RESUMEN
Protegrin 1 (PG-1) is a broad-spectrum antimicrobial peptide that contains 18 amino acid residues (RG GRLCYCRRRFCVCVGR) and has two intramolecular cystine disulfide bonds. To determine the minimal structure responsible for protegrin-mediated activity against Neisseria gonorrhoeae, we synthesized 15 protegrin variants and tested them against two well-characterized gonococcal strains. The MICs of PG-1 were 0.61 microM (1.31 microg/ml) for the serum-sensitive strain F 62 and 0.98 microM (2.11 microg/ml) for the serum-resistant strain FA 19. Six amino acid residues (Arg1, Gly2, Gly3, Arg4, Gly17, and Arg18) and either disulfide bond could be deleted from PG-1 without impairing its potency against strain F 62. In contrast, only Gly17 and Arg18 could be removed without decreasing its activity against FA 19. Protegrin congener 64a (PC-64a; LTYCRRRFCVTV), a variant of PG-1 with 12 amino acid residues and one disulfide bond, displayed MICs of 0.45 microM (0.68 microg/ml) for strain F 62 and 1.37 microM (2.07 microg/ml) for strain FA 19, which approximated those of intact PG-1. Serum-sensitive sac-1+ and sac-3+ transformants of N. gonorrhoeae FA 19 and two FA 19 derivatives with truncated lipooligosaccharide structures were more susceptible to PG-1 and variants with altered disulfide structures. These data suggest that structurally simpler protegrin variants, such as PC-64a, could be used as topical microbicides for N. gonorrhoeae. They also suggest that the cystine-stabilized antiparallel beta-sheet formed by PG-1 residues 5 to 16 is principally responsible for its activity against gonococci.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Neisseria gonorrhoeae/efectos de los fármacos , Proteínas/química , Proteínas/farmacología , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Péptidos Catiónicos Antimicrobianos , Secuencia de Carbohidratos , Disulfuros/química , Farmacorresistencia Microbiana , Lipopolisacáridos/química , Datos de Secuencia Molecular , Neisseria gonorrhoeae/genética , Proteínas/síntesis química , Relación Estructura-Actividad , Transformación BacterianaRESUMEN
Hemocytes from the invertebrate Styela clava, a solitary tunicate, contained a family of four alpha-helical antimicrobial peptides that were purified, sequenced and named clavanins A, B, C and D. Each clavanin contained 23 amino acid residues and was C-terminally amidated. The tunicate peptides resembled magainins in size, primary sequence and antibacterial activity. Synthetic clavanin A was prepared and displayed comparable antimicrobial activity to magainins and cecropins. The presence of alpha-helical antimicrobial peptides in the hemocytes of a urochordate suggests that such peptides are primeval effectors of innate immunity in the vertebrate lineage.
Asunto(s)
Antibacterianos/farmacología , Proteínas Sanguíneas/farmacología , Hemocitos/inmunología , Fragmentos de Péptidos/farmacología , Urocordados/inmunología , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Listeria monocytogenes/efectos de los fármacos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificaciónAsunto(s)
Antibacterianos/química , Proteínas Sanguíneas/química , Evolución Molecular , Sistema Inmunológico/inmunología , Péptidos/química , Secuencia de Aminoácidos , Animales , Antibacterianos/inmunología , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/inmunología , Defensinas , Humanos , Insectos/inmunología , Datos de Secuencia Molecular , Neutrófilos/inmunología , Péptidos/genética , Péptidos/inmunologíaRESUMEN
We tested 20 protegrins against Chlamydia trachomatis serovar L2 (L2/434/Bu). Five of the protegrins had native structures; the others included nonamidated, enantiomeric, and truncated variants and peptides with <2 disulfide bonds. Antichlamydial activity resided principally in residues 5 to 15 of native protegrin PG-1, and optimal activity required both intramolecular disulfide bonds.
