SEARCH · PubMed Health

Results for “alpha-Defensins”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources

PubMed1985

Primary structures of three human neutrophil defensins.

The primary structures of three human neutrophil antimicrobial peptides (HNP) were determined. The peptides, HNP-1, HNP-2, and HNP-3, which we have termed defensins, were rich in cystine, arginine, and aromatic residues, but were devoid of free sulfhydryl groups and carbohydrate moieties. They were 29-30 residues in length and identical in sequence in all but their amino terminal residues. The defensins were homologous in sequence to peptides of similar size and biological activity previously purified from rabbit polymorphonuclear leukocytes, but unrelated to other neutrophil proteins of known sequence. 11 amino acid residues of the human defensins, including all six cysteinyl residues, were invariantly conserved in the six rabbit members of this multigene peptide family. That similarly structured antimicrobial peptides are present in both rabbit and human leukocytes supports their purported role as cidal agents in phagocyte-mediated host defense.

Amino Acid Sequence↗

PubMed1988

Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations.

We tested the in vitro susceptibility of Candida albicans to three defensins from human neutrophilic granulocytes (HNP-1, 2, and 3), a homologous defensin from rabbit leukocytes (NP-1), and four unrelated cationic peptides. Although the primary amino acid sequences of HNP-1, 2, and 3 are identical except for a single amino-terminal amino acid alteration, HNP-1 and HNP-2 killed C. albicans but HNP-3 did not. C. albicans blastoconidia were protected from HNP-1 when incubations were performed in the absence of oxygen or in the presence of inhibitors that blocked both of its mitochondrial respiratory pathways. Neither anaerobiosis nor mitochondrial inhibitors substantially protected C. albicans exposed to NP-1, poly-L-arginine, poly-L-lysine, or mellitin. Human neutrophilic granulocyte defensin-mediated candidacidal activity was inhibited by both Mg2+ and Ca2+, and was unaffected by Fe2+. In contrast, Fe2+ inhibited the candidacidal activity of NP-1 and all of the model cationic peptides, whereas Mg2+ inhibited none of them. These data demonstrate that susceptibility of C. albicans to human defensins depends both on the ionic environment and on the metabolic state of the target cell. The latter finding suggests that leukocyte-mediated microbicidal mechanisms may manifest oxygen dependence for reasons unrelated to the production of reactive oxygen intermediates by the leukocyte.

Anaerobiosis↗

PubMed1989

Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity.

Defensins are small, cysteine-rich antimicrobial peptides that are abundant in human, rabbit, and guinea pig neutrophils (PMN). Three defensins (human neutrophil peptide defensin [HNP]-1, HNP-2, and HNP-3) constitute between 30 and 50% of the total protein in azurophil granules of human PMN. We examined the mechanism of HNP-mediated bactericidal activity against Escherichia coli ML-35 (i-, y-, z+) and its pBR322-transformed derivative, E. coli ML-35p. Under conditions that supported bactericidal activity, HNP-1 sequentially permeabilized the outer membrane (OM) and inner membrane (IM) of E. coli. Coincident with these events, bacterial synthesis of DNA, RNA, and protein ceased and the colony count fell. Although these events were closely coupled under standard assay conditions, OM permeabilization was partially dissociated from IM permeabilization when experiments were performed with E. coli that had been plasmolyzed by mannitol. Under such conditions, the rate and extent of bacterial death more closely paralled loss of IM integrity than OM permeabilization. Electron microscopy of E. coli that had been killed by defensins revealed the presence of striking electron-dense deposits in the periplasmic space and affixed to the OM. Overall, these studies show that HNP-mediated bactericidal activity against E. coli ML-35 is associated with sequential permeabilization of the OM and IM, and that inner membrane permeabilization appears to be the lethal event.

Blood Bactericidal Activity↗

PubMed1989

Monocyte-chemotactic activity of defensins from human neutrophils.

