Annexin I is stored within gelatinase granules of human neutrophil and mobilized on the cell surface upon adhesion but not phagocytosis

Annexin I, a member of the calcium- and phospholipid-binding annexin superfamily of proteins, is largely present in human neutrophils. To determine its exact intracellular distribution a combination of flow cytometry, confocal microscopy and electron microscopy analyses were performed on resting human neutrophils as well as on cells which had been activated. In resting neutrophils, annexin I was found to be present in small amounts in the nucleus, in the cytoplasm and partially also associated with the plasma membrane. The cytoplasmic pool of annexin I was predominant, and the protein was co-localized with gelatinase (marker of gelatinase granules), but not with human serum albumin or CD35 (markers of secretory vesicles), or with lysosomes. Electron microscopy showed the presence of annexin I inside the gelatinase granules. Neutrophil adhesion to monolayers of endothelial cells, but not phagocytosis of particles of opsonized zymosan, provoked an intense mobilization of annexin I, with a marked externalization on the outer leaflet of the plasma membrane. Remaining intracellular annexin I was also found in proximity of the plasma membrane. These results provide a novel mechanism for annexin I secretion from human neutrophils, which is via a degranulation event involving gelatinase granules.     

Glycosylation of natural human neutrophil gelatinase B and neutrophil gelatinase B-associated lipocalin.

Gelatinase B is a matrix metalloproteinase (MMP-9) involved in tissue remodeling, development, cancer, and inflammation. Neutrophils produce three major forms of (pro)gelatinase B: 92 kDa monomers, homodimers, and complexes of gelatinase B covalently bound to neutrophil gelatinase B-associated lipocalin (NGAL). In contrast to the case for other proteinases, little information about the glycosylation of any natural human MMP is available. Here, both gelatinase B and NGAL were purified from human peripheral blood neutrophils, and the entire contents of the released N- and O-glycan pools were analyzed simultaneously using recently developed high-performance liquid chromatography-based technology. The results are discussed within the context of the domain structure of gelatinase B and a molecular model of NGAL based on data from this study and the three-dimensional nuclear magnetic resonance (NMR) structure of the protein. More than 95% of the N-linked glycans attached to both gelatinase B and NGAL were partially sialylated, core-fucosylated biantennary structures with and without outer arm fucose. The O-linked glycans, which were estimated to comprise approximately 85% of the total sugars on gelatinase B, mainly consisted of type 2 cores with Galbeta1,4GlcNAc (lactosamine) extensions, with or without sialic acid or outer arm fucose. This paper also contains the first report of O-linked glycans attached to NGAL. Although both proteins were isolated from neutrophils and contained O-linked glycans mainly with type 2 cores, the glycans attached to individual serine/threonine residue(s) in NGAL were significantly smaller than those on gelatinase B. In contrast to NGAL, gelatinase B contains a region rich in Ser, Thr, and Pro typical of O-glycosylated mucin-like domains.     

The activation of human neutrophil gelatinase

To study the mechanisms of activation of human neutrophil gelatinase, the enzyme has been purified using a combination of chromatography on a DEAE-Sephacel and a gelatin-peptide-Sepharose column. On reducing SDS-polyacrylamide-gel electrophoresis the purified gelatinase ran as a single band of about 94,000 Da, and had a specific activity of 5624.4 units/mg of enzyme protein. When latent gelatinase was treated with trypsin, cathepsin G, neutrophil elastase, HgCl2 or urea, its activity was enhanced and the enzyme was processed and converted into species of the lower molecular mass. Upon activation, the protein band of 94,000 Da of reduced latent gelatinase underwent a decrease of about 6,000-12,000 Da. Formation of the species of lower molecular mass during urea activation could be blocked by the addition of EDTA.     

Proteomic analysis of human neutrophil granules

Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.     

Activation of the latent human neutrophil gelatinase by urea.

The mechanism of activation of the latent human neutrophil gelatinase by urea has been studied in greater detail. After dialysis of the latent gelatinase against increasing concentrations of urea a considerable increase of its activity was observed. Moreover, the results indicate a progressive conversion of the latent 94,000 Da gelatinase into a proteolytically active fragment of 80,000 Da, which was subsequently processed to a few species of lower molecular mass inactive against gelatin. This conversion was completely inhibited by EDTA, suggesting an autocatalytic reaction. The inhibition was reversed by Zn2+ or Co2+. Thus, urea alters both the enzymatic and physical characteristics of the latent gelatinase which suggests that conformational changes may induce autoactivation of the latent enzyme.     

Activation of human neutrophil gelatinase by endogenous serine proteinases.