Asunto(s)
Antibacterianos/farmacología , Chlamydia trachomatis/efectos de los fármacos , Proteínas/farmacología , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos , Línea Celular , Ratones , Datos de Secuencia Molecular , Proteínas/químicaRESUMEN
Protegrins are 2-kDa antimicrobial peptides that contain 16-18 amino acid residues and two intramolecular disulfide bonds. We studied the contribution of these disulfide bonds to the bactericidal activity of protegrins in physiological concentrations of NaCl by comparing protegrin PG-1 with variants that lacked one or both cysteine disulfides. Whereas the bactericidal and liposome-lytic properties of protegrin PG-1 were enhanced by adding 100 mM NaCl to the phosphate-buffered medium, NaCl addition strongly inhibited the effects of its linearized, disulfide-free variant, [A6, A8, A13, A15]protegrin-1. Whereas protegrin PG-1 manifested beta-sheet structure by CD (circular dichroism) and ATR-FTIR (attenuated-total-reflectance-Fourier-transform-infrared) spectroscopy in buffer or membrane-mimetic environments, [A6, A8, A13, A15]protegrin-1 manifested disordered structure in phosphate buffer and alpha-helical characteristics in membrane-mimetic environments. Both single-disulfide protegrin variants, [A8, A13]protegrin-1 and [A6, A15]protegrin-1, assumed beta-sheet conformations with liposomes that simulated bacterial membranes, and both retained substantial bactericidal activity when 100 mM NaCl was present. These findings demonstrate that the intramolecular disulfide bonds of protegrins are required for their antiparallel beta-sheet conformation in membrane-mimetic environments and for their potent antimicrobial activity in media containing NaCl concentrations comparable to those found in serum and extracellular fluids.
Asunto(s)
Antibacterianos/farmacología , Disulfuros/química , Proteínas/química , Proteínas/farmacología , Secuencia de Aminoácidos , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos , Bacterias/efectos de los fármacos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Fluoresceínas/metabolismo , Liposomas/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas/genética , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
BACKGROUND: The protegrins are a family of arginine- and cysteine-rich cationic peptides found in porcine leukocytes that exhibit a broad range of antimicrobial and antiviral activities. They are composed of 16-18 amino-acid residues including four cysteines, which form two disulfide linkages. To begin to understand the mechanism of action of these peptides, we set out to determine the structure of protegrin-1 (PG-1). RESULTS: We used two-dimensional homonuclear nuclear magnetic resonance spectroscopy to study the conformation of both natural and synthetic PG-1 under several conditions. A refined three-dimensional structure of synthetic PG-1 is presented. CONCLUSIONS: Both synthetic and natural protegrin-1 form a well-defined structure in solution composed primarily of a two-stranded antiparallel beta sheet, with strands connected by a beta turn. The structure of PG-1 suggests ways in which the peptide may interact with itself or other molecules to form the membrane pores and the large membrane-associated assemblages observed in protegrin-treated, gram-negative bacteria.
Asunto(s)
Antibacterianos/química , Proteínas/química , Secuencia de Aminoácidos , Animales , Antibacterianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos , Leucocitos/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas/genética , Proteínas/aislamiento & purificación , Soluciones , PorcinosRESUMEN
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.
Asunto(s)
Proteínas Sanguíneas/antagonistas & inhibidores , alfa-Defensinas , Secuencia de Aminoácidos , Animales , Actividad Bactericida de la Sangre , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Línea Celular , Citotoxicidad Inmunológica , ADN Complementario/genética , Defensinas , Humanos , Listeria monocytogenes/efectos de los fármacos , Datos de Secuencia Molecular , Neutrófilos/inmunología , Neutrófilos/metabolismo , Nucleopoliedrovirus/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Células Tumorales CultivadasRESUMEN
We developed a sensitive and quantitative radial diffusion method to ascertain the susceptibility of six strains of Neisseria gonorrhoeae to antimicrobial peptides derived from mammalian leukocytes. The test organisms included the well-characterized serum-resistant FA19 and serum-sensitive F62 strains plus four antibiotic-resistant clinical isolates. Although each N. gonorrhoeae strain was resistant to human neutrophil defensins, all six were exquisitely sensitive to protegrins, a family of small beta-sheet antimicrobial peptides recently identified in porcine leukocytes. Protegrin-treated N. gonorrhoeae became vacuolated and had striking membrane changes when viewed by transmission and scanning electron microscopy. Because low concentrations of protegrins can also inactivate Chlamydia trachomatis and human immunodeficiency virus, they show promise for development as topical agents to avert sexually transmitted diseases.
Asunto(s)
Neisseria gonorrhoeae/efectos de los fármacos , Proteínas/farmacología , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos , Proteínas Sanguíneas/farmacología , Defensinas , Difusión , Farmacorresistencia Microbiana , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Neisseria gonorrhoeae/ultraestructuraRESUMEN
We compared the susceptibilities of Chlamydia trachomatis elementary bodies (EBs) to human defensin HNP-2 and porcine protegrin PG-1, cysteine-rich beta-sheet antimicrobial peptides produced by mammalian leukocytes. Although both peptides protected McCoy cell monolayers from infection by chlamydial EBs, protegrins were especially potent. Protegrin-mediated inactivation of chlamydiae occurred rapidly, was relatively independent of the presence of serum, and was effective against serovars L2, D, and H. Protegrin-treated EBs showed striking morphological changes, with obvious damage to their limiting membranes and loss of their cytoplasmic contents and nucleoid. Their effectiveness against chlamydial EBs and other sexually transmitted pathogens combined with their relative lack of cytotoxicity suggests that protegrins and related molecules could serve as prototypes for topical agents to prevent sexually transmitted chlamydial infection.