We investigated the monocyte-chemotactic activity of fractionated extracts of human neutrophil granules. Monocyte-chemotactic activity was found predominantly in the defensin-containing fraction of the neutrophil granules. Purified preparations of each of the three human defensins (HNP-1, HNP-2, HNP-3) were then tested. HNP-1 demonstrated significant chemotactic activity for monocytes: Peak activity was seen at HNP-1 concentrations of 5 X 10(-9) M and was 49 +/- 20% (mean +/- SE, n = 9) of that elicited by 10(-8) M FMLP. HNP-2 (peak activity at 5 X 10(-9) M) was somewhat less active, yielding 19 +/- 10% (n = 11). HNP-3 failed to demonstrate chemotactic activity. Checkerboard analysis of monocyte response to HNP-1 and HNP-2 confirmed that their activity was chemotactic rather than chemokinetic. Neutrophils demonstrated a low level of response to defensins but this reaction was primarily chemokinetic. Defensins may play a role in the recruitment of monocytes by neutrophils into inflammatory sites.

Blood Proteins↗

PubMed1996

Intramolecular inhibition of human defensin HNP-1 by its propiece.

We examined mechanisms that protect host defense cells from their cytotoxic effector molecules. Human neutrophil peptides (HNP) 1-3 are microbicidal and cytotoxic defensins, initially synthesized as 94-amino acid preproHNP(1-94), cotranslationally proteolyzed to proHNP(20-94), then converted by removal of the anionic propiece to mature HNP(65-94)(HNP-1 and -3) and HNP(66-94) (HNP-2). We hypothesized that during synthesis and subcellular sorting the anionic propiece inhibits the cytotoxicity of the cationic defensin. We expressed preproHNP-1 cDNA in recombinant baculovirus-infected insect cells that secreted the normally transient proHNP-1(20-94) into the medium. Cyanogen bromide cleaved proHNP-1(20-94) at the fortuitously located Met64 to yield mature recombinant HNP-1(65-94) and unlinked propiece. Recombinant and native HNP-1 purified from PMN were identical as judged by mass spectrometry, retention time in reverse-phase high performance liquid chromatography, migration on acid-urea polyacrylamide gels, and reaction with a conformation-specific antibody. Recombinant and native HNP-1 had comparable microbicidal activity towards Listeria monocytogenes and were similarly potent in permeabilizing K562 leukemia cells, but proHNP-1(20-94) was virtually inactive in both assays. Addition of unlinked propiece (proHNP-1(20-64) with Met64-->homoserine) inhibited the bactericidal and cell-permeabilizing activity of mature HNP-1 in a dose-dependent manner. Linked, and to a lesser extent unlinked, propiece interfered with the binding of HNP-1 to target cells. The propiece thus acts as an efficient intramolecular inhibitor of defensin HNP-1 cytotoxicity.

Amino Acid Sequence↗

PubMed1993

The gene encoding the human corticostatin HP-4 precursor contains a recent 86-base duplication and is located on chromosome 8.

The human neutrophil-derived cationic peptide HP-4 exhibits corticostatic activity on adrenal cells and is an L-type calcium channel agonist at nanomolar concentrations. Complementary DNA clones encoding the HP-4 precursor have been isolated from a human bone marrow cDNA library by screening with oligonucleotide probes. The nucleotide sequence shares about 72% identity with the cDNA encoding defensin HP-1, but differs from it, and from other genes of this family characterized to date, by an extra 83-base segment. This extra segment is not adjacent to an intron and is apparently the result of a recent duplication within the coding region corresponding to most of the mature HP-4 peptide. The predicted amino acid sequence shows the HP-4 precursor structure to be typical of this family of molecules. By analysis of DNA from a pannel of hamster/human hybrid cell lines, the HP-4 gene was found to be on chromosome 8, as is the gene for human peptide HP-1. Comparison with the few sequences of other corticostatin/defensin genes available does not indicate distinct lineages of corticostatic and noncorticostatic peptides, since HP-1 and HP-4 cDNA sequences share more identity with each other than either shares with cDNAs encoding rabbit MCP-1 or MCP-2, or guinea pig GNCP-1.

Amino Acid Sequence↗

PubMed1992

[Effects of human defensin HNP-1 on the production of tumor necrosis factor-alpha by human blood monocytes in vitro].

The influence of human defensin HNP-1 on the synthesis of tumor necrosis factor-alpha (TNF-alpha) by human blood monocytes was studied. It was shown that TNF-alpha production by human monocytes activated by SAC or PMA was augmented in the presence of HNP-1 concentrations of 10(-8)-10(-9) M. HNP-1 alone induced no synthesis of TNF-alpha. High concentration of HNP-1 (10(-4) M) was cytotoxic for human monocytes.

