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PubMed2004

High concentrations of alpha-defensins in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome.

alpha-Defensins, antimicrobial peptides localized in neutrophils, participate in tissue damage through their cytotoxic effects in neutrophil-mediated pulmonary diseases. Neutrophils play an important role in the pathogenesis of acute respiratory distress syndrome (ARDS). We measured alpha-defensins levels in plasma and bronchoalveolar lavage fluid (BALF) of ARDS patients to assess the kinetics of alpha-defensins in ARDS. Plasma alpha-defensins levels were higher in ARDS patients than in control subjects, and BALF levels were also higher in ARDS patients than in control subjects. In ARDS, BALF alpha-defensins levels correlated with those of interleukin (IL)-8, and plasma alpha-defensins levels also correlated with Lung Injury Score. Peripheral neutrophil alpha-defensins contents were higher in ARDS than the control. IL-8 dose-dependently stimulated alpha-defensins release from cultured neutrophils and these levels were higher in ARDS than the control. Reverse-phase high performance liquid chromatography showed high plasma levels of pro-defensins, precursors of alpha-defensins from the bone marrow in ARDS, although alpha-defensins in peripheral and BALF neutrophils were mature type. In conclusion, high plasma alpha-defensins in ARDS patients result from the release of pro-defensins from bone marrow, rather than mature alpha-defensins from neutrophils that accumulate in the alveolar space. The alpha-defensins contents of peripheral neutrophils in ARDS are higher and easier to release than control.

Aged↗
PubMed1999

Defensins: key players or bystanders in infection, injury, and repair in the lung?

Antimicrobial peptides have been identified as key elements in the innate host defense against infection. Recent studies have indicated that the activity of antimicrobial peptides may be decreased in cystic fibrosis, suggesting a major role for these peptides in host defense against infection. One of the most intensively studied classes of antimicrobial peptides are defensins. Defensins comprise a family of cationic peptides that in human subjects can be divided into the alpha- and beta-defensin subfamilies. The alpha-defensins are produced by neutrophils and intestinal Paneth's cells, whereas beta-defensins are mainly produced by epithelial cells. Although studies on beta-defensins have so far focused on their antimicrobial activity, studies on alpha-defensins have suggested a role of these peptides in inflammation, wound repair, and specific immune responses. alpha-Defensins, which accumulate in airway secretions of patients with various chronic inflammatory lung disorders, were shown to be cytotoxic toward airway epithelial cells and to induce chemokine secretion in several cell types. Furthermore, the capacity of alpha-defensins to promote bacterial adherence to epithelial cells in vitro further supports a role for these peptides in the pathogenesis of chronic obstructive pulmonary disease and cystic fibrosis. Increased numbers of neutrophils are also present in the airways of patients with asthma, suggesting that neutrophils are involved in the pathogenesis of this disease. Because defensins are able to induce histamine release by mast cells and increase the airway hyperresponsiveness to histamine, it is tempting to speculate that defensins may also contribute to the inflammatory processes in asthma. Besides these proinflammatory effects, alpha-defensins may also display anti-inflammatory activities, including regulation of complement activation and proteinase inhibitor secretion. Finally, defensins may be involved in wound repair because defensins increase epithelial cell proliferation. Thus recent defensin research has revealed potential links between the innate and acquired immune system.

Anti-Infective Agents↗
PubMed1998

Enteric beta-defensin: molecular cloning and characterization of a gene with inducible intestinal epithelial cell expression associated with Cryptosporidium parvum infection.

