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PubMed1997

Defensin stimulates the binding of lipoprotein (a) to human vascular endothelial and smooth muscle cells.

There is evidence to suggest that elevated plasma levels of lipoprotein (a) [Lp(a)] represent a risk factor for the development of atherosclerotic vascular disease, but the mechanism by which this lipoprotein localizes to involved vessels is only partially understood. In view of studies suggesting a link between inflammation and atherosclerosis and our previous finding that leukocyte defensin modulates the interaction of plasminogen and tissue-type plasminogen activator with cultured human endothelial cells, we examined the effect of this peptide on the binding of Lp(a) to cultured vascular endothelium and vascular smooth muscle cells. Defensin increased the binding of Lp(a) to endothelial cells approximately fourfold and to smooth muscle cells approximately sixfold. Defensin caused a comparable increase in the amount of Lp(a) internalized by each cell type, but Lp(a) internalized as a consequence of defensin being present was not degraded, resulting in a marked increase in the total amount of cell-associated lipoprotein. Abundant defensin was found in endothelium and in intimal smooth muscle cells of atherosclerotic human cerebral arteries, regions also invested with Lp(a). These studies suggest that defensin released from activated or senescent neutrophils may contribute to the localization and persistence of Lp(a) in human vessels and thereby predispose to the development of atherosclerosis.

Adult↗
PubMed2006

Elevated BALF concentrations of alpha- and beta-defensins in patients with pulmonary alveolar proteinosis.

Defensins are endogenous antibiotics and regulators of inflammation, immunity and wound repair. Their concentrations are substantially increased in bronchoalveolar lavage fluid (BALF) of patients with infectious lung diseases. alpha-defensin (HAD) levels are also elevated in patients with idiopathic pulmonary fibrosis (IPF) and correlated with the decline in pulmonary function tests, suggesting the association of defensins with the pathogenesis of interstitial lung diseases. The aim of this study was to determine the profile of defensins in interstitial lung diseases. Serum and BALF levels of HAD and beta-defensin 1 and 2 (HBD-1, and -2) were measured by radioimmunoassay in 63 patients with interstitial lung diseases, including idiopathic pulmonary alveolar proteinosis (PAP), IPF, nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP) and pulmonary sarcoidosis, and in 9 healthy volunteers as controls. Levels of HAD in BALF of patients with PAP were significantly higher than those in controls and patients with COP and sarcoidosis. Serum levels of HAD in all groups were significantly higher than those in controls. Levels of HBD-1 and -2 in BALF of patients with PAP were extremely high in all subjects. Serum levels of HBD-1 were higher in all patient groups, with the exception of those with PAP, and those of HBD-2 were also higher in patients with IPF and sarcoidosis, compared with controls. BALF of PAP patients, but not IPF patients and controls, expressed antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Our findings suggest different kinetics of HAD and HBD-1 and -2 in serum and BALF of interstitial lung diseases and that these antimicrobial peptides in the airway lumen may contribute to prevention of bacterial airway infections in PAP.

Adult↗
PubMed2000

Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin.

A novel trypsin inhibitor was extracted from the seeds of Cassia fistula by a process successively involving soaking seeds in water, extraction of the seeds in methanol, and extraction of the cell wall material at high ionic strength. The protease inhibitor (PI) was subsequently purified by chromatography on carboxymethylcellulose, gel filtration and reversed phase HPLC (RP-HPLC). Electrospray ionization mass spectrometry (ESMS) of the oxidized from of the PI yielded an average molecular mass of 5458.6+/-0.8 Da. Edman sequencing of the PI yielded a full-length 50 amino acid sequence inferred to contain eight cysteines and with a calculated average molecular mass (fully oxidized form) of 5459.3 Da, in agreement with the observed mass. The C. fistula seed PI is homologous to the family of plant defensins (gamma-thionins), which have four disulfide linkages at highly conserved locations. The C. fistula PI inhibits trypsin (IC(50) 2 µM), and is the first known example of a plant defensin with protease inhibitory activity, suggesting a possible additional function for some members of this class of plant defensive proteins. C. fistula seeds also contain a 9378 Da lipid transfer protein (LTP) homologue, other LTPs, a 7117 Da protein copurifying with PI activity and a 5144 Da defensin which does not inhibit trypsin. The complete sequence of the 5144 Da defensin was determined by Edman sequencing, yielding a calculated average molecular mass (oxidized form) of 5144.1 Da, in agreement with the mass observed by ESMS. The likely trypsin inhibitory residue on the 5459 Da defensin is Lysine-25, the corresponding amino acid being Tyrosine-25 in the homologous 5144 Da defensin that is not a trypsin inhibitor.

