1, a plant defensin from Brassica rapa, confers resistance against brown planthopper (Nilaparvata lugens) in transgenic rices.

Plant defensins are small (5-10 kDa) basic peptides thought to be an important component of the defense pathway against fungal and/or bacterial pathogens. To understand the role of plant defensins in protecting plants against the brown planthopper, a type of insect herbivore, we isolated the Brassica rapa Defensin 1 (BrD1) gene and introduced it into rice (Oryza sativa L.) to produce stable transgenic plants. The BrD1 protein is homologous to other plant defensins and contains both an N-terminal endoplasmic reticulum signal sequence and a defensin domain, which are highly conserved in all plant defensins. Based on a phylogenetic analysis of the defensin domain of various plant defensins, we established that BrD1 belongs to a distinct subgroup of plant defensins. Relative to the wild type, transgenic rices expressing BrD1 exhibit strong resistance to brown planthopper nymphs and female adults. These results suggest that BrD1 exhibits insecticidal activity, and might be useful for developing cereal crop plants resistant to sap-sucking insects, such as the brown planthopper.

PDC1, a corn defensin peptide expressed in Escherichia coli and Pichia pastoris inhibits growth of Fusarium graminearum

Plant defensin corn 1 ( Pdc1) gene was amplified from corn genomic DNA with the primers designed from a corn EST sequence homologous to a barley defensin gene. The cloned gene contains two exons and an intron. The deduced 9 KDa PDC1 peptide has a sequence that is identical to corn γ2-zeathionin and has the typical features of a plant defensin, including a signal sequence of 35 amino acids, followed by a characteristic defensin domain of 47 amino acids containing 8 cysteines. The defensin protein was expressed from the cloned cDNA introduced into two different expression systems; prokaryotic, Escherichia coli and eukaryotic, yeast ( Pichia pastoris). The PDC1 protein was purified with a nickel resin column and was tested for its antifungal activities using the pathogen Fusarium graminearum. The protein expressed in both E. coli and P. pastoris had antifungal activity, however the protein expressed in P. pastoris was more efficient in inhibiting growth of F. graminearum. FTIR analysis of PDC1 protein expressed in the two expression systems showed that expression in P. pastoris gave a product with more β-sheets and less random unordered structure than when it was expressed in E. coli. In addition, removal of the His-tag used for purification increased the fungicidal activity of the PDC1 protein. The data presented here suggest that the defensin PDC1 peptide of corn could be effectively used to restrict the disease caused by F. graminearum.

Role of the polypeptide backbone and post-translational modifications in cross-reactivity of Art v 1, the major mugwort pollen allergen

Artemisia vulgaris (mugwort) is one of the main causes of late summer pollinosis in Europe, with >95% of patients sensitized to the glycoallergen Art v 1. Despite the importance of this allergen, little is known about its cross-reactive behavior. Here we investigated the occurrence of conserved Art v 1 antigenic determinants in sources known to display clinically relevant cross-reactivity with mugwort pollen. For this purpose, monoclonal antibodies specific for a cysteine-stabilized epitope of the Art v 1 defensin domain and for carbohydrates attached to the proline domain were produced by hybridoma and phage display technologies. Using polyclonal Art v 1-specific rabbit sera and antibodies against both the Art v 1 carbohydrate and polypeptide moieties, we could identify cross-reactive structures in pollen from botanically related Asteraceae weeds (Artemisia absinthium, Helianthus annuus and Ambrosia sp.). Homologous allergens were also recognized by IgE from mugwort-sensitized patients and the reactivity could be decreased by serum pre-incubation with natural and recombinant Art v 1. As no cross-reactive structures could be found in foods associated with mugwort pollinosis, we conclude that Art v 1 is poorly involved in mugwort cross-reactivity to food allergens.

Immune recognition of novel isoforms and domains of the mugwort pollen major allergen Art v 1

Allergen isoforms can differ in their IgE and T cell recognition patterns, and thus might have an impact on the selection of candidates for molecule-based diagnostic and therapeutic approaches. The present study aimed at the identification and characterization of isoforms of Art v 1, the mugwort pollen major allergen. In addition, single Art v 1 domains were physicochemically and immunologically characterized. For this purpose, the Art v 1 cDNA was radiolabeled and used to screen a mugwort pollen cDNA library. Positive clones were sequenced and used for the production of recombinant proteins in Escherichia coli using the pHIS-Parallel2 vector. Protein purification was performed by affinity- and ion exchange chromatography. Antibody binding to the recombinant proteins was determined by immunoblot, ELISA, cross-inhibition experiments, and mediator release assays. We could identify 7 Art v 1 isoforms differing in 1–6 amino acid residues. Interestingly, all amino acid variations were restricted to the proline domain carrying the molecule's post-translational modifications. No significant difference in IgG or IgE reactivity could be observed between Art v 1 isoforms and the defensin domain produced in E. coli. When expressed in E. coli, the proline domain was not recognized by Art v 1-specific antibodies. Our results demonstrated that the relevant IgE epitopes of Art v 1 are located on the defensin domain and suggest the involvement of carbohydrates in the allergenicity of natural Art v 1. Plant-based expression systems could help to reveal possibly different glycosylation patterns and IgE binding properties of Art v 1 isoforms. These findings have direct implications on the development of novel tools for mugwort pollen allergy diagnosis and therapy.

