RESUMO
All known naturally occurring linear cationic peptides adopt an amphipathic alpha-helical conformation upon binding to lipids as an initial step in the induction of cell leakage. We designed an 18-residue peptide, (KIGAKI)3-NH2, that has no amphipathic character as an alpha-helix but can form a highly amphipathic beta-sheet. When bound to lipids, (KIGAKI)3-NH2 did indeed form a beta-sheet structure as evidenced by Fourier transform infrared and circular dichroism spectroscopy. The antimicrobial activity of this peptide was compared with that of (KIAGKIA)3-NH2, and it was better than that of GMASKAGAIAGKIAKVALKAL-NH2 (PGLa) and (KLAGLAK)3-NH2, all of which form amphipathic alpha-helices when bound to membranes. (KIGAKI)3-NH2 was much less effective at inducing leakage in lipid vesicles composed of mixtures of the acidic lipid, phosphatidylglycerol, and the neutral lipid, phosphatidylcholine, as compared with the other peptides. However, when phosphatidylethanolamine replaced phosphatidylcholine, the lytic potency of PGLa and the alpha-helical model peptides was reduced, whereas that of (KIGAKI)3-NH2 was improved. Fluorescence experiments using analogs containing a single tryptophan residue showed significant differences between (KIGAKI)3-NH2 and the alpha-helical peptides in their interactions with lipid vesicles. Because the data suggest enhanced selectivity between bacterial and mammalian lipids, linear amphipathic beta-sheet peptides such as (KIGAKI)3-NH2 warrant further investigation as potential antimicrobial agents.
Assuntos
Antibacterianos/química , Bactérias/metabolismo , Metabolismo dos Lipídeos , Peptídeos/química , Sequência de Aminoácidos , Animais , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Fluoresceínas/metabolismo , Hemólise , Dados de Sequência Molecular , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Conformação Proteica , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Triptofano/químicaRESUMO
The interleukin 9 (IL-9) pathway has recently been associated with the asthmatic phenotype including an eosinophilic tissue inflammation. The mechanism by which IL-9 affects eosinophils (eos) is not known. To investigate whether this cytokine has a direct activity on the development of eos and eosinophilic inflammation, a model of thioglycolate-induced peritoneal inflammation was used in IL-9 transgenic (TG5) and background strain (FVB) mice. In this model, a transient eosinophilic infiltration in the peritoneal cavity was observed in FVB mice 12 to 24 hours after thioglycolate injection that coincided with peak IL-5 and IL-9 release. In contrast, TG5 mice developed a massive eosinophilia that persisted at high levels (81% of total cells) even 72 hours after thioglycolate injection. Release of eosinophilic major basic protein (MBP), IL-4, and IL-5 to the peritoneal cavity of these mice was significantly increased when compared with the control FVB strain. To study the mechanism by which IL-9 exerts its effect on eos, bone marrow or peritoneal cells were cultured in the presence of IL-5, IL-9, or their combination in vitro. IL-5 alone was able to generate significant numbers of eos in TG5 but not FVB mice, whereas a combination of IL-5 and IL-9 induced marked eosinophilia in both strains indicating a synergism between these 2 cytokines. These data suggest that IL-9 may promote and sustain eosinophilic inflammation via IL-5-driven eos maturation of precursors.
Assuntos
Quimiocinas CC , Quimiotaxia de Leucócito/efeitos dos fármacos , Eosinofilia/etiologia , Eosinófilos/efeitos dos fármacos , Interleucina-9/fisiologia , Peritonite/induzido quimicamente , Ribonucleases , Transferência Adotiva , Animais , Proteínas Sanguíneas/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Quimiocina CCL11 , Citocinas/metabolismo , Proteínas Granulares de Eosinófilos , Humanos , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Interleucina-9/genética , Interleucina-9/metabolismo , Interleucina-9/farmacologia , Linfócitos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Imunológicos , Infiltração de Neutrófilos/efeitos dos fármacos , Peritonite/sangue , Peritonite/complicações , Baço/citologia , Linfócitos T/transplante , Tioglicolatos/toxicidade , Fatores de TempoRESUMO
Magainins are cationic peptides with antimicrobial activity which were originally isolated from the skin of the African clawed frog (Xenopus laevis). Several synthetic derivatives of this class of peptides were evaluated for antiviral activity against herpes simplex virus, type 1 (HSV). Some of the peptides (MSI-102, -248, -420, -499/500 combination, -591, -594, and -1251) showed significant reduction of HSV plaque-forming units. The antiviral effect was enhanced when HSV was pretreated with the peptides prior to inoculation onto Vero monolayers, suggesting a direct effect on the virion. Most of the peptides with anti-HSV activity were lysine-rich, and the addition of octanoyl groups to the peptides appeared to enhance the antiviral effect.
