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At least 19 recordsLinked to original sources
PubMed1995

Solution structure of bovine neutrophil beta-defensin-12: the peptide fold of the beta-defensins is identical to that of the classical defensins.

The solution structure is reported for bovine neutrophil beta-defensin-12 (BNBD-12), a member of the beta-defensin family of antimicrobial peptides. Structural constraints in the form of proton-proton distances, dihedral angles, and hydrogen bond constraints were derived from two-dimensional, homonuclear magnetic resonance spectroscopy experiments. The three-dimensional structure of BNBD-12 was calculated using distance geometry and restrained molecular dynamics. An ensemble of structures with low NOE constraint violation energies revealed a precisely defined triple-stranded, antiparallel beta-sheet as the structural core of the peptide. The N-terminal beta-strand and three locally well-defined tight turns form a hydrophobic face. Conserved isoleucine and glycine residues form a beta-bulge structure which initiates a beta-hairpin secondary structure motif composed of the second and C-terminal beta-strands. The beta-hairpin contains numerous charged residues and forms the cationic face of BNBD-12. The N-terminal residues were found to be disordered, due to an absence of tertiary NOEs. The triple-stranded beta-sheet, the beta-bulge preceding the hairpin, and the cationic/hydrophobic amphiphilic character are definitive features of all defensin structures determined to date. Further, we predict that the tracheal antimicrobial peptide (TAP) and the recently described gallinacins will have tertiary structures similar to that of BNBD-12.

Amino Acid Sequence↗
PubMed1995

hBD-1: a novel beta-defensin from human plasma.

We report the isolation and characterization of a novel peptide with significant sequence homology to beta-defensins from human blood filtrate. The human beta-defensin-1 (hBD-1) is a short basic peptide of 36 amino acid residues. It contains six cysteines forming three intramolecular disulfide bonds. The molecular mass of hBD-1 is 3928.6 Da. Cloning of the specific cDNA confirmed the amino acid sequence of the native peptide. hBD-1 shares the nine conserved amino acids characteristic for beta-defensins from respiratory epithelial cells and neutrophils of cattle and chicken leukocytes. hBD-1 is present in nanomolar concentration in human plasma.

Amino Acid Sequence↗
PubMed1996

Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells.

We compared the expression of human alpha- and beta-defensins by various human tissues. mRNA for alpha-defensins HNP1-3, abundant in bone marrow, was detected in peripheral blood leukocytes, spleen and thymus by RT-PCR, which revealed alpha-defensins HD5 and HD6 only in the small intestine. In contrast, the pancreas and kidney expressed high levels of hBD-1 and lower levels of this beta-defensin were found in many organs by RT-PCR (salivary gland > trachea > prostate and placenta > thymus, testis, small intestine). hBD-1 mRNA was produced constitutively by cultured normal human epithelial cells derived from the trachea, bronchi, small airways and the mammary gland. These largely non-overlapping tissue distributions of human alpha- and beta-defensins suggest that hBD-1 may be positioned to defend epithelial cells and mucosae from infection, whereas expression of HNP1-3 in neutrophils and HD5 and HD6 in Paneth cells allows these alpha-defensins to participate in systemic and small intestinal host defenses, respectively.

Amino Acid Sequence↗
PubMed1993

Characterization of the disulfide motif in BNBD-12, an antimicrobial beta-defensin peptide from bovine neutrophils.

BNBD-12, a prototype beta-defensin peptide from bovine neutrophils, was chosen for determination of the disulfide motif in this family of tridisulfide antimicrobial peptides. Disulfide-containing fragments of BNBD-12 were generated by incubation with trypsin, and the amino acid composition of one tryptic fragment allowed for the assignment of one of the three disulfides. The remaining two disulfides, contained in a 16-residue tryptic oligopeptide, were characterized by amino acid analysis of fragments generated by a single round of Edman degradation which cleaved the Cys-Cys peptide bond present near the carboxyl terminus of BNBD-12. Cleavage of this bond produced two disulfide-containing oligopeptides, the compositions of which provided unambiguous assignments of the disulfides involved. The cystine motif in BNBD-12 differs from that of classical defensins, and indicates that the beta-defensins and defensins must have differently folded chains, though they share several functional properties.

