Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins.

Bac5 and Bac7, antibiotics of the bactenecin (proline/arginine-rich peptide) family, are stored as proforms in the large granules of bovine neutrophils [Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H. and Romeo, D. (1990) J. Cell Biol. 111, 1363-1371]. These proforms have been purified to homogeneity from granule extracts by immunoaffinity and reverse-phase chromatography. While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium with minimal inhibitory concentrations of 6-12 micrograms/ml, proBac5 and proBac7 do not affect the growth of the same microorganisms, even at 500 micrograms/ml. Previous investigations have suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a neutral serine protease stored in the azurophil granules. Purified proBac5 and proBac7 were thus treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass spectrometry, and N-terminal sequence analysis. Of the three proteases, only elastase is able to catalyze the stepwise cleavage of probactenecins into the corresponding mature peptides, which have the same mass, N-terminal sequence and antibiotic activity of authentic Bac5 and Bac7. These results point to the importance of cooperation between azurophils and large granules in mounting a defense reaction.     

Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules

Bactenecins are highly cationic polypeptides of bovine neutrophil granules and exert in vitro a potent antimicrobial activity. We have previously purified two bactenecins, designated in an abbreviated form Bac7 and Bac5 from their approximate molecular masses of 7 and 5 kD (Gennaro, R., B. Skerlavaj, and D. Romeo. 1989. Infect. Immun. 57:3142-3146). Here we have studied the biosynthesis, processing, and localization of precursors of Bac7 and Bac5 in bovine bone marrow cells of the myeloid lineage. In vitro translation directed by mRNA isolated from these cells has shown that the primary translation products are preprobactenecins of 23.5 and 21 kD, and are processed to polypeptides of 20 and 15.8 kD, respectively. The 20-kD polypeptide is the granule storage form of Bac7, or proBac7, as also demonstrated by Western blot analysis of lysates of peripheral neutrophils. Between 15 and 50 min from the beginning of its biosynthesis the 15.8-kD polypeptide is converted into the 15-kD granule storage form of Bac5, or proBac5. As shown by immunogold EM, proBac7 and proBac5 are sorted and targeted to the matrix of the so called large granules, which are the predominant organelles in the cytoplasm of bovine neutrophils and are the exclusive store of the nonoxidative antimicrobial system of these cells. Solubilization of granules with Triton X-100 with concomitant unmasking of proteases leads to cleavage of the proforms to Bac7 and Bac5. Experiments performed with protease inhibitors suggest that the proteolytic cleavage is catalyzed in detergent-solubilized neutrophils by neutral serine protease(s), very likely derived from the azurophil granules.     

Amino acid sequences of two proline-rich bactenecins. Antimicrobial peptides of bovine neutrophils

Bactenecins are highly cationic polypeptides of the large granules of bovine neutrophils, exerting in vitro a potent antimicrobial activity. Two bactenecins, with an approximate molecular weight of 7000 and 5000, called Bac7 and Bac5, are characterized by a high content of proline (greater than 45%) and arginine (greater than 23%) residues. Their complete amino acid sequences were determined by automated Edman degradation combined, in the case of Bac5, with plasma desorption mass spectrometry. Bac7 comprises 59 residues and includes three tandem repeats of a tetradecamer characterized by several Pro-Arg-Pro triplets spaced by single hydrophobic amino acids. Resolution of the primary structure of Bac5 required fragmentation with N-bromosuccinimide as well as digestion of the obtained C-terminal fragment with carboxypeptidases P and Y directly in the mass spectrometer. Bac5 comprises 42 amino acid residues with a repeated motif of Arg-Pro-Pro triplets also alternating with single apolar residues.     

Processing of Neutrophil α-Defensins Does Not Rely on Serine Proteases In Vivo

The α-defensins, human neutrophil peptides (HNPs) are the predominant antimicrobial peptides of neutrophil granules. They are synthesized in promyelocytes and myelocytes as proHNPs, but only processed in promyelocytes and stored as mature HNPs in azurophil granules. Despite decades of search, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unidentified. Thus, neither the enzyme that processes proHNPs nor the localization of processing has been identified. It has been hypothesized that proHNPs are processed by the serine proteases highly expressed in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which are able to process recombinant proHNP into HNP in vitro. We investigated whether serine proteases are in fact responsible for processing of proHNP in human bone marrow cells and in human and murine myeloid cell lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of ³⁵S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease proHNP processing in primary human bone marrow cells. Furthermore, introduction of NE, CG, and PR3 into murine promyelocytic cells did not enhance the proHNP processing capability. Finally, two patients suffering from Papillon–Lefèvre syndrome, who lack active neutrophil serine proteases, demonstrated normal levels of fully processed HNP in peripheral neutrophils. Contradicting earlier assumptions, our study found serine proteases dispensable for processing of proHNPs in vivo. This calls for study of other protease classes in the search for the proHNP processing protease(s).     