A growing body of evidence suggests that endogenous antibiotics contribute to the innate defense of mammalian mucosal surfaces. In the cow, beta-defensins constitute a large family of antibiotic peptides whose members have been previously isolated from the respiratory and oral mucosa, as well as circulating phagocytic cells. A novel bovine genomic clone with beta-defensin-related sequence [corrected] related to those of these alpha-defensins was isolated and characterized. The corresponding cDNA was isolated from a small intestinal library; its open reading frame predicts a deduced sequence of a novel beta-defensin, which we designate enteric beta-defensin (EBD). Northern blot analysis of a variety of bovine tissues revealed that EBD mRNA is highly expressed in the distal small intestine and colon, anatomic locations distinct from those for previously characterized beta-defensins. EBD mRNA was further localized by in situ hybridization to epithelial cells of the colon and small intestinal crypts. Infection of two calves with the intestinal parasite Cryptosporidium parvum induced 5- and 10-fold increases above control levels of EBD mRNA in intestinal tissues. An anchored-PCR strategy was used to identify other beta-defensin mRNAs expressed in the intestine. In addition to that of EBD, several low-abundance cDNAs which corresponded to other beta-defensin mRNAs were cloned. Most of these clones encoded previously characterized beta-defensins or closely related isoforms, but two encoded a previously uncharacterized prepro-beta-defensin. Northern blot evidence supported that all of these other beta-defensin genes are expressed at levels lower than that of the EBD gene in enteric tissue. Furthermore, some of these beta-defensin mRNAs were abundant in bone marrow, suggesting that in enteric tissue their expression may be in cells of hematopoietic origin. Extracts of small intestinal mucosa obtained from healthy cows have numerous active chromatographic fractions as determined by an antibacterial assay, and one peptide was partially purified. The peptide corresponded to one of the low-abundance cDNAs. This study provides evidence of beta-defensin expression in enteric tissue and that the mRNA encoding a major beta-defensin of enteric tissue, EBD, is inducibly expressed in enteric epithelial cells. These findings support the proposal that beta-defensins may contribute to host defense of enteric mucosa.

Amino Acid Sequence↗
PubMed2004

The novel antimicrobial peptide beta3-defensin is produced by the amnion: a possible role of the fetal membranes in innate immunity of the amniotic cavity.

BACKGROUND: Innate immunity evolved to eliminate microorganisms before, or after their entry into the tissues, but before enough antigen is available to activate an adaptive, immune response. Innate immunity is so successful that the majority of encountered microbes are neutralized. The beta-defensins are antimicrobial peptides produced by skin and mucosal surfaces and are an integral part of the innate immune system. The ability of the amnion cells, which are epithelial derivatives, to produce antimicrobial beta-defensins has not been explored. OBJECTIVE: This study was undertaken to test the hypothesis that amnion cells synthesize beta-defensins under either basal or stimulated conditions. METHODS: Amnion epithelial FL cells (ATCC CCL 62) were cultured in Ham's F12 and Dulbecco's modified Eagle medium plus 10% fetal calf serum until confluence, then replated into 24-well plates at 1.5 million cells per well. Cells from triplicate wells were harvested after 1, 3, 6, and 24 hours of exposure to microbial wall components (lipopolysaccharide [LPS]: 1 microg/mL or peptidoglycan [PG]: 10 microg/mL). Reverse transcription real-time polymerase chain reaction was performed with the use of human-specific primers for beta1, beta2, beta3, and beta4 defensins to compare basal messenger RNA (mRNA) levels of defensins and in response to treatment. beta-actin was used for standardization. Protein expression was investigated by immunofluorescence of the cells in culture, and by immunohistochemistry in paraffin sections of human fetal membranes from pregnancies with or without histologic chorioamnionitis. RESULTS: Amnion FL cells expressed mRNA for all known beta-defensins with beta3-defensin mRNA levels significantly higher compared with others ( P < .001, 1-way analysis of variance [ANOVA]). beta3 was the only beta-defensin whose mRNA was upregulated in response to the microbial mimics LPS (1-way ANOVA, P = .019) and PG (1-way ANOVA, P = .011). Immunofluorescence confirmed that beta3-defensin protein was present in cultured amnion cells, and upregulated in response to PG and LPS in distinct cells. Similarly, in tissue sections of human fetal membranes amnion epithelium was intensely positive for beta3-defensin protein by immunohistochemistry. Conspicuous beta3-defensin staining was also detected in the chorio-decidua. CONCLUSION: Amnion cells have the ability to produce beta-defensins. The beta3-defensin appears to be the predominant epithelial defensin expressed. Its induction by microbial mimics suggests that the amniotic epithelium may play a role in the innate immunity of the amniotic cavity.

Amnion↗
PubMed2002

Antibacterial peptide defensin is involved in midgut immunity of the soft tick, Ornithodoros moubata.