Journal Article↗
PubMed1999

Peptide localization and gene structure of cryptdin 4, a differentially expressed mouse paneth cell alpha-defensin.

Paneth cells in crypts of the small intestine express antimicrobial peptides, including alpha-defensins, termed cryptdins in mice. Of the known Paneth cell alpha-defensins, the cryptdin 4 gene is unique, because it is inactive in the duodenum and expressed at maximal levels in the distal small bowel (D. Darmoul and A. J. Ouellette, Am. J. Physiol. 271:G68-G74, 1996). With a cryptdin 4-specific antibody, immunohistochemical staining of ileal Paneth cells was strong and specific for cytoplasmic granules, demonstrating that this microbicidal peptide is a secretory product of Paneth cells in the distal small intestine. Consistent with the pattern of cryptdin 4 mRNA distribution along the length of the gut, the cryptdin 4 peptide was not detected in duodenum. Structurally, the cryptdin 4 gene resembles other Paneth cell alpha-defensin genes. Its two exons, transcriptional start site, intron, splice sites, and 3' flanking sequences are characteristic of the highly conserved mouse alpha-defensin genes. However, in the region upstream of the transcriptional initiation site, the cryptdin 4 gene contains a repeated 130-bp element that is unique to this alpha-defensin gene. Every independent cryptdin 4 genomic clone examined carries the repeated element, which contains putative recognition sequences for TF-IID-EIIA, cMyc-RS-1, and IgHC.2/CuE1.1; the repeat proximal to the start of transcription replaces DNA at the corresponding position in other mouse alpha-defensin genes. We speculate that this unique duplicated element may have a cis-acting regulatory role in the positional specificity of cryptdin 4 gene expression.

Amino Acid Sequence↗
PubMed1994

Biosynthesis of defensins and other antimicrobial peptides.

Defensins are small (about 30 amino acid residues) cationic antimicrobial peptides with a conserved framework of six disulphide-linked cysteines. Human defensin HNP-1 and the closely related HNP-3 are amphiphilic dimers that act in part by permeabilizing cell membranes. Defensin mRNAs, abundant in neutrophilic promyelocytes, certain non-human macrophages and Paneth cells, encode 94-100 amino acid prepropeptides. PreproHNP-1 is post-translationally processed to inactive proHNP-1 then to mature HNP-1 stored in granules. Bactenecin Bac-5 and perhaps other related neutrophil peptides are also synthesized as prepropeptides but are stored in granules as inactive propeptides. Their conserved cathelin-like propiece inhibits the cysteine protease, cathepsin L, and is removed only during granule release. Charge neutralization of mature peptide by the propiece is seen in both probactenecins and prodefensins. In contrast the propiece of cecropins is very short and proceropins are microbicidal. The pathways that convert myeloid preprodefensins to defensins are specific to myeloid cells but the signal for targeting to granules also functions in non-myeloid granulated cells. The truncation of the anionic propiece by deletion mutagenesis dramatically reduces defensin synthesis, suggesting that the propiece may assist in peptide stabilization, folding or subcellular transport. Despite some similarities in the mechanism of action of the various families of antimicrobial peptides, their precursors differ greatly, presumably owing to differing functions of the propieces.

Amino Acid Sequence↗
PubMed1993

The structure of neutrophil defensin genes.