Molecular Determinants for the Interaction of Human Neutrophil α Defensin 1 with its Propeptide

Human neutrophil α-defensins (HNPs) are cationic antimicrobial peptides that are synthesized in vivo as inactive precursors (proHNPs). Activation requires proteolytic excision of their anionic N-terminal inhibitory pro peptide. The pro peptide of proHNP1 also interacts specifically with and inhibits the antimicrobial activity of HNP1 inter-molecularly. In the light of the opposite net charges segregated in proHNP1, functional inhibition of the C-terminal defensin domain by its propeptide is generally thought to be of electrostatic nature. Using a battery of analogs of the propeptide and of proHNP1, we identified residues in the propeptide region important for HNP1 binding and inhibition. Only three anionic residues in the propeptide, Glu 15, Asp 20 and Glu 23, were modestly important for interactions with HNP1. By contrast, the hydrophobic residues in the central part of the propeptide, and the conserved hydrophobic motif Val 24Val 25Val 26Leu 28 in particular, were critical for HNP1 binding and inhibition. Neutralization of all negative charges in the propeptide only partially activated the bactericidal activity of proHNP1. Our data indicate that hydrophobic forces have a dominant role in mediating the interactions between HNP1 and its propeptide — a finding largely contrasting the commonly held view that the interactions are of an electrostatic nature.

Ha-DEF1, a sunflower defensin, induces cell death in Orobanche parasitic plants

Plant defensins are small basic peptides of 5-10 kDa and most of them exhibit antifungal activity. In a sunflower resistant to broomrape, among the three defensin encoding cDNA identified, SF18, SD2 and HaDef1, only HaDef1 presented a preferential root expression pattern and was induced upon infection by the root parasitic plant Orobanche cumana. The amino acid sequence deduced from HaDef1 coding sequence was composed of an endoplasmic reticulum signal sequence of 28 amino acids, a standard defensin domain of 50 amino-acid residues and an unusual C-terminal domain of 30 amino acids with a net positive charge. A 5.8 kDa recombinant mature Ha-DEF1 corresponding to the defensin domain was produced in Escherichia coli and was purified by means of a two-step chromatography procedure, Immobilized Metal Affinity Chromatography (IMAC) and Ion Exchange Chromatography. Investigation of in vitro antifungal activity of Ha-DEF1 showed a strong inhibition on Saccharomyces cerevisiae growth linked to a membrane permeabilization, and a morphogenetic activity on Alternaria brassicicola germ tube development, as already reported for some other plant defensins. Bioassays also revealed that Ha-DEF1 rapidly induced browning symptoms at the radicle apex of Orobanche seedlings but not of another parasitic plant, Striga hermonthica, nor of Arabidopsis thaliana. FDA vital staining showed that these browning areas corresponded to dead cells. These results demonstrate for the first time a lethal effect of defensins on plant cells. The potent mode of action of defensin in Orobanche cell death and the possible involvement in sunflower resistance are discussed.

Impact of Pro Segments on the Folding and Function of Human Neutrophil α-Defensins

Human neutrophil α-defensins (HNPs) are synthesized in vivo as inactive precursor proteins, i.e. preproHNPs. A series of sequential proteolytic events excise the N-terminal inhibitory pro peptide, leading to defensin maturation and storage in azurophilic granules. The anionic pro peptide, required for correct sub-cellular trafficking and sorting of proHNPs, inhibits the antimicrobial activity of cationic defensins, either inter or intra-molecularly, presumably through charge neutralization. To better understand the role of the pro peptide in the folding and functioning of α-defensins and/or pro α-defensins, we chemically attached the proHNP1 pro peptide or wtpro peptide and the following artificial pro segments to the N terminus of HNP1: polyethylene glycol (PEG), Arg 10 (polyR), Ser 10 (polyS), and crpro peptide, a charge-reversing mutant of the pro peptide where Arg/Lys residues were changed to Asp, and Asp/Glu residues to Lys. Comparative in vitro folding suggested that while all artificial pro segments chaperoned defensin folding, with PEG being the most efficient, the pro peptide catalyzed the folding of proHNPs likely through two independent mechanisms: solubilization of and interaction with the C-terminal defensin domain. Further, the N-terminal artificial pro segments dramatically altered the bactericidal activity of HNP1 against both Escherichia coli and Staphylococcus aureus. Surprisingly, crpro peptide and wtpro peptide showed similar properties with respect to intra-molecular and inter-molecular catalysis of defensin folding as well as α-defensin binding, although their binding modes appeared different. Our findings identify a dual chaperone activity of the pro peptide and may shed light on the molecular mechanisms by which pro α-defensins fold in vivo.

Determinants of Mouse Alpha‐Defensin Bactericidal Activity

Mouse Paneth cell cryptdins are expressed as inactive propeptides and are activated upon proteolysis by MMP-7. MMP-7 cleavage sites Ser43↓ Ile44, Ala53↓ Leu54, and Ser58↓ Leu59 are conserved in the cryptdin family. All three recombinant processing intermediates, proCrp4(44-92), proCrp4(54-92), and proCrp4(59-92), exhibit bactericidal activity, revealing that the activating cleavage step occurs at Ser43↓ Ile44. A recombinant proCrp4 molecule with all acidic residues upstream of Ser43↓ Ile44 mutated to Gly showed activity comparable to Crp4 in in vitro bactericidal assays. The conversion of acidic residues in the prosegment of proCrp4 to Gly shows an intramolecular interaction between acidic residues in the prosegment and cationic residues in the defensin domain is required for peptide inactivity. In addition, an examination of the α-defensin family reveals a ratio of 9:1 in favor of Arg compared to Lys. To investigate the prevalence of Arg in α-defensins, a Crp4 variant was produced recombinantly with all the Arg mutated to Lys. (R→K)-Crp4 was consistently less active than native Crp4 against a panel of bacteria tested in in vitro bactericidal assays. This is more apparent when (R→K)-Crp4 was assayed against bacteria with less susceptibility to α-defensins. This evidence supports the conclusion that the selection of Arg in α-defensins is to maintain optimal activity against pathogens.