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T Ganz

Publications and source records attributed to T Ganz.

At least 19 recordsLinked to original sources
PubMed1996

Defensin modulates tissue-type plasminogen activator and plasminogen binding to fibrin and endothelial cells.

Defensins are naturally occurring antimicrobial peptides that may participate in host defense against microorganisms. We previously reported that the amino acid sequence of leukocyte defensins resembles the lysine-binding site in the kringles of plasminogen and that defensin inhibits fibrinolysis mediated by tissue-type plasminogen activator (tPA) and plasminogen. In the present paper we analyze the mechanisms of this inhibition. Defensin binds specifically to cultured human umbilical vein endothelial cells (HUVEC) (half-maximal binding = 3 microM) as well as to fibrin. At saturating concentrations (5-10 microM), defensin stimulates the maximum binding of plasminogen to HUVEC and to fibrin approximately 10-fold. However, defensin inhibits plasminogen binding to both surfaces at concentrations >10 microM. Defensin also inhibits tPA and plasminogen-mediated fibrinolysis in a dose-dependent manner at all concentrations tested. Fibrinolysis is almost totally inhibited by 6 microM defensin, a concentration that stimulates the binding of plasminogen to fibrin. Discordance between the enhancement of plasminogen binding and its activation cannot be explained by an inhibitory effect of defensin on tPA binding nor by inhibition of plasmin activity, each of which occur only at higher concentrations. Rather, these results suggest that plasminogen bound to fibrin in the presence of defensin is less susceptible to activation by tPA.

Anti-Infective Agents↗
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↗
PubMed1995

Structures of genes for two cathelin-associated antimicrobial peptides: prophenin-2 and PR-39.

We characterized genes for prophenin (PF)-2 and PR-39, two cathelin-associated antimicrobial peptides found in porcine leukocytes. Both contained 4 exons and 3 introns and were compact, contiguous and highly homologous. Exons I-III encoded most of their cathelin domains. Exon IV specified the final few cathelin residues, including its conserved C-terminal valine, followed by the mature PR-39 peptide or a PF-2 precursor. The highly conserved 5' flanking sequences of this gene family contained NF-kappa B, IL-6, GM-CSF and NF-1 binding motifs and the introns were unusually conserved. These data suggest that the panoply of porcine cathelin-associated antimicrobial peptides arose relatively recently via gene reduplications and exon shuffling, and that in vivo expression of cathelin-associated antimicrobial peptides may respond to mediators generated early during infection.

Amino Acid Sequence↗
PubMed1995

The structure of porcine protegrin genes.

We cloned the genes of three protegrins, a family of cathelin-associated antimicrobial peptides originally isolated from porcine leukocytes. Each gene comprised 4 exons and 3 introns, wherein Exon I encoded the signal sequence and the first 37 amino acids of cathelin, Exons II and III contained 36 and 24 additional cathelin residues and Exon IV contained the final two cathelin residues followed by the protegrin sequence. This quadripartite gene structure helps explain how structurally diverse antimicrobial peptides can be expressed on common, cathelin-containing precursors. Southern blot probed with an oligonucleotide specific for protegrin genes suggested that several identical or nearly identical protegrin genes were densely clustered in the pig chromosome.

Amino Acid Sequence↗
PubMed1995

Murine 32D c13 cells--a transfectable model of phagocyte granule formation.

Granulocytes expose phagocytized microbes to microbicidal substances that are stored in cytoplasmic granules and delivered by fusion to the phagocytic vacuoles (phagosomes). To determine if the murine myeloid cell line 32D c13 is suitable as a genetic model of protein translocation to granules and phagosomes, we permanently transduced 32D c13 cells with human HNP-1 defensin cDNA, incubated them with opsonized zymosan, and immunostained them for human defensin HNP-1. Although their phagocytic rate was much slower than that of neutrophil granulocytes, 32D c13 cells ingested zymosan into vacuoles that accumulated most of the transgenic defensin. The 32D c13 cell line should be useful for studies of the targeting of proteins to granules and phagosomes.

Animals↗
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

Defensins and other endogenous peptide antibiotics of vertebrates.

Gene-encoded peptide antibiotics are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. Their de novo synthesis or release from storage sites can be induced rapidly, which makes them particularly important in the initial phases of resistance to microbial invasion. The endogenous antimicrobial peptides of animals are products of single genes and are synthesized as preproproteins. Multistep processing yields the mature peptide, which generally acts by inducing microbial membrane permeabilization. Several families of antimicrobial peptides have been identified that differ with respect to the presence of disulfide linkages, amino acid composition, structural conformation, and spectrum of activity. The arginine-rich three disulfide-containing beta-sheet defensins are remarkably abundant and widely distributed in animals and plants. The antibiotic peptides of higher eukaryotes merit further study for their role in natural immunity and their potential as novel therapeutic compounds.

Amino Acid Sequence↗
PubMed1995

Defensins.

Defensins are a family of small cationic, antibiotic peptides that contain six cysteines in disulfide linkage. The peptides are abundant in phagocytes and small intestinal mucosa of humans and other mammals and in the hemolymph of insects. They contribute to host defense against microbes and may participate in tissue inflammation and endocrine regulation during infection. Bioengineered defensins are potentially useful as prophylactic and therapeutic agents in infections.

Amino Acid Sequence↗
PubMed1995

Human neutrophil defensin and serpins form complexes and inactivate each other.