Defensins are a family of microbicidal peptides abundant in the granules of mammalian neutrophils, in rabbit alveolar macrophages, and in human and murine intestinal Paneth cells. We cloned and sequenced the genes of three neutrophil-specific defensins. Human HNP-1 and HNP-3 are nearly identical and rabbit NP-3a is closely related. The four known neutrophil-specific defensin genes are strikingly similar in the structure and organization of their three exons and two introns, but the three defensin genes expressed in macrophages (MCP-1 and -2) or Paneth cells (HD-5) are organized differently: HD-5 had only two exons, and MCP-1 and -2 have a comparatively short first intron. The diverse genomic organization of defensin genes may contribute to their cell-specific expression.

Amino Acid Sequence↗
PubMed1997

The mouse genome encodes a single homolog of the antimicrobial peptide human beta-defensin 1.

The cysteine-rich beta-defensin peptides are broad-spectrum bactericidal agents expressed in epithelial and myeloid tissues. The human beta-defensin-1 (hBD-1) gene maps adjacent to the human alpha-defensin cluster and is expressed in the respiratory, gastrointestinal and genitourinary tracts. Here, we characterize a mouse beta-defensin gene (mBD-1) which is: (1) closely related to hBD-1 both in sequence and gene organization; (2) expressed at high levels in the mouse kidney and at lower levels in brain, heart, lung, uterus, spleen, skeletal muscle, stomach, and small intestine; and (3) maps to mouse chromosome 8 at or near the location of the mouse alpha-defensin genes. These data indicate that mBD-1 is a close homolog of hBD-1, and suggest that analysis of its role in mouse host defense may provide significant insights into human epithelial innate immunity.

Amino Acid Sequence↗
PubMed1992

NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1.

The solution structure of two homologous naturally occurring antimicrobial peptides, rabbit defensin NP-2 and human defensin HNP-1, have been determined by two-dimensional nuclear magnetic resonance spectroscopy, distance geometry, and restrained molecular dynamics calculations. The structure of these defensins consists of an antiparallel beta-sheet in a hairpin conformation, a short region of triple-stranded beta-sheet, several tight turns, and a loop region that has a well-defined local structure but with a global orientation that is not well-defined with respect to the rest of the molecule. The solution structures of these two peptides are compared with the solution and crystal structures of two other homologous defensins. The structures for the defensins are also compared with known structures of other naturally occurring antimicrobial peptides.

Animals↗
PubMed1999

Cloning and expression of bovine neutrophil beta-defensins. Biosynthetic profile during neutrophilic maturation and localization of mature peptide to novel cytoplasmic dense granules.

beta-Defensins are microbicidal peptides implicated in host defense functions of phagocytic leukocytes and certain surface epithelial cells. Here we investigated the genetic structures and cellular expression of BNBD-4, -12, and -13, three prototypic bovine neutrophil beta-defensins. Characterization of the corresponding cDNAs indicated that BNBD-4 (41 residues) derives from a 63-amino acid prepropeptide and that BNBD-12 (38 residues) and BNBD-13 (42 residues) derive from a common 60-amino acid precursor (BNBD-12/13). The peptides were found to be encoded by two-exon genes that are closely related to bovine epithelial beta-defensin genes. BNBD-4 and BNBD-12/13 mRNAs were most abundant in bone marrow, but were expressed differentially in certain non-myeloid tissues. In situ hybridization and immunohistochemical studies demonstrated that BNBD-4 synthesis is completed early in myelopoiesis. BNBD-12 was localized exclusively to the novel dense granules, organelles that also contain precursors of cathelicidins, antimicrobial peptides that undergo proteolytic processing during phagocytosis. In contrast to cathelicidins, Western blot analyses revealed that mature beta-defensins are the predominant organellar form in myeloid cells. Stimulation of neutrophils with phorbol myristate acetate induced secretion of BNBD-12, indicating that it is co-secreted with pro-cathelicidins. The exocytosis of BNBD-12 by activated neutrophils reveals different mobilization pathways for myeloid alpha- and beta-defensins.