Assuntos
Antivirais/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Herpesvirus Humano 1/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/química , Células Vero , Ensaio de Placa ViralRESUMO
Starting from the sequences of magainin 2 analogs, peptides with slightly increased hydrophobic moment (mu) but retained other structural parameters were designed. Circular dichroism investigations revealed that all peptides adopt an alpha-helical conformation when bound to phospholipid vesicles. Analogs with increased mu were considerably more active in permeabilizing vesicles mainly composed of zwitterionic lipid. In addition, the antibacterial and hemolytic activities of these analogs were enhanced. Correlation of permeabilization and binding indicated that the activity increase is predominantly caused by an increased membrane affinity of the peptides due to strengthened hydrophobic interactions.
Assuntos
Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos , Membrana Celular/metabolismo , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas de Xenopus , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Permeabilidade da Membrana Celular , Humanos , Metabolismo dos Lipídeos , Magaininas , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
To investigate the influence of the angle subtended by the positively charged helix face on membrane activity, six amphipathic alpha-helical peptides with angles between 80 degrees and 180 degrees, but with retained hydrophobicity, hydrophobic moment, and positive overall charge, were designed starting from the sequence of the antibacterial peptide magainin 2. CD investigations revealed that all analogs are in an alpha-helical conformation in vesicle suspension. The ability of the peptides to induce dye release from negatively charged phosphatidylglycerol (PG) vesicles decreased with increasing angle. However, peptides with a large angle of positively charged residues (140-180 degrees) exhibited a considerably higher permeabilizing activity at zwitterionic phosphatidylcholine (PC) and mixed PC/PG (3:1) vesicles than analogs with a small angle (80-120 degrees). In addition, analogs with large angles were more active in antibacterial and hemolytic assays. The antibacterial specificity of these analogs was decreased. Binding investigations showed that peptide binding is favored by a large angle and a high content of negatively charged phospholipid. In contrast, a small angle and a low negative membrane charge enhanced the membrane-permeabilizing efficiency of the bound peptide fraction. All analogs stabilized the bilayer phase of phosphatidylethanolamine over the inverted hexagonal phase. Therefore, a class L mechanism of permeabilization can be excluded. Furthermore, the analogs do not act by the induction of positive curvature strain or by a "carpet-like" mechanism. Our results are in accordance with a pore mechanism: The membrane-permeabilizing efficiency of analogs with enhanced angle of positively charged residues is reduced due to electrostatic repulsion between adjacent helices within the pore, thus resulting in a decreased pore-forming probability and/or pore destabilization.