Blood Bactericidal Activity↗

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↗

PubMed1991

The isolation and identification of multiple forms of the neutrophil granule peptides from human leukemic cells.

HP-1 is a 30-residue cysteine- and arginine-rich peptide of the human neutrophil primary granule and is the most abundant human representative of the family of peptides variously called defensins and corticostatins. Peptides belonging to this family have many biological activities including the non-oxidative destruction of ingested microorganisms, the inhibition of adrenocorticotropin-stimulated synthesis of glucocorticoids, monocyte chemotaxis, the non-cytolytic inhibition of [3H]thymidine incorporation in HL-60 promyelocyte-like cells and the stimulation of nifedipine-sensitive calcium channels. Using a combination of reversed-phase and size-exclusion high performance liquid chromatography and an HP-1 radio-immunoassay, three immunoreactive peptides were detected and isolated from the promyelocyte-like cell line, HL-60, and from leukocytes of patients with chronic myelogenous and chronic lymphocytic leukemias. One of these peptides was HP-1 itself. A second was identified by gas-phase Edman microsequencing as an amino-terminally extended fragment of the HP-1 precursor which we call HP1-56. The third is likely to arise from enzymatic cleavage of the precursor at a dibasic site. Of the leukemic cells the greatest amount of HP1-56 relative to HP-1 was found in cells from a patient in myeloblastic crisis but overall the richest source of HP1-56 relative to HP-1 was found to be in fetal lung tissue. HP1-56 is difficult to detect in normal peripheral neutrophils and its presence in cells that are actively biosynthesizing primary granule components such as HL-60 may make it useful for studying the biosynthesis of granule polypeptides, their ontogeny, and possibly as a marker protein for leukemic diseases.

Amino Acid Sequence↗

PubMed1989

Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family.

Primary (azurophil) granules of neutrophils contain proteins which play a major role in the killing and digestion of bacteria in the phagolysosome. We have isolated and characterized a novel antimicrobial peptide from the azurophil granule fraction of discontinuous Percoll gradients. We have named this peptide human neutrophil peptide 4 (HNP-4) based on its structural similarity to a group of antimicrobial polypeptides known as defensins (HNP 1-3). Using size exclusion and reverse-phase high performance liquid chromatography, HNP-4 was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. The amino acid sequence determined from isolated HNP-4 and from tryptic fragments of reduced and alkylated peptide is: NH2-Val-Cys-Ser-Cys-Arg-Leu-Val-Phe-Cys-Arg-Arg-Thr-Glu- Leu-Arg-Val-Gly-Asn-Cys-Leu-Ile-Gly-Gly-Val-Ser-Phe-Thr-Tyr-Cys-Cys-Thr- Arg-Val - COOH. Based on this sequence, HNP-4 has a calculated molecular weight of 3715 and a theoretical pI of 8.61. HNP-4 shows structural similarity to the family of three human defensins. HNP-4 and the defensins have identical cysteine backbones and, like the defensins, HNP-4 is rich in arginine (15.2 mol %). However, the amino acids at 22 of the 33 positions differ between HNP-4 and human defensins. Further, HNP-4 is significantly more hydrophobic than the defensins, as determined by its retention time on reverse-phase high performance liquid chromatography. In vitro, purified HNP-4 was shown to kill Escherichia coli, Streptococcus faecalis, and Candida albicans. Compared to a mixture of the other human defensins, HNP-4 was found to be approximately 100 times more potent against E. coli and four times more potent against both S. faecalis and C. albicans.

Amino Acid Sequence↗

PubMed1989

Determination of the disulfide array in the human defensin HNP-2. A covalently cyclized peptide.