Amino Acid Sequence↗
PubMed1993

Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils.

A new family of cysteine-rich antimicrobial peptides from bovine neutrophils was isolated and characterized. Thirteen structurally homologous peptides were purified to homogeneity from a granule-rich cytoplasmic fraction of purified blood neutrophils. The complete sequences of the peptides were determined by a combination of enzymatic digestion, Edman degradation, and additional biochemical characterization of the carboxyl termini. The peptides are characterized by a highly cationic 38-42-residue chain which includes 6 invariantly spaced cysteines which form three disulfides. They share a highly conserved consensus sequence which is also found in a recently described epithelial antimicrobial peptide from bovine trachea. The in vitro antibacterial activities of the 13 neutrophil peptides, determined in assays using Staphylococcus aureus and Escherichia coli as test organisms, demonstrated that each peptide possessed antimicrobial activity, and that several were as active as the most potent neutrophil defensin, rabbit NP-1. Though the structural and functional attributes of the bovine neutrophil peptides are similar to those of defensins, the two peptide families are distinguished by their unique consensus sequences and additionally by differing tridisulfide motifs. We therefore propose that this new defensin-like antimicrobial peptide family be named beta-defensins.

Amino Acid Sequence↗
PubMed1996

Coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha.

Peptides with potent broad-spectrum antibiotic activity have been identified in many animal species. Recent investigations have demonstrated that epithelial cells are a site of antibiotic peptide expression, suggesting that these peptides contribute to host defense at mucosal surfaces. Expression of tracheal antimicrobial peptide (TAP), a member of the beta-defensin family of peptides, is inducible in cultured tracheal epithelial cells (TEC) upon challenge with bacterial lipopolysaccharide (LPS) (G. Diamond, J.P. Russell, and C.L. Bevins, Proc. Natl. Acad. Sci. USA, in press). In this study, an anchored reverse transcriptase PCR strategy was used to determine if TAP was the sole beta-defensin isoform expressed upon stimulation of the cells with LPS. In addition to TAP, a second class of cDNA clones which encoded lingual antimicrobial peptide (LAP), a beta-defensin peptide recently isolated from a different mucosal site, the bovine tongue, was identified (B.S. Schonwetter, E.D. Stolzenberg, and M. Zasloff, Science 267:1645-1648, 1995). Northern (RNA) blot analysis demonstrated in vivo expression of LAP mRNA in tracheal mucosa. Levels of LAP mRNA were higher in cultured TEC challenged with either LPS or tumor necrosis factor alpha than in control cells. Thus, a response of TEC exposed to inflammatory mediators is induction of antibiotic-encoding genes, including both TAP and LAP. This work complements the in vivo studies of Schonwetter et al. (cited above), which showed elevated levels of LAP mRNA in squamous epithelial cells of the tongue near sites of tissue injury and inflammation, by suggesting possible mediators of the in vivo observation. Together these lines of investigations support the hypothesis that inducible expression of endogenous antibiotic peptides by inflammatory mediators characterizes local defense of mammalian mucosal surfaces.

Amino Acid Sequence↗
PubMed1994

Gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes.

We purified three homologous antimicrobial peptides ('gallinacins') from chicken leukocytes, examined their antimicrobial activity in vitro, and established their primary sequences by a combination of gas phase microsequencing and on-line LC-ESI-MS analysis of endo- and exoprotease peptide digests. The peptides contained 36-39 amino acid residues, were relatively cationic due to their numerous lysine and arginine residues, and each contained 3 intramolecular cystine disulfide bonds. Gallinacins showed primary sequence homology to the recently delineated beta-defensin family, heretofore found only in the respiratory epithelial cells and neutrophils of cattle, suggesting that beta-defensins originated at least 250 million years ago, before avian and mammalian lineages diverged. The 9 invariant residues (6 cysteines, 2 glycines and 1 proline) common to avian gallinacins and bovine beta-defensins are likely to constitute the essential primary structural motif of this ancient family of host-defense peptides.

Amino Acid Sequence↗
PubMed1996

Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica.

Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum. To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 x gav, and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity. Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 6 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 microM ZnCl2 (zinc saline solution) induced killing of P. haemolytica and other bacteria comparable to defensins and beta-defensins [minimal bactericidal concentration (MBC)50 range, 0.01-0.06 mM] but not in 0.14 M NaCl/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC50 range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC50 values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide. P. haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min. Ultrastructurally, cells contained flocculated intracellular constituents. In contrast to cationic defensins and beta-defensins, surfactant-associated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution. They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.

Amino Acid Sequence↗
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↗
PubMed1995

Epithelial antibiotics induced at sites of inflammation.

The role of antimicrobial peptides in epithelial defense is not fully understood. An epithelial beta-defensin, lingual antimicrobial peptide (LAP), was isolated from bovine tongue and the corresponding complementary DNA cloned. LAP showed a broad spectrum of antibacterial and antifungal activities. LAP messenger RNA abundance was markedly increased in the epithelium surrounding naturally occurring tongue lesions. This increase coincided with the cellular hallmarks of acute and chronic inflammation in the underlying lamina propria, supporting a role for epithelial antimicrobial peptides as integral components of the inflammatory response.

Amino Acid Sequence↗
PubMed1994

Isolation of antimicrobial peptides from avian heterophils.

Five bactericidal peptides (chicken heterophil peptides CHP1 and CHP2; turkey heterophil peptides THP1, THP2, and THP3) were purified from avian heterophil granules. All peptides were cationic and rich in cysteine, arginine, and lysine. The complete amino acid sequence, consisting of 39 amino acids, was determined for CHP1. This peptide had a molecular weight of 4481 as determined by mass spectrometry. Partial NH2-terminal amino acid sequences were obtained for the remaining peptides. Both chicken peptides and THP1 shared sequence homology at 22 residues and a cysteine motif which was similar to that of bovine beta-defensins. THP2 and THP3 were homologous to each other but were not homologous to the other three and had a unique cysteine motif. Peptides CHP1, CHP2, and THP1 killed Staphylococcus aureus and Escherichia coli in vitro, whereas THP2 and THP3 killed only S. aureus in vitro.

Amino Acid Sequence↗
PubMed2026

Comparative Genomics-Guided Epitope Prioritization and in Silico Design of a Multi-Epitope DNA Vaccine Candidate Against Megalocytivirus pagrus 1.

Megalocytivirus pagrus 1 infection is a World Organisation for Animal Health-listed aquatic animal disease caused by a virus species comprising the RSIV, ISKNV, and TRBIV genogroups. Here, we integrated comparative genomics and immunoinformatics to prioritize a multi-epitope protein construct, pMEV, and to design a DNA vaccine candidate encoding it, with emphasis on RSIV-type infection relevant to rock bream aquaculture. Analysis of 61 complete genomes identified 28 core gene clusters, from which myristoylated membrane protein (MMP) and major capsid protein (MCP) were prioritized as source antigens for epitope screening. Four cytotoxic T-cell, five helper T-cell, and five linear B-cell epitope candidates were selected based on sequence-based screening and exploratory peptide-MHC docking. The selected epitopes were assembled with rock bream beta-defensin-3, PADRE, and peptide linkers to generate the 283-aa pMEV construct. Sequence-based physicochemical analyses indicated properties relevant to subsequent structural and expression-based evaluation, while computationally refined structural modeling identified nine putative conformational B-cell epitope regions. TLR3 docking, normal mode analysis, and a 200-ns molecular dynamics simulation characterized the structural behavior of the selected computational complex without inferring receptor activation. C-ImmSim further generated model-dependent generic humoral and helper T-cell-associated response patterns within a mammalian-based simulation framework. Finally, the pMEV coding sequence was codon-optimized and incorporated into an in silico pcDNA3.1(+)-based DNA vaccine design. Collectively, this study provides a comparative genomics-guided framework for prioritizing an experimentally testable multi-epitope DNA vaccine candidate against M. pagrus 1, while construct expression, immunogenicity, and protective efficacy remain to be evaluated experimentally.

Animals↗
PubMed1994

Defensins.