Amino Acid Sequence↗
PubMed1996

Evaluation of susceptibility of gram-positive and -negative bacteria to human defensins by using radial diffusion assay.

Defensins are small cationic bactericidal peptides present abundantly in the granules of polymorphonuclear neutrophils (PMNs). Human PMNs contain four defensins termed HNP-1 to HNP-4. We used a new assay system in agar plates, the radial diffusion assay, to evaluate the effects of human defensins against gram-positive and -negative bacteria. A crude mixture of HNP-1, -2, and -3 (crude HNPs) was purified from human PMN extracts by reversed-phase high-pressure liquid chromatography (RP-HPLC). The different components were later separated by RP-HPLC and gel permeation chromatography. We compared the antibacterial activities of purified HNP-1, -2, and -3 against Escherichia coli, Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus, and methicillin-resistant S. aureus strains using the radial diffusion assay. The antibacterial activities of HNP-1 and HNP-2 against all strains tested were similar to those of the crude HNPs, but the activity of HNP-3 was less than those of the other defensins. To quantitate the activities of HNPs against different bacteria, we defined the minimal dose of crude HNPs forming a detectable clear zone around the bacteria as the minimal inhibitory dose (MID) and determined the MIDs for 10 strains of E. coli, 12 strains of P. aeruginosa, 10 strains of methicillin-susceptible S. aureus, and 12 strains of methicillin-resistant S. aureus isolates, including clinical isolates. In general, the MIDs of the HNPs were similar against similar bacterial species. However, the MIDs for P. aeruginosa were higher than those for the other organisms tested. The radial diffusion assay is suitable as a screening test for measuring the susceptibilities of isolates to defensins, because it is sensitive and simple and has good reproducibility.

Anti-Bacterial Agents↗
PubMed2004

Theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry.

We tested the ability of 20 synthetic theta defensins to protect cells from infection by type 1 and type 2 herpes simplex viruses (HSV-1 and -2, respectively). The peptides included rhesus theta defensins (RTDs) 1 to 3, originally isolated from rhesus macaque leukocytes, and three peptides (retrocyclins 1 to 3) whose sequences were inferred from human theta-defensin (DEFT) pseudogenes. We also tested 14 retrocyclin analogues, including the retro, enantio, and retroenantio forms of retrocyclin 1. Retrocyclins 1 and 2 and RTD 3 protected cervical epithelial cells from infection by both HSV serotypes, but only retrocyclin 2 did so without causing cytotoxicity or requiring preincubation with the virus. Surface plasmon resonance studies revealed that retrocyclin 2 bound to immobilized HSV-2 glycoprotein B (gB2) with high affinity (K(d), 13.3 nM) and that it did not bind to enzymatically deglycosylated gB2. Temperature shift experiments indicated that retrocyclin 2 and human alpha defensins human neutrophil peptide 1 (HNP 1) to HNP 3 protected human cells from HSV-2 by different mechanisms. Retrocyclin 2 blocked viral attachment, and its addition during the binding or penetration phases of HSV-2 infection markedly diminished nuclear translocation of VP16 and expression of ICP4. In contrast, HNPs 1 to 3 had little effect on binding but reduced both VP16 transport and ICP4 expression if added during the postbinding (penetration) period. We recently reported that theta defensins are miniature lectins that bind gp120 of human immunodeficiency virus type 1 (HIV-1) with high affinity and inhibit the entry of R5 and X4 isolates of HIV-1. Given its small size (18 residues), minimal cytotoxicity, lack of activity against vaginal lactobacilli, and effectiveness against both HSV-2 and HIV-1, retrocyclin 2 provides an intriguing prototype for future topical microbicide development.

Amino Acid Sequence↗
PubMed2003

Defensin pattern in chronic gastritis: HBD-2 is differentially expressed with respect to Helicobacter pylori status.