防御素研究进展

防御素是广泛分布于自然界中的广谱性抗菌多肽,在生物体内由特殊的细胞合成,经过一系列的加工后分泌表达出功能分子,由于其抗菌活性高,且作用范围广,引起了人们极大的兴趣,目前通过对其天然表达细胞的培养、合成肽和基因表达等手段制备,并进一步对其作用机理进行了探讨,但临床应用仍未见报道。     

Original involvement of antimicrobial peptides in mussel innate immunity

Recently, the existence and extended diversity of antimicrobial peptides has been revealed in two mussel species. These molecules are classified into four groups according to common features of their primary structure: defensins, mytilins, myticins and mytimycin. In Mytilus galloprovincialis, gene structure reveals synthesis as precursors in circulating hemocytes. Synthesised even in absence of challenge, the precursors mature and the peptides are stored in granules as active forms. The different peptides are engaged in the destruction of bacteria inside phagocytes, before being released into hemolymph to participate in systemic responses. Such involvement in anti-infectious responses is unique, and apparently more related to those of mammalian phagocytes than to those of insects.     

High fidelity processing and activation of the human α-defensin HNP1 precursor by neutrophil elastase and proteinase 3

The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1-4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages ("folded proHNP1") and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro.     

Linear bactenecin analogs with cell selectivity and anti‐endotoxic activity

Bactenecin (Bac) is a 12‐residue disulfide‐linked antimicrobial peptide isolated from the granules of bovine neutrophils. In this study, to develop novel linear Bac analogs with cell selectivity and anti‐endotoxic activity, we designed and synthesized a series of linear Bac analogs with amino acid substitution in Cys^3,11 and/or Val^6,7 of Bac. Among Bac analogs, some analogs (Bac‐W, Bac‐KW, Bac‐L, Bac‐KL, Bac‐LW, and Bac‐KLW) with higher hydrophobicity showed the amalgamated property of cell selectivity and anti‐endotoxic activity. Furthermore, Bac‐W, Bac‐KW, Bac‐LW, and Bac‐KLW showed serum stability comparable with that of disulfide‐bonded Bac. Therefore, these Bac analogs (Bac‐W, Bac‐KW, Bac‐LW, and Bac‐KLW) can serve as promising antibiotics for the development of therapeutic agents for treatment against endotoxic shock and bacterial infection. In addition, our results suggest that a little increase in hydrophobicity may be responsible for the decreased cell selectivity of the multiple Arg‐containing peptides (Bac‐W, Bac‐L, and Bac‐LW) over the multiple Lys‐containing peptides (Bac‐KW, Bac‐KL, and Bac‐KLW). Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Defensins in granules of phagocytic and non-phagocytic cells

Antimicrobial proteins stored in lysosome-like granules of neutrophils and macrophages probably play an important role in killing phagocytosed microbes after delivery to the phagolysosome. Among the granules' antimicrobial armamentarium are defensins, peptides that kill a broad spectrum of microorganisms in vitro. Antimicrobial defensins were recently also isolated from non-phagocytic granulocytes of the mouse small intestinal epithelium, from where they are secreted into the lumen to function extracellularly. Clarification of the antimicrobial mechanisms of defensins in intracellular and extracellular environments will provide a key to understanding peptide-mediated host defence.     

Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens

A key target of many intracellular pathogens is the macrophage. Although macrophages can generate antimicrobial activity, neutrophils have been shown to have a key role in host defense, presumably by their preformed granules containing antimicrobial agents. Yet the mechanism by which neutrophils can mediate antimicrobial activity against intracellular pathogens such as Mycobacterium tuberculosis has been a long-standing enigma. We demonstrate that apoptotic neutrophils and purified granules inhibit the growth of extracellular mycobacteria. Phagocytosis of apoptotic neutrophils by macrophages results in decreased viability of intracellular M. tuberculosis. Concomitant with uptake of apoptotic neutrophils, granule contents traffic to early endosomes, and colocalize with mycobacteria. Uptake of purified granules alone decreased growth of intracellular mycobacteria. Therefore, the transfer of antimicrobial peptides from neutrophils to macrophages provides a cooperative defense strategy between innate immune cells against intracellular pathogens and may complement other pathways that involve delivery of antimicrobial peptides to macrophages.     