Asunto(s)
Antibacterianos/farmacología , Proteínas Sanguíneas/farmacología , Chlamydia trachomatis/efectos de los fármacos , Proteínas/farmacología , alfa-Defensinas , Animales , Péptidos Catiónicos Antimicrobianos , Defensinas , Humanos , Pruebas de Sensibilidad Microbiana , ConejosRESUMEN
We determined the cysteine connectivity of protegrin PG-2, a leukocyte-derived antimicrobial peptide, by performing sequential enzyme digestions with chymotrypsin and thermolysin, and monitoring each digest by direct liquid chromatography-electrospray mass spectrometric analysis. This approach resolved the disulphide pairing pattern unambiguously with only picomolar amounts of PG-2. The inferred cysteine connectivity was confirmed by traditional amino acid composition analyses using nanomolar amounts of the protegrin. The results suggest that protegrins will assume a tachyplesin-like, disulphide-stabilized anti-parallel beta-sheet configuration in solution.
Asunto(s)
Antiinfecciosos/química , Proteínas/química , Aminoácidos/análisis , Animales , Antiinfecciosos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos , Cromatografía Líquida de Alta Presión , Quimotripsina , Cisteína/química , Disulfuros/química , Leucocitos/citología , Espectrometría de Masas , Estructura Molecular , Estructura Secundaria de Proteína , Proteínas/aislamiento & purificación , Soluciones , Porcinos , TermolisinaRESUMEN
We purified and characterized an unusual antimicrobial peptide, prophenin-1 (PF-1), from porcine leukocytes. The peptide had a mass of 8,683 and contained 79 residues, including 42 (53.2%) prolines and 15 (19.0%) phenylalanines. Its N-terminal 60 residues consisted of three perfect and three nearly perfect repeats of a decamer, FPPPNFPGPR. Prophenin-1 was encoded on a cathelin-containing precursor and showed substantially more activity against E. coli, a Gram-negative bacterium, than against Listeria monocytogenes, a Gram-positive organism, in vitro.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Leucocitos/química , Proteínas/química , Proteínas/farmacología , Secuencia de Aminoácidos , Animales , Antibacterianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos , Escherichia coli/efectos de los fármacos , Listeria monocytogenes/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Peso Molecular , Proteínas/aislamiento & purificación , Porcinos/sangreRESUMEN
Cathepsin G is a neutral serine protease of the granzyme B family which is found in human PMN, cells known to be important in the defense of the periodontium against periodontal bacteria. We propose that cathepsin G serves as a "pro-antibiotic" containing peptide domains which express selective antibiotic properties. In this study, we used HPLC to separate the low-molecular-weight peptides derived from the ultrafiltrate of a granule extract from unstimulated PMN. One of the peptides exhibited intense bactericidal activity as determined by radial diffusion overlay assay (against Escherichia coli ML-35P), an amino-terminal sequence "RVSSFLPWIR...", and a 3.1-kDa molecular mass determined by electrospray ionization-mass spectrometry. The sequence and mass are consistent with the C-terminus of cathepsin G deduced by cDNA analysis. These findings support the hypothesis that antibiotic peptides derived from cathepsin G occur naturally in human PMN. Since this is the first naturally occurring antibiotic peptide derived from cathepsin G, we designate it "CG-1".
