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PubMed1998

Antimicrobial peptide expression is developmentally regulated in the ovine gastrointestinal tract.

Antimicrobial peptides are abundant components of the innate immune system present in species throughout the plant and animal kingdoms. In mammals, these immune peptides have been localized to epithelial tissues of the pig, mouse, rat, cow and human gastrointestinal tracts. We have identified in sheep two members of the beta-defensin antimicrobial peptide gene family that are expressed in a unique pattern throughout the gastrointestinal tract. Sheep beta-defensin 1 mRNA is the most prevalent from tongue to colon with the exception of the distal ileum, where beta-defensin 2 mRNA predominates. Sheep beta-defensin expression varies significantly between animals and is developmentally regulated both pre- and postnatally. These changes in antimicrobial peptide expression may correlate with anatomical differentiation as well as physiologic adaptations to extra-uterine life.

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↗
PubMed1998

Localization and genomic organization of sheep antimicrobial peptide genes.

Antimicrobial peptides are an abundant and diverse component of animal innate immunity. Within mammalian species, defensins and cathelicidins are the two principal antimicrobial peptide families. We identified and sequenced ten new sheep genes which encode potential antimicrobial peptides including two beta-defensins and eight cathelicidins. We mapped the two-exon beta-defensin genes to sheep chromosome 26 and the four-exon cathelicidin genes to sheep chromosome 19 using sheep-hamster somatic cell hybrids in conjunction with flow-sorted sheep chromosomes. These assignments confirm homology between sheep, cattle, mouse, and human antimicrobial peptide gene families. Contig construction for the sheep cathelicidin gene family demonstrates that three genes, OaDodeA, OaDodeB, and OaMAP-34, are present head-to-tail in a 14.5 kb region, and that four proline/arginine-rich genes, OaBac5, OaBac7.5, OaBac11, and OaBac6, are arranged head-to-tail in a region covering 30.5 kb. This richly diverse family of sheep cathelicidin peptides is encoded in a gene array which may reflect the mechanism of its evolution.

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↗
PubMed1996

Production of active bovine tracheal antimicrobial peptide in milk of transgenic mice.

Tracheal antimicrobial peptide (TAP) is a member of the beta-defensin family of antibiotic peptides found in the tracheal mucosa of the cow. TAP gene expression in the bovine airway is inducible by lipopolysaccharide and inflammatory mediators, suggesting that it functions to protect the upper airway from infection. Limited availability of bovine TAP (bTAP) has precluded investigation of its potential utility in agriculture and medicine. To overcome this problem, transgenic mice expressing bTAP using an expression vector driven by control sequences from the murine whey acidic protein (WAP) gene have been generated. The WAP/bTAP transcript was detected in RNA isolated from mammary tissue of transgenic females. bTAP was purified to homogeneity from milk via acid precipitation, reverse-phase HPLC, and ion-exchange chromatography. This milk-derived bTAP had antimicrobial activity against Escherichia coli. Amino-terminal peptide sequencing confirmed the identity of this material as a bTAP isoform. bTAP available from a mammary gland bioreactor will allow evaluation of bTAP for use as an antibiotic in agriculture and medicine.

Amino Acid Sequence↗
PubMed1997

Epithelial antibiotic induced in states of disease.

Epithelial defensins provide an active defense against the external microbial environment. We investigated the distribution and expression of this class of antimicrobial peptides in normal cattle and in animals in varying states of disease. beta-defensin mRNA was found to be widely expressed in numerous exposed epithelia but was found at higher levels in tissues that are constantly exposed to and colonized by microorganisms. We observed induction in ileal mucosa during chronic infection with Mycobacterium paratuberculosis and in bronchial epithelium after acute infection with Pasteurella haemolytica. It has been proposed that expression of antimicrobial peptides is an integral component of the inflammatory response. The results reported here support this hypothesis and suggest that epithelial defensins provide a rapidly mobilized local defense against infectious organisms.

Animals↗
PubMed1996

Six antimicrobial peptide genes of the cathelicidin family map to bovine chromosome 22q24 by fluorescence in situ hybridization.

Six phage clones containing gene members of the family of antimicrobial peptides named cathelicidins, were mapped to bovine chromosome 22q24, by means of fluorescence in situ hybridization. The mapping data suggest the clustering of cathelicidins into a CATHL@ locus, in a similar manner as for beta-defensins, another family of antimicrobial peptides, defining the locus DEFB@ mapped to 27q13-->q14.

Animals↗
PubMed1996

Systematic isolation of circulating human peptides: the concept of peptide trapping.

The structural determination of circulating human peptides is essential to determine their correct posttranslationally processed form. Human hemofiltrate from patients with end stage renal disease is accessible in large quantities and is used as a source for the preparation of circulating peptides. After complete peptide extraction from hemofiltrate, a systematic separation with different chromatographic techniques is achieved. Single peptides are selected according to their mass and chromatographic elution position. Following chromatographic purification, amino acid sequence analysis is performed in combination with data base search. The identification of circulating peptides leads to numerous fragments resulting from cleavage of larger plasma proteins as well as to the discovery of new peptide hormones. The results obtained so far give insight into the degradation of plasma proteins such as fibrinogen, which results in the generation of fragments with biological activity themselves and in the identification of a novel cytokine HCC-1, the first member of beta-defensins in humans, hBD-1, and different peptides not present in any data base.

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↗