Assuntos
Antibacterianos/química , Lipossomos/química , Peptídeos/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Fluoresceínas , Modelos Estruturais , Dados de Sequência Molecular , Peptídeos/síntese química , Fosfatidilcolinas , FosfatidilgliceróisRESUMO
Polyphemusin is a broad-spectrum antimicrobial peptide isolated from hemocytes of the North American horseshoe crab Limulus polyphemus. To date the polyphemusin used for scientific analyses has been purified from the natural materials or obtained by chemical synthesis. We report here the recombinant expression in Escherichia coli, and subsequent purification, of a polyphemusin analogue (rLim1). To prevent toxicity of the antimicrobial peptide in the highly susceptible E. coli host, we used a carboxy-terminal fusion protein cloning strategy provided by a maltose-binding protein (MBP) gene fusion system (New England Biolabs). Antimicrobial activity of recombinant polyphemusin was similar to that seen with amidated native polyphemusin peptide. When rLim1 was tested for antibiotic activity against the apicomplexan protozoan oyster pathogen Perkinsus marinus, complete inhibition was observed at 12 micrograms/ml, and partial inhibition at 8 micrograms/ml.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Apicomplexa/efeitos dos fármacos , Proteínas de Escherichia coli , Proteínas de Transporte de Monossacarídeos , Ostreidae/parasitologia , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Sequência de Bases , Proteínas de Transporte/genética , Clonagem Molecular , Expressão Gênica , Caranguejos Ferradura/química , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/efeitos dos fármacos , Peptídeos/genética , Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an alpha-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic analogs for negatively charged membranes. Likewise, the peptide affinity to PC-rich membranes increases in the order of hydrophobicity. Correlation of peptide binding and membrane permeabilization of PC/PG (3:1) vesicles revealed that the observed differences in peptide activity on membranes of low negative surface charge are mainly caused by the different binding affinities. The antibacterial and hemolytic activity of the peptides increases with enhanced hydrophobicity. A strong correlation was found between the hemolytic effect and the bilayer-permeabilizing activity against PC-rich vesicles. Whereas the antibacterial specificity of the more hydrophobic analogs is retained for Escherichia coli, the specificity for Pseudomonas aeruginosa decreases with increasing hydrophobicity.
Assuntos
Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos , Bicamadas Lipídicas/metabolismo , Peptídeos/metabolismo , Proteínas de Xenopus , Antibacterianos/química , Antibacterianos/farmacologia , Dicroísmo Circular , Fluoresceínas/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Hemólise , Humanos , Magaininas , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/farmacologia , Permeabilidade , Fosfatidilcolinas , Fosfatidilgliceróis , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
The hydrophobicity (H), hydrophobic moment (mu) and the angle subtended by the positively charged helix face (phi) of a set of model and magainin 2 amide peptides with conserved charge and helix propensity have been shown to be effective modulators of antibacterial and haemolytic activity. Except peptides of low hydrophobicity which are inactive, changing the parameters has little influence on the activity against Gram-negative bacteria, thus revealing the dominance of electrostatic interactions for the effect. However, the increase of H, mu and phi substantially enhances haemolytic and Gram-positive antibacterial activity and is related to a reduction of peptide specificity for Gram-negative bacteria.
Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Magainins are cationic, membrane-active peptides which show broad-spectrum antimicrobial activity. We have investigated the secondary structure and location of an analogue of magainin 2 in synthetic phospholipid bilayers using a combination of Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Ala19-magainin 2 amide exhibits both alpha-helix and beta-sheet secondary structures in lipid bilayers containing either dipalmitoylphosphatidylglycerol (DPPG) or a 1:1 molar mixture of DPPG and dipalmitoylphosphatidylcholine (DPPC). The combination of FTIR and solid-state NMR results suggests that there are two populations of peptide. The secondary structure of one population is alpha-helix while that of the other population is beta-sheet. We demonstrate that the solid-state NMR technique, rotational-echo double resonance (REDOR), can be used to measure both intra- and intermolecular dipole-dipole interactions in membrane-bound peptides. Our REDOR experiments indicate that alpha-helical Ala19-magainin 2 amide is bound near the phospholipid head groups.
Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos , Bicamadas Lipídicas/química , Peptídeos/química , Peptídeos/metabolismo , Fosfolipídeos/química , Estrutura Secundária de Proteína , Proteínas de Xenopus , Sequência de Aminoácidos , Escherichia coli/efeitos dos fármacos , Bicamadas Lipídicas/metabolismo , Magaininas , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peptídeos/farmacologia , Fosfolipídeos/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus aureus/efeitos dos fármacos , TemperaturaRESUMO
Host defense peptides are widely distributed in nature, being found in species from bacteria to humans. The structures of these peptides from insects, horseshoe crabs, frogs, and mammals are known to have the common features of a net cationic charge due to the presence of multiple Arg and Lys residues and in most cases the ability to form amphipathic structures. These properties are important for the mechanism of action that is thought to be a nonreceptor-mediated interaction with the anionic phospholipids of the target cell followed by incorporation into the membrane and disruption of the membrane structure. Host defense peptides have been shown to have broad spectrum antimicrobial activity, able to kill most strains of bacteria as well as some fungi, protozoa, and in addition, many types of tumor cells. Specificity for pathogenic cells over host cells is thought to be due to the composition of the cell membranes, with an increased proportion of anionic phospholipids making the pathogen more susceptible and the presence of cholesterol making the host membranes more resistant. Structure-activity relationship studies have been performed on insect cecropins and apidaecins, horseshoe crab tachyplesins and polyphemusins, and the frog magainins, CPFs (caerulein precursor fragments) and PGLa. In general, changes that increased the basicity and stabilized the amphipathic structure have increased the antimicrobial activity; however, as the peptides become more hydrophobic the degree of specificity decreases. One magainin-2 analogue, MSI-78, has been developed by Magainin Pharmaceuticals as a topical antiinfective and is presently in clinical trials for the treatment of infected diabetic foot ulcers.
Assuntos
Antibacterianos/química , Hormônios de Inseto/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Dípteros , Caranguejos Ferradura , Hormônios de Inseto/farmacologia , Insetos , Mamíferos , Dados de Sequência Molecular , Mariposas , Peptídeos/farmacologia , Ranidae , Homologia de Sequência de AminoácidosRESUMO
Magainin peptides and model amphipathic peptides exhibit antibiotic activity and are also cytolytic for transformed human cells. Here we demonstrate in vitro that MSI-511 (an all-D amino-acid model magainin peptide) and MSI-130 (a margainin analogue) were more lytic for 17 human melanomas than for normal melanocytes. Melanomas established s.c. in athymic nude mice and then injected once with the peptide MSI-511 completely disappeared in 6 out of 9 animals, whereas a control peptide had no effect. Murine skin at the tumor injection site was initially affected, but healed within 2 weeks with minimal scarring. Similarly, accelerated healing was seen in human skin grafted to SCID mice and injected with MSI-511. Our results indicate that lytic magainin peptides can be used for local tumor therapy with minimal long-term damage to normal tissues.
Assuntos
Antineoplásicos/uso terapêutico , Melanoma/tratamento farmacológico , Peptídeos/uso terapêutico , Animais , Peptídeos Catiônicos Antimicrobianos , Feminino , Humanos , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pele/efeitos dos fármacos , Transplante de Pele , Células Tumorais CultivadasRESUMO
Antimicrobial peptides comprise a diverse class of molecules used in host defense by plants, insects, and animals. In this study we have isolated a novel antimicrobial peptide from the skin of the bullfrog, Rana catesbeiana. This 20 amino acid peptide, which we have termed Ranalexin, has the amino acid sequence: NH2-Phe-Leu-Gly-Gly-Leu-Ile-Lys-Ile-Val-Pro-Ala-Met-Ile-Cys-Ala-Val-Thr- Lys-Lys - Cys-COOH, and it contains a single intramolecular disulfide bond which forms a heptapeptide ring within the molecule. Structurally, Ranalexin resembles the bacterial antibiotic, polymyxin, which contains a similar heptapeptide ring. We have also cloned the cDNA for Ranalexin from a metamorphic R. catesbeiana tadpole cDNA library. Based on the cDNA sequence, it appears that Ranalexin is initially synthesized as a propeptide with a putative signal sequence and an acidic amino acid-rich region at its amino-terminal end. Interestingly, the putative signal sequence of the Ranalexin cDNA is strikingly similar to the signal sequence of opioid peptide precursors isolated from the skin of the South American frogs Phyllomedusa sauvagei and Phyllomedusa bicolor. Northern blot analysis and in situ hybridization experiments demonstrated that Ranalexin mRNA is first expressed in R. catesbeiana skin at metamorphosis and continues to be expressed into adulthood.