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Sanguíneas/biosíntesis , Catepsinas/química , Proteínas de la Membrana , Neutrófilos/química , Péptidos , Secuencia de Aminoácidos , Antibacterianos/análisis , Proteínas Bacterianas/química , Proteínas Sanguíneas/química , Catepsina G , Gránulos Citoplasmáticos/química , Humanos , Datos de Secuencia Molecular , Neutrófilos/citología , Profármacos , Serina EndopeptidasasRESUMEN
We purified a molecule from the murine small intestine that killed both Escherichia coli and Listeria monocytogenes, and identified it as intestinal phospholipase A2 (iPLA2) by NH2-terminal sequencing and enzymatic measurements. The ability of iPLA2 to kill. L. monocytogenes was greatly enhanced by 5 mM calcium, inhibited by EGTA and abolished after reduction and alkylation, suggesting that enzymatic activity was required for iPLA2-mediated bactericidal activity. A mouse-avirulent phoP mutant, S. typhimurium 7953S, was 3.5-fold more susceptible to iPLA2 than its isogenic virulent parent, S. typhimurium 14028S (estimated minimal bactericidal concentrations 12.7 +/- 0.5 micrograms/ml vs. 43.9 +/- 4.5 micrograms/ml P < 0.001). Overall, these findings identify iPLA2 as part of the antimicrobial arsenal that equips Paneth cells to protect the small intestinal crypts from microbial invasion. Because iPLA2 is identical to Type 2 phospholipase A2 molecules found in other sites, including spleen, platelets and inflammatory exudate cells, this enzyme may also contribute to antibacterial defenses elsewhere in the body.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Mucosa Intestinal/enzimología , Intestino Delgado/enzimología , Listeria monocytogenes/efectos de los fármacos , Fosfolipasas A/farmacología , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Calcio/farmacología , Relación Dosis-Respuesta a Droga , Yema de Huevo , Ácido Egtácico/farmacología , Femenino , Mucosa Intestinal/citología , Intestino Delgado/citología , Ratones , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/farmacología , Fosfolipasas A/química , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A2RESUMEN
We purified three peptides ("cryptdins") from the small intestines of mice, established their primary amino acid sequences and examined their antimicrobial activity. Their primary sequences revealed approximately 50% identity to a group of antimicrobial defensins that we had previously isolated from the granules of rat neutrophils. In addition to their ability to kill Gram-positive (L. monocytogenes) and Gram-negative bacteria (E. coli and S. typhimurium) in vitro, the peptides were much more active against an avirulent (phoP) S. typhimurium strain than against its isogenic, mouse-virulent progenitor. Overall, these data suggest that endogenous antimicrobial peptides produced by Paneth cells may protect small intestinal crypts, which are critical sites of epithelial cell renewal, from invasion by autochthonous flora or by perorally acquired potential pathogens, such as Listeria and Salmonella.
Asunto(s)
Antibacterianos/aislamiento & purificación , Intestino Delgado/química , Precursores de Proteínas/fisiología , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Cromatografía Líquida de Alta Presión , Escherichia coli/efectos de los fármacos , Genes , Intestino Delgado/citología , Intestino Delgado/microbiología , Listeria monocytogenes/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Precursores de Proteínas/aislamiento & purificación , Precursores de Proteínas/farmacología , Ratas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Virulencia/genéticaRESUMEN
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.
Asunto(s)
Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos , Proteínas Aviares , Pollos/inmunología , Leucocitos/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Actividad Bactericida de la Sangre , Proteínas Sanguíneas/química , Cromatografía Líquida de Alta Presión , Cisteína/química , Defensinas , Datos de Secuencia Molecular , Péptidos/farmacología , Alineación de Secuencia , Homología de Secuencia de AminoácidoAsunto(s)
Antibacterianos/farmacología , Proteínas Sanguíneas/aislamiento & purificación , Proteínas Sanguíneas/farmacología , Escherichia coli/efectos de los fármacos , Neutrófilos/fisiología , Secuencia de Aminoácidos , Antibacterianos/aislamiento & purificación , Técnicas Bacteriológicas , Actividad Bactericida de la Sangre , Proteínas Sanguíneas/química , Separación Celular/métodos , Cromatografía en Gel/métodos , Cromatografía Líquida de Alta Presión/métodos , Gránulos Citoplasmáticos/química , Defensinas , Escherichia coli/crecimiento & desarrollo , Humanos , Indicadores y Reactivos , Pruebas de Sensibilidad Microbiana/métodos , Datos de Secuencia Molecular , Neutrófilos/citología , Homología de Secuencia de AminoácidoRESUMEN