Two defensin genes A and B were previously demonstrated to be up-regulated by blood feeding in the soft tick, Ornithodoros moubata [Nakajima et al. (2001) Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae). Insect Biochem Mol Biol 31: 747-751]. In this study, two defensin isoforms C and D similar to defensins A and B were newly cloned. A total of four defensins have been identified in O. moubata. All four Ornithodoros defensins are coded as prepro-defensins. Ornithodoros defensin genes consist of four exons and three introns, an organization reported in mussel defensins but not insect defensins. Ornithodoros defensin C and D genes are predominantly expressed in the midgut and up-regulated in response to blood feeding. The mature peptide of the previously cloned Ornithodoros defensin A was purified from the midgut lumen, indicating defensin is secreted into the midgut. These findings confirm the involvement of Ornithodoros defensin in midgut immunity.

Amino Acid Sequence↗
PubMed2006

Crystal structures of human alpha-defensins HNP4, HD5, and HD6.

Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.

Amino Acid Sequence↗
PubMed2006

Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized alpha/beta motifs.

Insect defensins containing cysteine-stabilized alpha/beta motifs (Cs-alpha/beta defensin) are cationic, inducible antibacterial peptides involved in humoral defence against pathogens. To examine trends in molecular evolution of these antimicrobial peptides, sequences similar to the well-characterized Cs-alpha/beta defensin peptide of Anopheles gambiae, using six cysteine residues as landmarks, were retrieved from genomic and protein databases. These sequences were derived from different orders of insects. Genes of insect Cs-alpha/beta defensin appear to constitute a multigene family in which the copy number varies between insect species. Phylogenetic analysis of these sequences revealed two main lineages, one group comprising mainly lepidopteran insects and a second, comprising Hemiptera, Coleoptera, Diptera and Hymenoptera insects. Moreover, the topology of the phylogram indicated dipteran Cs-alpha/beta defensins are diverse, suggesting diversity in immune mechanisms in this order of insects. Overall evolutionary analysis indicated marked diversification and expansion of mature defensin isoforms within the species of mosquitoes relative to non-mosquito defensins, implying the presence of finely tuned immune responses to counter pathogens. The observed higher synonymous substitution rate relative to the nonsynonymous rate in almost all the regions of Cs-alpha/beta defensin of mosquitoes suggests that these peptides are predominately under purifying selection. The maximum-likelihood models of codon substitution indicated selective pressure at different amino acid sites in mosquito mature Cs-alpha/beta defensins is differ and are undergoing adaptive evolution in comparison to non-mosquito Cs-alpha/beta defensins, for which such selection was inconspicuous; this suggests the acquisition of selective advantage of the Cs-alpha/beta defensins in the former group. Finally, this study represents the most detailed report on the evolutionary strategies of Cs-alpha/beta defensins of mosquitoes in particular and insects in general, and indicates that insect Cs-alpha/beta defensins have evolved by duplication followed by divergence, to produce a diverse set of paralogues.

Amino Acid Motifs↗
PubMed2001

Expression of human beta-defensin-1 promotes differentiation of keratinocytes.

Epithelial cells have been shown to express the antibiotic peptides human beta-defensins-1 and 2. While beta-defensin-2 is known to be up-regulated by bacterial factors and proinflammatory mediators, the expression of beta-defensin-1 does not appear to be affected by these mediators. To determine the regulation and function of beta-defensin-1 we analyzed its expression upon stimulation of inflammatory mediators in vitro and ex vivo. In immortalized human cell lines (HaCaT) and nasal polyps beta-defensin-1 was not induced upon incubation with bacteria or proinflammatory mediators, suggesting that the inertness of beta-defensin-1 expression levels is not the result of the shortcoming of HaCaT cells. As proliferation and regeneration play an important role at sites of inflammation, we examined the expression level of beta-defensin-1 in relation to the differentiation and proliferation of HaCaT cells. beta-defensin-1 mRNA levels remained low during proliferation but were highly induced upon differentiation. In contrast, beta-defensin-2 expression was unaffected under these conditions. To examine the function of beta-defensin-1 in cellular proliferation and differentiation processes beta-defensin-1 was overexpressed in keratinocytes. Protein expression analysis of the differentiation marker keratin 10 revealed that its expression is highly induced in the presence of increased concentrations of beta-defensin-1. Hence our data indicate that high expression of beta-defensin-1 promotes cell differentiation processes of keratinocytes.