Assuntos
Anti-Infecciosos/química , Peptídeos Cíclicos/química , Polimixinas/química , Pele/química , Sequência de Aminoácidos , Animais , Anti-Infecciosos/isolamento & purificação , Sequência de Bases , DNA Complementar , Dissulfetos/química , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos Cíclicos/isolamento & purificação , RNA Mensageiro/metabolismo , Rana catesbeiana , Homologia de Sequência de AminoácidosRESUMO
Linear helical channel-forming peptides structurally similar to the Xenopus-derived antibiotic, Magainin2-amide, were synthesized. Because activity resides in the physicochemical properties of the peptides, an all-D-amino acid as well as an all-L-amino acid sequence were tested for anticancer activity. In vitro activity against carcinoma cells and in vivo efficacy against four murine ascites tumors were determined. The novel peptides proved to have enhanced potency in vitro and in vivo as compared to the parent compound. The 50% inhibitory concentrations against A549 cells for the all-D, the all-L, and Magainin2 were 6, 10, and 110 micrograms/ml, respectively. All three peptides had activity against P388 leukemia, S180 ascites, and a spontaneous ovarian tumor when injected i.p. Increase in life span of over 100% was produced for the analogues in the latter two models. The maximally effective concentrations for the analogues were 20 to 25 mg/kg while Magainin2 required 50-60 mg/kg for in vivo efficacy. The all-D-amino acid peptide, MSI-238, proved as effective as doxorubicin at a more advanced stage of the ovarian tumor and this activity may be attributed to its resistance to proteolytic degradation. Therefore, this class of amphiphilic alpha-helical cationic peptides has potential in the peritoneal treatment of ovarian cancer.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Antineoplásicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Peptídeos/farmacologia , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Leucemia L1210/tratamento farmacológico , Leucemia L1210/patologia , Leucemia P388/tratamento farmacológico , Leucemia P388/patologia , Magaininas , Masculino , Camundongos , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Neoplasias Experimentais/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Peptídeos/farmacocinética , Peptídeos/toxicidade , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/patologia , Sarcoma 180/tratamento farmacológico , Sarcoma 180/patologia , Teratoma/tratamento farmacológico , Teratoma/patologiaRESUMO
Cytotoxic T lymphocytes (CTL) play an important role in limiting viral infections and in eradicating virus from host tissues. Recent progress in understanding the processing and presentation of viral antigens to CTL indicates that the CTL antigen receptor recognizes peptides derived from viral proteins that are bound to an antigen binding groove present in class I major histocompatibility complex (MHC) molecules. In understanding CTL anti-viral responses and in creating vaccines designed to elicit CTL responses, it is critical to identify the portions of viral proteins that bind class I molecules and are recognized by T cell receptors. Previous findings have indicated that a significant portion of the CTL response of H-2d mice to influenza virus is specific for one of the viral polymerases (PB2). To identify the region of PB2 naturally processed and presented by influenza virus-infected mouse cells to CTL, 31 PB2 peptides of 9-16 residues in length were chosen and chemically synthesized. Two peptides, PB2, residues 146-159 and 187-195, were found to sensitize histocompatible target cells for recognition by influenza virus-specific CTL. When CTL were generated to individual viral proteins using influenza-vaccinia recombinant viruses, we found, to our surprise, that PB2-specific CTL failed to recognize cells sensitized with PB2 peptides 146-159 and 187-195. Further analysis showed that these PB2 peptides were, in fact, recognized by nucleoprotein (NP)-specific CTL generated by NP-vac virus priming and influenza A virus stimulation, or NP peptide stimulation in vitro of NP-vac or influenza A-primed CTL. These results demonstrate that while screening peptide libraries one cannot assume that positive peptides necessarily identify the viral protein to which the CTL response is directed.