HNP-2 is a 29-residue peptide present in human neutrophils and is a member of the defensin family of antimicrobial peptides. All defensins contain an invariant disulfide infrastructure comprised of 6 half-cystine residues. The disulfide structure of HNP-2 was determined using a novel method to identify the cross-links involving the amino- and carboxyl-terminal cysteine residues. A derivative of HNP-2 was synthesized by covalent modification of the terminal cysteine residues. This derivative was purified, characterized, and subjected to exhaustive proteolytic digestion. Characterization of purified proteolytic fragments by amino acid analysis and/or sequence analysis identified an oligopeptide containing all 6 cystine residues. This oligopeptide was subjected to a single cycle of Edman degradation to cleave the peptide bond linking 2 adjacent cysteines. Purification and characterization of the Edman reaction products allowed for assignment of the disulfide array in HNP-2, revealing a cystine motif unique to the defensin peptide family. Further, the covalent structure of HNP-2 was found to be cyclic as one disulfide links the amino- and carboxyl-terminal cysteine residues. HNP-2 is the only polypeptide known to possess such a configuration.

Amino Acid Sequence↗

PubMed1986

In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes.

We examined the activity of defensins, cysteine-rich cationic peptides that are abundant in the cytoplasmic granules of human and rabbit granulocytes, against various tumor targets. The three human defensins, HNP-1, HNP-2, and HNP-3, lysed human and murine targets in chromium release and dye exclusion assays. Defensin-mediated tumor cell lysis was concentration-dependent, inhibited by serum, and dependent on temperature-sensitive events. Lysis was first detected by three hours of incubation and it reached a plateau between eight and 14 hours. In vitro exposure of murine teratocarcinoma cells to HNP 1-3 abrogated their oncogenicity in vivo. Nonmalignant target cells were also susceptible to defensin-mediated lysis. Four rabbit granulocyte defensins exerted marked (NP-1, NP-2) or moderate (NP-3a, NP-3b) cytotoxic activity, whereas defensin NP-5 was not cytotoxic. When tumor cells were incubated with human defensins in combination with hydrogen peroxide, synergistic cytotoxicity was detected. As defensins are released from granulocytes by various stimuli, their release could contribute to extracellular cytotoxicity which is independent of reactive oxygen intermediates.

Animals↗

PubMedDate not supplied

[In vitro effects of peptides of the corticostatin/defensin family on production of mitogen-induced cytokines].

Cytokines are autocrine, paracrine and endocrine glycoproteins that interact with specific cell receptors and have pleiotropic effects. Increasing evidence indicates that cytokines, immune interferon (IFN-gamma) and interleukin 6 (IL-6) among others, modulate hypothalamic-pituitary-adrenal function. Corticostatins/defensins are a family of cationic peptides recently isolated from phagocytic cells of myeloid lineage. Four peptides have been isolated from human neutrophils: HP-1, 2, 3 and 4. As defensins they participate in immunosurveillance against viruses, bacteria and fungi. Some members of the family are also able to inhibit ACTH-induced steroidogenesis. Among human peptides, only HP-4 is corticostatic. We previously demonstrated that HP-1 and HP-4 inhibit in vitro the spontaneous and cytokine-inducible natural killer activity of human peripheral blood mononuclear cells (PBMC) and potentiate cortisol-dependent inhibition. The present work was carried out to determine whether two human corticostatins/defensins, HP-1 and HP-4, were able to modulate in vitro IFN-gamma and IL-6 production by human PBMC stimulated with phytohemagglutinin or Concanavalin A. IFN-gamma was titrated using biological assay with WISH cells as indicators and vesicular stomatitis virus as the challenge virus. IL-6 was measured by means of enzyme amplified sensitivity immunoassay. Both HP-1 and HP-4 significantly reduced cytokine production. Our data indicate that HP-1 and HP-4 are novel modulators of lymphocyte functions in vitro. Their depressing properties on ACTH-induced steroidogenesis and on cytokine production add complexity to neuroendocrine-immune circuits involving hypothalamic-pituitary-adrenal function.

Anti-Bacterial Agents↗

PubMed1995

The pro region of human neutrophil defensin contains a motif that is essential for normal subcellular sorting.