Blotting, Western↗
PubMed2006

Bioinformatic and expression analysis of novel porcine beta-defensins.

Beta-defensins are a major group of mammalian antimicrobial peptides. Although more than 30 beta-defensins have been identified in humans, only one porcine beta-defensin has been reported. In this article we report the identification and initial characterization of 11 novel porcine beta-defensins (pBD). Using bioinformatic approaches, we screened 287,821 porcine expressed sequence tags for similarity of their predicted peptides to known human beta-defensins and identified full-length or partial sequences for the 11 novel pBDs. Similar to the previously identified pBD1, all of these peptides have a consensus beta-defensin motif. A differential expression pattern for these newly identified genes was found. For example, unlike most beta-defensins, pBD2 and pBD3 were expressed in bone marrow and in other lymphoid tissues including thymus, spleen, lymph nodes, duodenum, and liver. Including pBD2 and pBD3, six porcine beta-defensins were expressed in lung and skin. Several newly identified porcine beta-defensins, including pBD123, pBD125, and pBD129, were expressed in male reproductive tissues, including lobuli testis and some segments of the epididymis. Phylogenetic analysis indicates that in most cases the evolutionary relationship between individual porcine beta-defensins and their human orthologs is closer than the relationship among beta-defensins in the same species. These findings establish the existence of multiple porcine beta-defensins and suggest that the pig may be an ideal model for the characterization of beta-defensin diversity and function.

Amino Acid Sequence↗
PubMed2003

Antibacterial activity and mechanism of action of tick defensin against Gram-positive bacteria.

Defensins are a major group of antimicrobial peptides and are found widely in vertebrates, invertebrates and plants. Invertebrate defensins have been identified from insects, scorpions, mussels and ticks. In this study, chemically synthesized tick defensin was used to further investigate the activity spectrum and mode of action of natural tick defensin. Synthetic tick defensin showed antibacterial activity against many Gram-positive bacteria but not Gram-negative bacteria and low hemolytic activity, characteristic of invertebrate defensins. Furthermore, bactericidal activity against pathogenic Gram-positive bacteria including Bacillus cereus, Enterococcus faecalis and methicillin-resistant Staphylococcus aureus was observed. However, more than 30 min was necessary for tick defensin to completely kill bacteria. The interaction of tick defensin with the bacterial cytoplasmic membrane and its ability to disrupt the membrane potential was analyzed. Tick defensin was able to disrupt the membrane potential over a period of 30-60 min consistent with its relatively slow killing. Transmission electron microscopy of Micrococcus luteus treated with tick defensin showed lysis of the cytoplasmic membrane and leakage of cellular cytoplasmic contents. These findings suggest that the primary mechanism of action of tick defensin is bacterial cytoplasmic membrane lysis. In addition, incomplete cell division with multiple cross-wall formation was occasionally seen in tick defensin-treated bacteria showing pleiotropic secondary effects of tick defensin.

Amino Acid Sequence↗
PubMed2006

Discovery of multiple beta-defensin like homologues in teleost fish.

Using a database mining approach, multiple defensin like genes have been discovered for the first time in fish, in species including zebrafish Danio rerio and the pufferfish, Takifugu rubripes and Tetraodon nigroviridis. They share the common features of vertebrate defensins, including small size, net cationic charge, and six conserved cysteines in the mature region. Based on their cysteine arrangement, the identified fish defensin like peptides resemble beta-defensin family members in birds and mammals. Computing modelling detected three beta-strands in all three zebrafish defensins and an extra N-terminal alpha-helix in one of the peptides. The coding regions of the fish genes contain three exons and two introns, the same as avian defensin genes. In zebrafish and tetraodon, two defensin genes identified are located in the same chromosome. An additional locus containing a third defensin gene has also been found in a different chromosome in zebrafish, demonstrating that multiple defensin loci may be present in fish. Comparative studies suggest that beta-defensins may represent the primitive form of the defensin family, which expanded during evolution by gene or genome duplication. In healthy zebrafish, constitutive expression of defensins was detected by RT-PCR in gill, gonad, gut, kidney, muscle, skin and spleen but the levels and patterns varied for individual defensin genes.