Assuntos
Vírus da Influenza A/imunologia , Nucleoproteínas/imunologia , Peptídeos/imunologia , Proteínas de Ligação a RNA , Linfócitos T Citotóxicos/imunologia , Proteínas do Core Viral/imunologia , Proteínas Virais/imunologia , Algoritmos , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Reações Cruzadas , Citotoxicidade Imunológica , Feminino , Imunidade Celular , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Peptídeos/genética , RNA Polimerase Dependente de RNA , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/genéticaRESUMO
Distinct combinations of class II major histocompatibility complex (MHC) alpha and beta chains show widely varying efficiencies of cell surface expression in transfected cells. Previous studies have analyzed the regions of the class II chains that are critically involved in this phenomenon of variable expression and have shown a predominant effect of the NH2-terminal domains comprising the peptide-binding site. The present experiments attempt to identify the post-translational defects responsible for this variation in surface class II molecule expression for both interisotypic alpha/beta combinations failing to give rise to any detectable cell membrane molecules (e.g., E alpha A beta k) and intraisotypic pairs with inefficient surface expression (e.g., A alpha d A beta k). The results of metabolic labeling and immunoprecipitation experiments using L cell transfectants demonstrate that in both of these cases, the alpha and beta chains form substantial amounts of stable intracellular dimers. However, the isotype- and allele-mismatched combinations do not show the typical post-translational increases in molecular weight that accompany maturation of the N-linked glycans of class II MHC molecules. Studies with endoglycosidase H reveal that no or little progression to endoglycosidase H resistance occurs for these mismatched dimers. These data are consistent with active or passive retention of relatively stable and long-lived mismatched dimers in a pre-medial-Golgi compartment, possibly in the endoplasmic reticulum itself. This retention accounts for the absent or poor surface expression of these alpha/beta combinations, and suggests that conformational effects of the mismatching in the NH2-terminal domain results in a failure of class II molecules to undergo efficient intracellular transport.
Assuntos
Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II/genética , Alelos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Membrana Celular/imunologia , Regulação da Expressão Gênica , Complexo de Golgi/fisiologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Células L/imunologia , Leucina/metabolismo , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Processamento de Proteína Pós-Traducional , TransfecçãoRESUMO
The magainin peptides of Xenopus laevis are broad-spectrum antimicrobial agents. Upon discharge from the skin glands, these basic, amphipathic peptides are each further processed at a single Xaa-Lys bond into half-peptides by a cosecreted protease. We describe the characterization and purification to homogeneity of this endopeptidase from Xenopus skin. The enzyme is a metalloprotease 110 kd in size. Analyses of substrate specificity revealed that the endopeptidase recognizes peptides that share the ability to adopt an amphipathic, alpha-helical motif composed of at least 12 residues, with one face strongly hydrophobic. Cleavage occurs on the amino side of a specific lysine that must be precisely positioned relative to the hydrophobic face of the alpha helix. This enzyme, which we propose to call "magaininase," represents a novel class of endopeptidases that hydrolyzes peptides on the basis of specific secondary structure rather than primary amino acid sequence.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Endopeptidases/metabolismo , Proteínas de Xenopus , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Endopeptidases/isolamento & purificação , Magaininas , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Extracts of the bovine tracheal mucosa have an abundant peptide with potent antimicrobial activity. The 38-amino acid peptide, which we have named tracheal antimicrobial peptide (TAP), was isolated by a sequential use of size-exclusion, ion-exchange, and reverse-phase chromatographic fractionations using antimicrobial activity as a functional assay. The yield was approximately 2 micrograms/g of wet mucosa. The complete peptide sequence was determined by a combination of peptide and cDNA analysis. The amino acid sequence of TAP is H-Asn-Pro-Val-Ser-Cys-Val-Arg-Asn-Lys-Gly-Ile-Cys-Val-Pro-Ile-Arg-Cys-Pr o- Gly-Ser-Met-Lys-Gln-Ile-Gly-Thr-Cys-Val-Gly-Arg-Ala-Val-Lys-Cys-Cys-Arg- Lys-Lys - OH. Mass spectral analysis of the isolated peptide was consistent with this sequence and indicated the participation of six cysteine residues in the formation of intramolecular disulfide bonds. The size, basic charge, and presence of three intramolecular disulfide bonds is similar to, but clearly distinct from, the defensins, a well-characterized class of antimicrobial peptides from mammalian circulating phagocytic cells. The putative TAP precursor is predicted to be relatively small (64 amino