Microbiostatic mechanisms may contribute substantially to host defense against infection by certain microbes. We studied the candidastatic activity of human neutrophils, neutrophil cytosol, and neutrophil-derived "calprotectin," a cytosolic protein complex comprised of two subunits, MRP8 and MRP14. Intact neutrophils, neutrophil lysates (prepared by ultrasonic disruption, freezing and thawing, or nonionic detergent extraction), and granule-depleted neutrophil cytosol were effective in restricting the growth of Candida albicans in a nutrient-rich tissue culture medium, RPMI 1640. Neither a subcellular fraction enriched in neutrophil granules nor selected purified granule components (lactoferrin, myeloperoxidase, cathepsin G, leukocyte elastase, lysozyme, and defensins) exerted candidastatic activity in this medium. Gel filtration liquid chromatography, anion exchange FPLC, and SDS-PAGE showed that the fungistatic factor in neutrophil cytosol was associated with the calprotectin complex. Its antifungal effects included restriction of yeast phase and mycelial growth and inhibition of glucose incorporation by yeast phase cultures. The antifungal effects of calprotectin were sustained for over 120 h and were inhibited by zinc. However, studies with 65Zn-enriched RPMI suggested that the candidastatic effects of calprotectin were not mediated by sequestration or binding of zinc. After reversed phase HPLC, calprotectin fractions containing MRP14 exhibited fungistatic activity, whereas fractions depleted of MRP14 but enriched for MRP8 lacked fungistatic activity. The results support a potentially significant role for the calprotectin complex of neutrophil cytosol in antifungal defense and suggest that MRP14 is of key importance in that activity.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Moléculas de Adhesión Celular Neuronal/farmacología , Neutrófilos/inmunología , Moléculas de Adhesión Celular Neuronal/aislamiento & purificación , Citosol/química , Humanos , Complejo de Antígeno L1 de Leucocito , Monocitos/inmunología , Neutrófilos/química , Zinc/farmacologíaRESUMEN
Porcine leukocytes contained three homologous peptides, PG-1, 2 and 3, that manifested potent microbicidal activity against Escherichia coli, Listeria monocytogenes and Candida albicans in vitro. The peptides ('protegrins') were composed of 16 (PG-2) or 18 amino acid residues, and, like tachyplesins (broad-spectrum antibiotic peptides of horseshoe crab hemocytes), they contained two intramolecular cystine disulfide bonds. Considerably smaller than defensins, protegrins nevertheless showed substantial homology to them, especially to the 'corticostatic' rabbit defensin, NP-3a. The relatively simple structure of protegrins should provide useful prototypes for constructing congeners with selectively enhanced host defense activities.
Asunto(s)
Antiinfecciosos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/aislamiento & purificación , Proteínas de Unión al ADN/química , Leucocitos/química , Péptidos Cíclicos , alfa-Defensinas , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Proteínas Sanguíneas/farmacología , Cromatografía Líquida de Alta Presión , Proteínas de Unión al ADN/farmacología , Defensinas , Electroforesis en Gel de Poliacrilamida , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/farmacología , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
Equine neutrophil antimicrobial peptide 2 (eNAP-2), a recently described antimicrobial peptide isolated from equine neutrophils, was found to selectively inactivate microbial serine proteases (subtilisin A and proteinase K) without inhibiting mammalian serine proteases (human neutrophil elastase, human cathepsin G, and bovine pancreatic trypsin). Although the primary structure of eNAP-2 resembled that of several known antiproteases that belong to the 4-disulfide core peptide family, this pattern of selectivity is unique. eNAP-2 formed a noncovalent complex with native subtilisin A or proteinase K but did not associate with these enzymes if they had been treated with phenylmethylsulfonyl fluoride, a serine protease inhibitor. The eNAP-2-microbial protease complex was disrupted by boiling or by exposure to low pH. We suggest that eNAP-2 exerted selective antiproteinase activity by binding tightly but noncovalently to the active site of subtilisin A or proteinase K. Since microbial exoproteases may act as virulence factors, the combined antimicrobial and antiprotease activities of eNAP-2 could allow it to play an important role in neutrophil-mediated antimicrobial defenses.
Asunto(s)
Antiinfecciosos/farmacología , Proteínas Sanguíneas/farmacología , Neutrófilos/química , Inhibidores de Serina Proteinasa/farmacología , Animales , Endopeptidasa K , Caballos , Serina Endopeptidasas/metabolismo , Subtilisinas/antagonistas & inhibidoresRESUMEN
Three murine microbicidal proteins (MUMPs) were purified from cells of the murine macrophage cell line RAW264.7 that had been activated by gamma interferon. Similar proteins were also present in nonactivated RAW264.7 cells, in cells of the murine macrophage cell line J774A.1, and in resident and activated murine peritoneal macrophages. MUMP-1, MUMP-2, and MUMP-3 killed Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Mycobacterium fortuitum, and Cryptococcus neoformans in vitro. MUMP-1 resembled an H1 histone but was unusual because its N-terminal residue (serine) was not N acetylated. Although MUMP-2 was N terminally blocked, its high lysine/arginine ratio and its reactivity with an antibody to H1 histones suggested that it also belonged to the H1 histone family. MUMP-3 was identical to histone H2B in 30 of 30 amino-terminal residues. Although the antimicrobial properties of histones have been recognized for decades, this is the first evidence that such proteins may endow the lysosomal apparatus of macrophages with nonoxidative antimicrobial potential. Other MUMPs, including some with a more restricted antimicrobial spectrum and one that appeared to be induced in RAW264.7 cells after gamma interferon stimulation, were noted but remain to be characterized.