牛抗菌肽Bac7-Bac5融合基因在大肠杆菌中的过表达，纯化及抑菌活性

牛抗菌肽Bac7和Bac5是一种线性阳离子小分子多肽,在机体天然免疫和获得性免疫中都发挥着重要作用。本研究根据Gen bank中公布的牛抗菌肽bac7和bac5成熟肽基因序列,人工合成了融合基因Bac7-Bac5片段,克隆于原核表达载体pET32(a+)中构建了重组表达载体（pET-B7-B5）,将其转化于E coli BL21(DE3) 中实现了重组蛋白B7-B5（rB7-B5）的过表达,表达的rB7-B5以包涵体形式存在,rB7-B5表达量约占细菌总蛋白的36.6％,分子量大小为33kD,与预测大小相符。以经Ni亲和层析柱纯化和多步透析法复性的rB7-B5,对猪胸膜肺炎放线杆菌和耐药性大肠杆菌具有很好的抑菌活性,本研究为新型抗菌制剂的研制和开发奠定了基础。     

Post-translational amino acid racemization in the frog skin peptide deltorphin I in the secretion granules of cutaneous serous glands

The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, alpha-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a D-amino acid, deltorphin I (Tyr-DAla-Phe-Asp-Val-Val-Gly NH2). Its biological activity is due to the racemization of L-Ala2 to D-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the L- to D-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.     

Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action.

Defensins are endogenous antimicrobial peptides stored in neutrophil granules. Here we report that a panel of defensins from human, rat, guinea pig, and rabbit neutrophils all have histamine-releasing activity, degranulating rat peritoneal mast cells with EC50 ranging from 70 to 2500 nM, and between 45 and 60% of the total histamine released. The EC50 for defensin-induced histamine secretion correlates with their net basic charge at neutral pH. There is no correlation between histamine release and antimicrobial potency. Degranulation induced by defensins has characteristics similar to those of activation by substance P. The maximum percent histamine release is achieved in <10 s, and it can be markedly inhibited by pertussis toxin (100 ng/ml) and by pretreatment of mast cells with neuraminidase. These properties differ from those for degranulation induced by IgE-dependent Ag stimulation and by the calcium ionophore A23187. GTPase activity, a measure of G protein activation, was induced in a membrane fraction from mast cells following treatment with defensin. Thus, neutrophil defensins are potent mast cell secretagogues that act in a manner similar to substance P and 48/80, through a rapid G protein-dependent response that is mechanistically distinct from Ag/IgE-dependent mast cell activation. Defensins may provide important pathways for communication between neutrophils and mast cells in defenses against microbial agents and in acute inflammatory responses.     

SEQUENTIAL DEGRANULATION OF THE TWO TYPES OF POLYMORPHONUCLEAR LEUKOCYTE GRANULES DURING PHAGOCYTOSIS OF MICROORGANISMS

The sequential discharge of neutrophilic polymorphonuclear leukocyte (PMN) granules—azurophils and specifics—was investigated by electron microscopy and cytochemistry. Thus the enzyme content of PMN phagocytic vacuoles was determined at brief intervals after phagocytosis of bacteria, utilizing peroxidase as a marker enzyme for azurophil granules, and alkaline phosphatase for specifics. At 30 s, approximately half the phagocytic vacuoles were reactive for alkaline phosphatase, whereas none contained peroxidase. Peroxidase-containing vacuoles were rarely seen at 1 min, but by 3 min, vacuoles containing both enzymes were consistently present. Alkaline phosphatase was found in both small and large vacuoles, whereas peroxidase was visible only in large ones. By 10 min, very big phagocytic vacuoles containing considerable amounts of reaction product for both enzymes were evident. These observations indicate that the two types of PMN granules discharge in a sequential manner, specific granules fusing with the vacuole before azurophils. In an earlier paper, we reported that the pH of phagocytic vacuoles drops to 6.5 within 3 min and to ~4 within 7–15 min. Substances known to be present in specific granules (alkaline phosphatase, lysozyme, and lactoferrin) function best at neutral or alkaline pH, whereas most of those contained in azurophil granules (i.e., peroxidase and the lysosomal enzymes) have pH optima in the acid range. Hence the sequence of granule discharge roughly parallels the change in pH, thereby providing optimal conditions for coordinated activity of granule contents.     

Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils

We used ultrastructural autoradiographic and cytochemical methods to localize esterase activities in unstimulated guinea pig basophils and in basophils undergoing degranulation or recovery from degranulation. We used tritium-labeled diisopropylfluorophosphate (DFP) as a probe for serine enzymes and localized this probe by ultrastructural autoradiography to cytoplasmic granules of immature or mature unstimulated basophils, as well as to granules released by degranulating basophils. Ultrastructural cytochemistry using alpha naphthyl acetate (ANA) as substrate localized nonspecific esterase activity to extruded granules, either within the interiors of degranulation sacs or within granules completely separated from degranulating basophils. Extruded granules retained their esterase activity for as long as 24 hr after antigen-induced degranulation. The plasma membranes of unstimulated or degranulating basophils, as well as of basophils recovering from degranulation, displayed prominent cell surface ANA esterase ectoenzyme activity. Lipid bodies, organelles present in the cytoplasm of both control and recovering basophils, were also alpha naphthyl acetate esterase (ANAE)-positive. Thus, cytochemical and autoradiographic techniques localized esterase and/or [3H]-DFP-binding activities to cytoplasmic granules, lipid bodies, and cell surface of basophils, and these enzyme activities persisted during both degranulation and recovery from degranulation.     

Linkage between neutrophil degranulation and calcium discharge

Calcium flux across organelle and plasma membranes is an important event in neutrophil activation. We measured calcium discharge into the media from neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine after treatment with cytochalasin b. Cytochalasin markedly potentiated calcium efflux from stimulated neutrophils, and similarly promoted release of lysosomal enzymes into the media. Colchicine neither reproduced nor modified the cytochalasin effect. Neutrophil cytoplasts discharged very little calcium in response to stimulation, and discharge was not significantly altered by cytochalasin b. These findings indicate that neutrophil degranulation is accompanied by efflux of calcium into the media, and suggest that the neutrophil granules constitute a source of mobilizable calcium which could be used to modify the extracellular microenvironment.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Specific granules of human eosinophils have lysosomal characteristics: presence of lysosome-associated membrane proteins and acidification upon cellular activation

Eosinophils possess characteristic specific granules. Their content may be important during host defense but it can also cause damage after release at sites of inflammation. We investigated possible lysosomal characteristics of these granules. Lysosome-associated membrane protein (LAMP)-1 and 2, were detected by Western blot, subcellular fractionation, and immunoelectron microscopy (IEM) and were localized to the membrane of specific granules and in vesicles of the cytoplasm, separate from secretory vesicles. No binding of mannose 6-phosphate receptor to proteins of specific granules could be detected, indicating that they are dephosphorylated and mature. Cellular activation by interleukin-5 caused acidification of specific granules, as detected by pH-dependent probes. The acidification was inhibited by concanamycin A (inhibitor of vacuolar H(+)-ATPase). Activation of eosinophils by serum-treated zymosan (STZ) caused degranulation into STZ-containing phagosomes and incorporation of LAMPs to their membranes. In conclusion, specific granules of eosinophils can be regarded as specialized primary lysosomes, a feature that may be important for their function and integrity.     

CYTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASE ACTIVITY IN GRANULE FRACTIONS FROM RABBIT POLYMORPHONUCLEAR LEUKOCYTES

When rabbit peritoneal exudates (97% polymorphonuclear [PMN] leukocytes, 2% mononuclear cells) were fractionated by zonal sedimentation or isopycnic centrifugation, four fractions (A, B, C, and D) were obtained, as reported earlier. "A" consisted largely of PMN azurophil granules, "B" of PMN specific granules, and "D" of membranous elements. The source of the more heterogeneous "C" fraction (containing acid hydrolases) was uncertain. To gain further information on the nature of this fraction, cytochemical tests for acid phosphatase (AcPase) were carried out on the starting cells and on the fractions. In intact PMN, lead phosphate reaction product was found in Golgi complexes, perinuclear cisternae, and some azurophil granules (immature forms or disrupted mature forms) of a few cells. The specifics and the intact azurophils were not reactive. Reaction product was also found within Golgi cisternae, secondary lysosomes, and some of the azurophil granules of mononuclear cells. Observations on the A and B fractions confirmed those in situ regarding the localization of reaction product in disrupted PMN azurophils, its absence from specifics, and the latency of the enzyme activity in intact azurophils. In the C fraction, AcPase was found in three structures (a) Golgi cisternae, (b) dense bodies, and (c) small pleomorphic granules Comparison with the starting cells indicates that the Golgi complexes are probably derived from both PMN leukocytes and mononuclear cells, whereas the remaining elements resemble (in size, shape, and density) secondary lysosomes and azurophil granules of mononuclear cells. The results indicate that the bulk of the cytochemically detectable AcPase present in the C fraction is derived from mononuclear cells, rather than from PMN leukocytes