Defensins, antimicrobial and cytotoxic peptides of neutrophils, bind to and are inactivated by blood proteins. We identified defensin interactions with alpha 1-proteinase inhibitor (alpha 1-PI), alpha 1-antichymotrypsin (alpha 1-ACT), alpha 2-antiplasmin (alpha 2-AP), and antithrombin III (AT III) and examined defensin binding to alpha 1-PI and alpha 1-ACT in more detail. Defensin interactions with either alpha 1-PI or alpha 1-ACT were not affected by iodoacetamide or high salt concentration. Preincubation of alpha 1-ACT or alpha 1-PI with increasing concentrations of defensin resulted in a progressive decrease of antiprotease activity of both inhibitors against cathepsin G and antiprotease activity of alpha 1-PI against human neutrophil elastase. At higher concentrations, defensin also ablated the inhibitory effect of normal human serum on cathepsin G and human neutrophil elastase. Both alpha 1-PI and alpha 1-ACT inhibited defensin cytotoxicity toward the human lung carcinoma cell line A549, whereas the elastase inhibitor antileukoprotease did not. Complex interactions between serpins and defensin may have a role in regulating inflammatory processes.

Blood Proteins↗
PubMed1994

Identification of defensin binding to C1 complement.

In human serum we found strong defensin binding to the complexes of activated C1 complement (C1) and C1 inhibitor (C1i). Purified C1q, activated C1 tetramer (r2s2) and C1i did not bind defensin. When r2s2 was dissociated by EDTA, only the activated C1s (C1s) bound defensin. Binding of defensins to C1 complement represents a newly recognized bridge between the complement- and phagocyte-mediated host defenses, and a potential mechanism for protecting infected tissue from cytotoxic injury by defensin.

Animals↗
PubMed1994

Defensins: a family of antimicrobial and cytotoxic peptides.

Defensins are antimicrobial and cytotoxic peptides that contain 29-35 amino acid residues, including 6 invariant cysteines that form 3 intramolecular disulfide bonds. They constitute more than 5% of the total cellular protein of human and rabbit neutrophils (PMN), and are also produced by rabbit lung macrophages and by murine and human small intestinal Paneth cells. Defensins exerted antimicrobial effects in vitro against many Gram-positive and Gram-negative bacteria, fungi, mycobacteria and some enveloped viruses, and were cytotoxic to a wide range of normal and malignant targets, including cells resistant to TNF-alpha and NK-cytolytic factor. Human and rabbit defensins formed voltage-sensitive channels in a variety of planar lipid bilayers when a negative voltage of approximately 70-90 mV was applied to the contralateral side. These channels showed modest anion selectivity and their formation was strongly influenced by defensin concentration. Although most other channel-forming peptides have prominent alpha-helical domains, the structure of defensin molecules is primarily composed of antiparallel beta-sheets. Studies with various prokaryotic and eukaryotic cells provided convincing evidence that defensins killed these targets by forming voltage-regulated channels in the susceptible cell's membrane. The broad spectrum of defensin-susceptible targets and the abundance of defensins in specialized host defense cells of the blood, lungs and intestinal tract suggest that defensins could play a significant role in innate immunity to infection and neoplasia.

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↗
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↗
PubMed1994

Inhibition of intracellular Histoplasma capsulatum replication by murine macrophages that produce human defensin.

Although purified defensins are effective microbicides in vitro, their operation within intact phagocytes has not been established. To address this question, we inserted cDNA encoding human defensin HNP-1 into a pBabe/neo retroviral vector and transduced it into RAW 264.7 cells, a murine macrophage line that lacks endogenous defensins. We isolated five independent clones of HNP-1-transduced cells, all of which secreted prodefensin and contained small amounts of fully processed HNP-1. The two clones that produced the largest amounts of defensin (clones 5 and 14), together with wild-type RAW cells and pBabe/neo-transduced RAW cells (control), were used for the present study. All cells were grown in Dulbecco's modified Eagle's medium-F12 medium that contained 10% heat-inactivated fetal bovine serum and gentamicin. The medium used for the transduced cells contained aminoglycoside G418 in lieu of gentamicin. Both wild-type and transduced cells were placed in antibiotic-free medium 96 h prior to challenge with a yeast-phase strain of Histoplasma capsulatum. Phagocytosis of yeast cells was allowed to proceed for 90 min and was followed by washing and further incubation for 18.5 h. Whereas the phagocytic index did not differ significantly among the four cell populations under study, the mean level of intracellular growth of H. capsulatum in the defensin-transduced RAW cells was significantly lower than those observed for any other cell types (P < 0.05). These findings constitute the first instance of xenogeneic expression of an antimicrobial peptide by phagocytes and suggest that macrophages can be armed with defensins to enhance their ability to restrict certain intracellular pathogens.

Animals↗
PubMed1993

Posttranslational processing and targeting of transgenic human defensin in murine granulocyte, macrophage, fibroblast, and pituitary adenoma cell lines.

Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal constituents of the neutrophil's azurophil granules. To determine the tissue specificity of posttranslational processing and subcellular targeting of defensins, the cDNA for a 94 aa human preprodefensin was transduced into murine cell lines (NIH 3T3 embryonic fibroblasts, AtT-20 pituitary adenoma, J774.1 and RAW 264.7 macrophages, and 32D and 32D cl3 granulocytes) using retroviral vectors. All transduced cell types expressed and to a variable extent constitutively secreted a 75 aa prodefensin formed by the removal of the amino terminal signal sequence. In AtT-20 cells, the 75 aa form accumulated intracellularly in granules and was releasable by secretagogues. Proteolytic processing to mature defensins was seen only in myeloid cells (J774.1, RAW 264.7, 32D, and 32D cl3). Newly formed mature defensin was rapidly degraded in J774.1 and RAW 264.7 macrophages, but accumulated stably in multivesicular bodies in 32D cells and in cytoplasmic granules of 32D cl3 cells. Our data suggest that the enzymatic and transport machinery required to process preprodefensin to mature defensin and to store it in cytoplasmic granules is a specialized feature of cells of granulocytic lineage.

3T3 Cells↗
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↗