Amino Acid Sequence↗
PubMed1998

Characterization of beta-defensin prepropeptide mRNA from chicken and turkey bone marrow.

Four avian beta-defension prepropeptide cDNA sequences [gallinacins: Gal 1 (synonym CHP 1, chicken heterophil peptide 1), and Gal 2; turkey heterophil peptides: THP 1 and THP 2] were amplified from chicken or turkey bone marrow mRNA samples, respectively. Partial chicken beta-defensin cDNA sequences were obtained using degenerate primers based on chicken peptide sequences (Gal 1/CHP 1 and Gal 2). The complete cDNA sequences of the chicken beta-defensins were then determined by designing specific intrapeptidal primers, from the newly acquired sequence, and pairing one primer with a specific poly A primer tail sequence (3' end) and the other primer with an adapter primer in a 5' rapid amplification of cDNA ends (RACE) reaction. The two, turkey beta-defensins were amplified from turkey marrow using primers designed from chicken beta-defensin preproregions. The complete amino acid sequences for the prepropeptides were deduced for all four avian beta-defensins. Previously, only partial mature peptide sequences for the turkey beta-defensins and complete mature peptide sequences for the chicken beta-defensins were known. All sequences obtained translated accurately to complete and partial amino acid sequences reported for beta-defensins purified from chicken and turkey heterophil granules except for one additional amino acid for Gal 1/CHP 1. The four deduced beta-defensin proregions lack the long, negatively charged propiece reported in classical defensin proregions. These regions are thought to stabilize and inactivate the positively charged mature peptide and target the propeptide to the storage granule. Instead, these beta-defensin proregions are shorter and similar to storage granule-free beta-defensins proregions reported for bovine tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP). These are the first prepropeptide beta-defensins from leukocyte granules to be completely characterized.

Amino Acid Sequence↗
PubMed1999

Levels of antimicrobial molecules defensin and lactoferrin are elevated in the cerebrospinal fluid of children with meningitis.

OBJECTIVE: To measure levels of defensins and lactoferrin in the cerebrospinal fluid (CSF) of children with meningitis. STUDY DESIGN. Prospective descriptive study involving children undergoing lumbar puncture during evaluation for meningitis. METHODS: CSF concentrations of defensins and lactoferrin were determined using enzyme-linked immunosorbent assays on 19 children with bacterial meningitis, 31 children with aseptic meningitis, and 32 control children found to have normal CSF during evaluation for meningitis. Pertinent clinical and laboratory data were gathered on all children. RESULTS: CSF concentrations of both defensins and lactoferrin were elevated markedly in children with bacterial and aseptic meningitis, compared with control children. No control subject had detectable levels of defensins in the CSF. Lactoferrin was undetectable in the CSF of 31 of 32 control subjects. Defensin and lactoferrin levels were significantly higher in the CSF of children with bacterial meningitis than in those with aseptic meningitis. Defensin levels in the CSF of children with bacterial meningitis ranged from 128 ng/mL to 99 430 ng/mL with a mean of 30 311 ng/mL (SD +/- 28 865) and a median of 23 042 ng/mL. Defensin levels in the CSF of children with aseptic meningitis ranged from 0 ng/mL to 1675 ng/mL with a mean of 227 ng/mL (SD +/- 433) and a median of 23 ng/mL. A significant correlation was found between defensin levels in the CSF and the total leukocyte count and the absolute neutrophil count in the CSF of children with bacterial meningitis. Lactoferrin levels in the CSF of children with bacterial meningitis ranged from 184 ng/mL to 31 412 ng/mL with a mean of 13 209 ng/mL (SD +/- 9644) and a median of 10 382 ng/mL. Lactoferrin levels in the CSF of children with aseptic meningitis ranged from 0 ng/mL to 2715 ng/mL with a mean of 1042 ng/mL (SD +/- 878) and a median of 852 ng/mL. No correlation was found between lactoferrin level in the CSF and the total leukocyte count or the absolute neutrophil count in the CSF of children with bacterial meningitis. In our study population, the sum total of CSF defensins and lactoferrin was found to be highly sensitive and specific in delineating bacterial from aseptic meningitis when compared with standard CSF studies. CONCLUSIONS: Significant elevations of defensins and lactoferrin, indicative of endogenous local antimicrobial peptide and polypeptide release, are found in the CSF of children with meningitis. We speculate that elevations in these antimicrobial molecules may reflect the intensity of the host response. Defensins seem to parallel neutrophil activation more closely than lactoferrin. Cumulative levels of CSF defensins and lactoferrin clearly distinguished bacterial meningitis from aseptic meningitis and control patients. Further investigation is warranted to determine the usefulness of measuring defensins and lactoferrin as a diagnostic tool and therapeutic monitor in the evaluation of children with meningitis.