Defensins are widely distributed and abundant 3-4 kDa antimicrobial peptides that are variable cationic and contain six disulfide-paired cysteines. Three structurally distinct peptide families have been identified: 'classical' defensins, beta-defensins and insect defensins. In many animal species, defensin genes are found in clusters with substantial sequence variability outside the core disulfide-linked cysteines. Defensin peptides have been found in the granules of phagocytes and intestinal Paneth cells, on epithelial surfaces of the intestine and the trachea, and in the hemolymph of insects. They are produced from larger precursors by stepwise, tissue-specific, proteolytic processing, a production resembling that of peptide hormones. Microbes in the phagocytic vacuoles of granulocytes and certain macrophages encounter high concentrations of defensins. Increased transcription of defensin genes and stimulus-dependent release of pre-synthesized defensin-containing cytoplasmic granules contribute to the local antimicrobial response.

Amino Acid Sequence↗
PubMed1991

Bactericidal potency and mechanistic specificity of neutrophil defensins against bovine mastitis pathogens.

Two neutrophil defensins, rabbit peptides NP-1 and NP-5, were examined for their in vitro antibacterial activity against a panel of Gram-positive and Gram-negative organisms isolated from mastitic cows. Incubation for 60 minutes with 5 micrograms/ml of NP-1 in 10 mM sodium phosphate buffer resulted in substantial killing (greater than 95%) of all organisms tested. Although NP-5 was virtually inactive under these same conditions, supplementation of the incubation mixture with dilute nutrient media sensitized otherwise resistant organisms to this defensin. For both NP-1 and NP-5, bactericidal activity was dependent on time and concentration. Our findings demonstrate that the spectrum of defensin bactericidal activities include clinically important bovine pathogens. Further, the data demonstrate that NP-1 and NP-5, peptides which are homologous in 18 of their 33 residue positions, possess distinct mechanisms of action.

Amino Acid Sequence↗
PubMed1991

A major human epididymis-specific cDNA encodes a protein with sequence homology to extracellular proteinase inhibitors.

The amino acid sequence of a major human epididymis-specific protein was deduced from the nucleotide sequence of its cloned cDNA. The encoded product showed characteristics of a secretory protein, with a signal peptide followed by a small (approximately 10-kDa), acidic (pI 4.3), and cysteine-rich polypeptide. The positions of half-cysteines suggested that it was a two-domain member of the family of 'four-disulfide core' proteins to which a number of proteinase inhibitors belong. Southern blot analyses of human genomic DNA showed that the transcripts originated from a single copy gene. Northern blot and in situ transcript hybridization specifically localized the HE4 (human epididymis gene product) mRNA to the epithelial cells of the epididymal duct, predominantly within the distal sections. A possible function in sperm maturation as indicated by amino acid similarities to extracellular proteinase inhibitors of genital tract mucous secretions is discussed in the context of its tissue-specific transcription.

Aged↗
PubMed1993

Tissue-specific gene expression as an indicator of epididymis-specific functional status in the boar, bull and stallion.

cDNA probes derived from genes expressed specifically in the human epididymis were used to examine gene expression in the epididymides of boar, bull and stallion by Northern hybridization. Two probes for the HE1 and HE4 gene products were found to recognize tissue-specific transcripts in all three species, with a regionally differential distribution within the epididymis. Additionally, antibodies recognizing the HE4 protein were shown to react specifically in the epididymis of the boar and bull. An extensive study of the boar showed that, whereas mRNA for the HE1-homologue was up-regulated markedly only at puberty, the HE4-homologue was already present at moderate levels prepubertally. The distribution of the HE1-homologue changed at sexual maturity from a maximum in the cauda epididymis in the 3-4-week-old pig, to a maximum in the corpus/caput region in the adult, while the shift was in the opposite direction for the HE4-homologue. Evidently, gene expression is not fixed regionally through epididymal development in this species. The abdominal epididymis of a hemicryptorchid pig also showed a differential change in expression for the two gene products by comparison with the scrotal testis from the same animal. The results suggest that the HE1 and HE4 gene homologues may be sensitive markers for physiological changes within the mammalian epididymis, and that the boar could prove a useful model to examine the regulation of these human epididymal transcripts.

Animals↗