BACKGROUND/AIMS: Recent reports have suggested that Helicobacter pylori infection induces the mucosal antibiotic peptide human beta defensin 2 (HBD-2). Therefore, the present study investigated mRNA and peptide expression of four different defensins in the upper gastrointestinal tract in patients with H pylori positive and negative chronic gastritis. MATERIALS/METHODS: Biopsies from the oesophagus to the duodenum were taken during routine gastroscopy in 71 individuals. Total RNA was extracted and the reverse transcription polymerase chain reaction was performed with primers for human defensins 5 and 6 (HD-5/6) or HBD-1 and HBD-2. Paraffin wax embedded tissue from gastric resections was tested for HD-5, HBD-1, and HBD-2 by immunohistochemistry. RESULTS: Helicobacter pylori colonisation was associated with an increased percentage of positive biopsies with respect to HBD-2 in the corpus (p < 0.05). Helicobacter pylori had no impact on the gastric expression of HD-5 and HBD-1, whereas HD-6 was increased in the fundus. The abundant expression of alpha defensins in the duodenum and beta defensins in the oesophagus served as a positive control in each individual. Immunohistochemical analysis confirmed the presence of the HD-5, HBD-1, and HBD-2 peptides in gastric resection specimens. CONCLUSIONS: The recently described induction of HBD-2 upon H pylori infection was confirmed in a clinical setting of chronic gastritis. This phenomenon may be mediated by components of the pathogen itself or may occur secondary to immune events in chronic inflammation.

Adult↗
PubMed1997

Human airway epithelia express a beta-defensin.

Recent evidence suggests that the surface fluid secreted by human airway epithelial cells contains antimicrobial activity. The identification of the factor(s) responsible for the antibacterial activity in airway surface fluid may bring new insights into lung defense mechanisms and the pathogenesis of disease. Defensins are small cationic peptides with broad-spectrum antimicrobial activity, and are therefore candidates for the bacterial killing factor in airway surface fluid. They are produced in mucosal epithelia and neutrophils of several species. To date no defensins have been isolated from human airway epithelia; however, such peptides are produced by bovine airway epithelia. A cDNA fragment for a human beta-defensin-1 (hBD-1) was recently cloned from the kidney. We cloned the full-length coding sequence for hBD-1 from human airway epithelia. The amino acid sequence for hBD-1 shared homology with the signal/propiece and mature peptides of other beta-defensins, including bovine tracheal antimicrobial peptide and lingual antimicrobial peptide. By Northern analysis, a single approximately 400-nt hBD-1 transcript was expressed in airway epithelia. Ribonuclease protection analysis demonstrated that hBD-1 transcripts were more abundant in the conducting airways than the gas exchange regions of human lung and that the expression was developmentally regulated. Human beta-defensin-1 may contribute to the antimicrobial activity of airway surface fluid and play a role in the mucosal defenses of the lung.

Adult↗
PubMed2002

[Glucocorticoid hormones in immunomodulating effect of defensins].

Defensins are a family of antimicrobial cationic peptides localized mainly in neutrophile granulocytes. The defensins are known to display corticostatic activity by means of suppression of stress- and ACTH-induced rise in corticosterone level in the blood. The present study examines influence of defensin fractions with different corticostatic activity on suppressor functions of T-lymphocytes. It was shown that corticostatic effects of defensins are revealed under an increased level of glucocorticoids in the blood after hydrocortisone application. Defensins were found to limit the inhibitory action of glucocorticoids on the suppressor functions of T-lymphocytes. After adrenalectomy this phenomenon was not observed.

Adjuvants, Immunologic↗
PubMed1992

[Investigation of microbicidal activity of neutrophil defensins against leptospires].