Anti-Infective Agents↗
PubMed1993

Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis.

We measured concentrations of defensins (human neutrophil peptides) in the plasma of healthy volunteers and patients with sepsis and meningitis. When a sensitive enzyme immunoassay was used, defensins were detected in plasma samples from 13 of 24 healthy blood donors, with a mean +/- SD of 42 +/- 53 ng/ml. Defensin levels in plasma samples from seven patients with sepsis at the onset of disease ranged from 900 ng/ml to 170,000 ng/ml. In 10 patients with meningitis in the initial phase of disease, plasma defensin concentrations ranged from 120 ng/ml to 910 ng/ml. Defensin concentrations in the plasma of both patient groups were significantly higher than those in healthy blood donors (p << 0.01), and patients with sepsis had higher defensin levels than patients with meningitis (p < 0.01). Defensin levels were significantly (p < 0.01) lower after the beginning of specific antibiotic therapy. Defensin concentrations in the plasma of patients with sepsis and meningitis correlated only weakly (r = 0.38) with blood neutrophil count. In vitro studies of defensin added to plasma indicated that all defensin was bound to plasma proteins. At high concentrations (1000 micrograms/ml), defensins caused precipitation of plasma proteins. Because plasma defensin levels may reflect neutrophil activation at sites of infection and inflammation, studies of the clinical utility of defensin ELISA are indicated.

Bacteremia↗
PubMed2003

Expression of antimicrobial defensins in the male reproductive tract of rats, mice, and humans.

Defensins are antimicrobial peptides that play a major role in innate immunity. Using reverse transcriptase-polymerase chain reaction, immunochemistry, or both, we performed a search of all presently known defensins in rat testis, epididymis, and isolated testicular cells; in mouse testis and epididymis; and in human testis and ejaculates. In the rat, all alpha- and beta-defensins except RNP-4 were expressed within the testis, whereas alpha-defensins RNP1-2, RNP-4, and beta-defensins RBD-1 and RBD-2 were present within the epididymis. In the mouse, the cryptdin transcripts CRS1C, mBD-1, and mBD-2 were detected in the testis and epididymis, whereas mBD-3 and mBD-4 were expressed only in the epididymis, and CRS4C was absent in both organs. In the human testis, transcripts for four known defensins were expressed with the consistent exception of HBD-2 and HBD-3. In rat interstitial tissue, resident macrophages expressed most of the defensins studied, whereas Leydig cells produced only RBD-2. In contrast, all studied defensins except RNP-4 were present in the seminiferous tubules. Within these tubules, peritubular and Sertoli cells expressed most of the studied alpha- and beta-defensins, whereas spermatogonia displayed only alpha-defensins, but at relatively high levels. Meiotic pachytene spermatocytes expressed only beta-defensins, whereas postmeiotic spermatids and their cytoplasmic lobes displayed both types. In humans, the HBD-1 peptide was expressed mainly in the germ line from pachytene spermatocytes to late spermatids. The peptide was also present in ejaculated spermatozoa and seminal plasma, where multiple soluble forms were present. Finally, high salt concentration or dithiothreitol-sensitive cationic extracts from human seminal plasma were indeed found to display antimicrobial activity. We conclude that the male reproductive tract produces defensins that most probably assume an important, innate organ defense system against pathogens.

Animals↗
PubMed1995

A novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity.