Human defensins (human neutrophil peptides) HNP 1-3 are 29-30 amino acid antibiotic and cytotoxic peptides highly abundant in the cytoplasmic granules of polymorphonuclear leukocytes. The peptides are produced from 94 amino acid (aa) prepropeptides by proteolytic cleavage of the signal sequence and stepwise removal of the 44-45 aa anionic propiece. To study the role of the propiece, we constructed five in-frame deletions in preproHNP-1 cDNA between the signal peptidase site and the amino-terminus of the mature defensin region (aa 21-64). The wild type HNP-1 cDNA and the deletion mutants were ligated into the pBabe-Neo retroviral vector, expressed in GP+E86 packaging derivative of NIH 3T3 cells, then transduced into the 32D cl3 granulocytic cell line. For each construction and both cell lines, we measured the accumulation of the various defensin forms in cells and media by 24-hour labeling or pulse-chase with 35S-cysteine- and immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Deletions in the amino-terminal two-fifths of the propiece, delta 21-28 and delta 21-38, had only minor effects on defensin biosynthesis in both cell lines and did not interfere with the accumulation of mature defensin in the granules of 32D cl3 cells. Deletions in the carboxyterminal three-fifths of the propiece (delta 21-51 and delta 21-64) diminished net defensin synthesis, blocked constitutive secretion of prodefensin in both cell lines, and interfered with defensin accumulation in cytoplasmic granules of 32D cl3 cells. These effects were reproduced by the smaller deletion delta 40-51, which contains highly conserved secondary structure. The propiece segment 40-51 appears to be essential for the subcellular trafficking and sorting of HNP-1 defensin.

3T3 Cells↗

PubMed1995

The subcellular localization of defensins and myeloperoxidase in human neutrophils: immunocytochemical evidence for azurophil granule heterogeneity.

We used immunogold electron microscopy to examine the distribution of human defensins 1-3 (HNP1-3) and myeloperoxidase (MPO) in the cytoplasmic granules of thin-sectioned, normal human neutrophils. The presence of MPO, defensins, or both was noted in 388 (62.3%) of 623 granule profiles. Whereas MPO and defensins colocalized in 32.5% of such granules, 32.7% demonstrated only MPO and 34.8% demonstrated only defensins. The staining densities of MPO and HNP1-3 showed a low correlation, even when both molecules were present in the same granules (r = 0.09), indicating that they were not present in fixed relative proportions even when colocalized. The low partial correlation coefficient (r = -0.23) between MPO and HNP1-3 for the 388 labeled granules controlling for granule area suggested that the deposition of MPO and defensins might even be negatively correlated. The distribution of MPO and defensins within the cytoplasmic granules of human neutrophils followed a "non-colocalization and multi-compartmental" model that conflicts with the classical notion of azurophil granules as a homogeneous population of organelles marked by the presence of MPO.

Blood Proteins↗

PubMed1994

A marked decrease in defensin mRNA in the only case of congenital neutrophil-specific granule deficiency reported in Japan.

A deficiency of defensins (human neutrophil peptides, HNP) has been previously demonstrated in two individuals with congenital neutrophil-specific granule deficiency (SGD), but its genetic basis is not well understood. We have studied another case of SGD, the only case reported in Japan, to elucidate the molecular basis of defensin deficiency. Using Western blot analysis, we showed for the first time that HNP-4, a novel human defensin, was also deficient in the patient's neutrophils. Northern blot analysis demonstrated that the defensin mRNA was absent in the bone marrow cells of the patient. Limited Southern blot analysis, using a defensin cDNA probe, did not reveal any differences in fragment patterns of the genomic DNA between the patient and the control. We propose that the defensin deficiency in our patient is a consequence of a marked decrease in defensin mRNA in neutrophil precursors. Our findings were consistent with a previous speculation suggesting that the SGD defect occurs at the level of transcription.

Adult↗

PubMed1993

[Activity of defensins from human neutrophils against Mycobacterium avium complex].

We examined the activity of defensins from human neutrophils against Mycobacterium avium complex (MAC). MAC were cultured in the presence of defensins at 37 degrees C for 4-48 hours. After incubation, colony counts were enumerated by colony-forming units (CFU). Human neutrophil peptide-1 (HNP-1) at 5 micrograms/ml had the ability to kill MAC. We tested 8 strains of MAC. HNP-1 was successful in killing every strain, although the degree of killing varied from strain to strain. We also found that HNP-2 and HNP-3 were as effective in killing MAC as HNP-1. We suggest that defensins play an important role in the host defenses against MAC.

Blood Bactericidal Activity↗