Defensins play an important role in oxygen-independent microbicidal mechanisms of neutrophils. They are effective against many bacteria, fungi and enveloped viruses. However, the effect of defensins upon leptospires has not been studied. In the present report, human defensins (i.e. HNP, a mixture of HNP1, HNP2 and HNP3 were prepared from human polymorphonuclear neutrophils by chromatography on Sephadex G-100 and then on Biogel P-10. Rabbit defensin NP1 was purified from rabbit peritoneal granulocytes by preparative acid urea polyacrylamide gel electrophoresis. By using the most-probable-number procedure, HNP and NP1 were tested in vitro for the killing of leptospira interrogans serogroup icteroheamorrhagiae serovar lai strain 017. NP1 was highly effective. When leptospires of strain 017 were incubated with 1 microgram/ml of NP1 at 30 degrees C for 4 hours, > 99% of these organisms were killed. HNP was less potent than NP1, and at 50 micrograms/ml, it killed > 90% of leptospires. As is also the case for the killing of bacteria, NP1 was active against leptospires in nutrient-free buffer, whereas HNP required the addition of glucose. The data suggest that defensins could play a major role in the killing of leptospires by neutrophils.

Animals↗
PubMed1992

Characterization of defensin precursors in mature human neutrophils.

Human defensins HNP-1 and -3 are broad spectrum antimicrobial peptides that are synthesized by human neutrophils as 94 amino acid (aa) precursors that require proteolytic removal of 64 amino-terminal residues to produce the mature defensins. Recent studies have shown that the early proteolytic processing events include two sequential cleavages, each removing 19 amino-terminal aa residues, that yield 75 aa and 56 aa prodefensins, respectively. The subsequent processing steps that convert these 56 aa prodefensins to mature (30 aa) HNP-1 and HNP-3 are not yet known. We identified four new defensin precursors in mature normal neutrophils. The most abundant of these were two 39 aa forms that resulted from the monobasic endoproteolytic cleavage of proHNP-1 and proHNP-3. The presence of two proline residues in the vicinity of this newly defined scission site suggested that this cleavage might be "proline-directed." Smaller amounts of the 34 aa and 32 aa prodefensin forms were also found. It remains to be established if these 39, 34, and 32 aa prodefensins are obligate intermediates in the prodefensin processing pathway, or arise from side reactions. In either event, because these prodefensin intermediates accounted for only 0.25% of the total defensin content, proteolytic conversion of 56 aa prodefensins to mature defensins appears to be a highly efficient process.

Amino Acid Sequence↗
PubMed1995

Neutrophilic defensins penetrate the blood-brain barrier.

Defensins are small, cationic, cyclic peptides that are abundantly stored in granules of neutrophils. Defensins non-specifically interact with membranes by forming weakly ion-selective pores. Here we demonstrate immunolocalization of defensin-secreting cells in human brain. Defensins, secreted by activated granulocytes, apparently are not prevented by the blood-brain barrier (BBB) from diffusing across cerebral endothelium to penetrate the neuropil for a considerable distance from the granulocyte. This is in contrast to other neutrophil proteins like the granule-associated enzyme elastase or the cytosolic protein MRP-14, which are strictly localized to the cytoplasm or granules of neutrophils. Thus, defensins, known chemokinetic and chemotactic molecules, display a unique distribution at BBB sites.

Antibodies, Monoclonal↗
PubMed1993

Defensins promote fusion and lysis of negatively charged membranes.

Defensins, a family of cationic peptides isolated from mammalian granulocytes and believed to permeabilize membranes, were tested for their ability to cause fusion and lysis of liposomes. Unlike alpha-helical peptides whose lytic effects have been extensively studied, the defensins consist primarily of beta-sheet. Defensins fuse and lyse negatively charged liposomes but display reduced activity with neutral liposomes. These and other experiments suggest that fusion and lysis is mediated primarily by electrostatic forces and to a lesser extent, by hydrophobic interactions. Circular dichroism and fluorescence spectroscopy of native defensins indicate that the amphiphilic beta-sheet structure is maintained throughout the fusion process. Taken together, these results support the idea that protein-mediated membrane fusion depends not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form a three-dimensional amphiphilic structure, which promotes the efficient mixing of the lipids between membranes. A molecular model for membrane fusion by defensins is presented, which takes into account the contributions of electrostatic forces, hydrophobic interactions, and structural amphiphilicity.

1,2-Dipalmitoylphosphatidylcholine↗