acids), and the mature peptide resides at the extreme carboxyl terminus and is bracketed by a short putative propeptide region and an inframe stop codon. The mRNA encoding this peptide is more abundant in the respiratory mucosa than in whole lung tissue. The purified peptide had antibacterial activity in vitro against Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, and Pseudomonas aeruginosa. In addition, the peptide was active against Candida albicans, indicating a broad spectrum of activity. This peptide appears to be, based on structure and activity, a member of a group of cysteine-rich, cationic, antimicrobial peptides found in animals, insects, and plants. The isolation of TAP from the mammalian respiratory mucosa may provide insight into our understanding of host defense of this vital tissue.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Peptídeos/genética , Peptídeos/isolamento & purificação , Traqueia/química , Sequência de Aminoácidos , Bactérias/efeitos dos fármacos , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA/genética , Expressão Gênica , Dados de Sequência Molecular , Oligonucleotídeos/química , RNA Mensageiro/genética , Traqueia/imunologia , Traqueia/microbiologiaRESUMO
To test for the assembly of human MHC class II molecules having an alpha chain from one isotype (HLA-DR, -DQ, or -DP) and the beta chain of another (mixed isotypic pairs), murine fibroblasts were transfected with expressible cDNAs encoding the different class II alpha and beta chains. A rapid and efficient transient transfection system was developed using a polyoma virus-based vector. Typically, 30-50% of cells transfected using this system expressed high levels of class II molecules on their surface, but only with matched isotypic pairs. Biochemical analysis of cells transfected with matched or mixed isotypic pairs of the DR and DP molecules revealed that only matched chains could pair efficiently inside the cell. Thus, the lack of expression of the two mixed isotypic pairs is due to inefficient primary assembly of the class II molecule and not to a processing or transport defect. To define what region of the beta chains controlled their assembly with alpha chains, a series of chimeric cDNA molecules containing both DR and DP beta chain sequences were constructed. Expression of these chimeric beta chains with DR and DP alpha chains was determined by cytofluorimetry and biochemical analysis. Both alpha chains paired with beta chains in which only the beta 1 domain was isotypically matched. In contrast, the pattern of expression of chimeras made at other points within the beta 1 domain was different for DR and DP. These data show that different areas of primary sequence are important for the assembly of different human class II isotypes, and suggest that HLA-DR and -DP molecules have different secondary or tertiary structures in their NH2-terminal domains.
Assuntos
Antígenos HLA-DP/genética , Antígenos HLA-DR/genética , Animais , DNA/análise , Antígenos HLA-DP/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Camundongos , Conformação Proteica , TransfecçãoRESUMO
Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Epitopos/análise , Antígenos H-2/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Clonais , Genes MHC Classe I , Antígenos H-2/genética , Antígeno de Histocompatibilidade H-2D , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Mapeamento de Peptídeos , Peptídeos/síntese químicaRESUMO
Cell surface-expressed receptors are often multichain complexes. One of these, the T cell receptor (TcR) alpha/beta-CD3 complex, is known to contain at least seven chains: the alpha and beta TcR chains plus the gamma, delta, epsilon and two zeta chains from the CD3 complex (alpha beta gamma delta epsilon zeta 2). To gain insight into the structure of the complex we have used anti-peptide antisera specific for the individual subunits of the complex, and nonionic and ionic detergents to determine subunit interactions within the complex. Four closely associated pairs of chains could be identified: alpha beta, zeta 2, gamma epsilon and delta epsilon. Interactions between the TcR alpha beta and either gamma epsilon or delta epsilon could be observed in the apparent absence of other CD3 chains. Furthermore, a hierarchy in the strength of the association between the TcR and the individual CD3 chains could be distinguished: TcR epsilon greater than TcR delta greater than TcR gamma. The zeta 2 dimer could only be detected in "intact" TcR-CD3 complexes shedding no light on possible interactions with either the TcR or CD3-gamma, delta and epsilon chains. Finally, cross-linking experiments suggest a close spatial relationship between the TcR alpha beta and both the CD3-gamma and CD3-epsilon chains. The results demonstrate that the methods used give valuable information on subunit interactions in a cell surface-expressed receptor complex and suggest a TcR-CD3 complex in which two epsilon chains are present, one linked to gamma and the other to delta. The data further indicate that gamma epsilon and delta epsilon complexes interact directly with the TcR chains. Based on the observations a model for the structure of the TcR-CD3 is presented and discussed.