Hemocytes of the horseshoe crab (limulus) contain a family of arthropodous peptide antibiotics, termed the tachyplesin family, and antibacterial protein, called anti-LPS factor, of which the former is located in the small (S) granules and the latter in the large (L) granules of the hemocytes. In our ongoing studies on granular components, we have identified here a novel defensin-like substance present in both L- and S-granules. This substance strongly inhibits the growth of Gram-negative and -positive bacteria, and fungi, such as Candida albicans. The isolated substance, tentatively termed "big defensin," consists of 79 amino acid residues, of which the COOH-terminal 37 residues have a sequence similar to those of mammalian neutrophil-derived defensins, especially rat defensin. Characterization of the disulfide motif in big defensin indicated that the disulfide array is identical to that of beta-defensins from bovine neutrophils. One clear structural difference is that the limulus hemocyte-derived big defensin has an extension of the NH2-terminal hydrophobic sequence with 35 amino acid residues followed by the COOH-terminal cationic defensin portion. This amphipathic nature of big defensin seems likely to be associated with its potent antibacterial activity. Furthermore, antibacterial activities of the NH2-terminal hydrophobic region and the COOH-terminal defensin portion separated by tryptic digestion are significantly different: the former displays a more potent activity against Gram-positive bacteria, whereas the latter is more potent against Gram-negative bacteria. Big defensin, therefore, may prove to represent a new class of defensin family possessing two functional domains with different antimicrobial activities.

Amino Acid Sequence↗
PubMed2004

Rapid evolution and diversification of mammalian alpha-defensins as revealed by comparative analysis of rodent and primate genes.

Mammalian alpha-defensins constitute a family of cysteine-rich, cationic antimicrobial peptides produced by phagocytes and intestinal Paneth cells, playing an important role in innate host defense. Following comprehensive computational searches, here we report the discovery of complete repertoires of the alpha-defensin gene family in the human, chimpanzee, rat, and mouse with new genes identified in each species. The human genome was found to encode a cluster of 10 distinct alpha-defensin genes and pseudogenes expanding 132 kb continuously on chromosome 8p23. Such alpha-defensin loci are also conserved in the syntenic chromosomal regions of chimpanzee, rat, and mouse. Phylogenetic analyses showed formation of two distinct clusters with primate alpha-defensins forming one cluster and rodent enteric alpha-defensins forming the other cluster. Species-specific clustering of genes is evident in nonprimate species but not in the primates. Phylogenetically distinct subsets of alpha-defensins also exist in each species, with most subsets containing multiple members. In addition, natural selection appears to have acted to diversify the functionally active mature defensin region but not signal or prosegment sequences. We concluded that mammalian alpha-defensin genes may have evolved from two separate ancestors originated from beta-defensins. The current repertoires of the alpha-defensin gene family in each species are primarily a result of repeated gene duplication and positive diversifying selection after divergence of mammalian species from each other, except for the primate genes, which were evolved prior to the separation of the primate species. We argue that the presence of multiple, divergent subsets of alpha-defensins in each species may help animals to better cope with different microbial challenges in the ecological niches which they inhabit.

Amino Acid Sequence↗
PubMed2005

Evolution of caprine and ovine beta-defensin genes.

Defensins comprise an important family of anti-microbial peptides. Among vertebrates, numerous defensin genes have been detected, but their evolutionary background is still discussed. We investigated the molecular evolution and variability of beta-defensins of Caprini via sequence analyses of defensin introns. Screening of several domestic and wild species of Caprini revealed a total of 13 discrete beta-defensin coding sequences, with three of them described before this study. Phylogenetic analyses revealed that the array of newly described defensin genes is of monophyletic origin and has arisen in numerous independent duplication events after separation of the ancestral defensins. As a result of that scenario, recent defensin genes are distributed in a species-specific manner. Values of synonymous and non-synonymous substitutions demonstrated that both modes of evolutionary pressure, positive as well as negative selection, have acted. In addition, conservation of some beta-defensin exons is demonstrated. Discrimination of certain beta-defensin genes was possible only due to intron-specific differences. Therefore, sequence analyses restricted to the exons would result in underestimation of the number of beta-defensin genes. Our study shows that for reconstruction of the phylogenetic history data of defensin introns are more appropriated. Comparisons among the amino acid sequences show moderate substitutions without changing the net charge of the mature peptides.

Amino Acid Sequence↗