The role of serine proteinases and oxidants in the activation of gelatinase released from human neutrophils was investigated. Gelatinase was measured by its ability to degrade both gelatin and native glomerular basement-membrane type IV collagen. When fMet-Leu-Phe or phorbol 12-myristate 13-acetate was used to stimulate the neutrophils, no gelatinase activity was measured in the absence of a mercurial activator, indicating that the enzyme was released entirely in latent form. However, when fMet-Leu-Phe-stimulated cells were treated with cytochalasin B, 50-70% of the maximal gelatinase activity was released. Activation was blocked by the serine-proteinase inhibitor phenylmethanesulphonyl fluoride and a specific inhibitor of neutrophil elastase, but was not affected by an inhibitor of cathepsin G. Addition of catalase or azide to prevent oxidative reactions did not affect activation of gelatinase under any conditions of stimulation, indicating that oxidants were not involved in activation. Our results imply that oxidative activation of gelatinase does not occur readily. However, neutrophil serine proteinases, particularly elastase, provide an alternative and apparently more efficient mechanism of activation.     

The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes.

Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.     

Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase

Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.     

Elastase in the different primary granules of the human neutrophil

Elastase in the human neutrophil is associated with various subpopulations of primary granules of different density. The proportion of this enzyme that is extracted with acetate pH 4.2 and cetyltrimethylammonium bromide varies in the different subpopulations. Nevertheless, the electrophoretic mobility and relative proportions of elastase isoenzymes is the same in both extracts from the different subpopulations. On stimulation of neutrophils with N-formylmethionylleucylphenylalanine, elastase is not released from the least dense subpopulation, whereas other two subpopulations do undergo degranulation to approximately the same extent. However, the release of elastase from these two denser granules differs after they are isolated and treated with calcium.     

Annexin I-mediated vesicular aggregation: mechanism and role in human neutrophils.

Whole cytosol isolated from human neutrophils was found to accelerate the Ca(2+)-dependent fusion of phospholipid vesicles with neutrophil plasma membranes as measured by several fluorescence resonance energy transfer lipid dilution assays or by the fate of an encapsulated aqueous soluble fluorophore. The Ca2+ (threshold of 2-10 microM) and protein concentration dependencies for fusion mediated by purified human neutrophil annexin I (lipocortin I), recombinant annexin I and des(1-9)annexin I showed behavior similar to that of whole cytosol. A monoclonal antibody against the N-terminal region of annexin I strongly inhibited the action of isolated annexins as well as whole cytosol, indicating that annexin I is the major activity of this type in whole neutrophil cytosol and that it functions even in this complex mixture of proteins. Residual Ca(2+)-dependent fusion activity in the absence of cytosol or annexin I was not inhibited by several antibodies against annexin I, implicating an as yet unknown protein. Kinetic analysis of liposomal fusion showed that annexin I, as in the case of synexin, accelerates aggregation of vesicles but not the actual fusion event per se. The disposition of annexin I in liposomal aggregates was studied by monitoring binding of the protein with a pyrene-phospholipid and by simultaneously monitoring vesicular aggregation by turbidity. An antibody to the N-terminus of annexin I inhibited vesicular aggregation but not binding, suggesting that initial binding of annexin I is similar to that of annexin V. A relatively small proportion of the bound annexin was involved in intervesicular linkage, and no exchange of bound annexin to subsequently added vesicles was observed. The lack of extensive contact between lipids of aggregated vesicles was supported by a lack of energy transfer between phospholipid probes on separate aggregating vesicles. Covalent linkage of maleimidyl or photoaffinity phospholipid derivatives with annexin I in vesicular aggregates did not allow complete disaggregation of vesicles by EDTA, suggesting that monomers of annexin I can contact two membranes simultaneously at the point of intervesicular linkage. These data are discussed in terms of possible models for the structure of this site.     

Cytotoxicity for tumor cells of cationic proteins from human neutrophil granules

Cationic proteins purified from human polymorphonuclear leukocyte granules exert a cytotoxic effect on mammalian tumor cells. This effect is time and concentration dependent, is inhibited by the anionic agent heparin, and is enhanced by preheating the cationic proteins. Other strongly basic proteins (histone, protamine) also exhibited cytotoxic activity. Myeloperoxidase isolated from human leukocytes is cytotoxic when combined with H2O2 and chloride. Under these conditions, the potency of the myeloperoxidase-mediated system is greater than that of the cationic proteins.     

Identification of phosphoproteins associated with human neutrophil granules following chemotactic peptide stimulation

Regulated exocytosis of neutrophil intracellular storage granules is necessary for neutrophil participation in the inflammatory response. The signal transduction pathways that participate in neutrophil exocytosis are complex and poorly defined. Several protein kinases, including p38 MAPK and the nonreceptor tyrosine kinases, Hck and Fgr, participate in this response. However, the downstream targets of these kinases that regulate exocytosis are unknown. The present study combined a novel inhibitor of neutrophil exocytosis with proteomic techniques to identify phosphopeptides and phosphoproteins from a population of gelatinase and specific granules isolated from unstimulated and fMLF-stimulated neutrophils. To prevent loss of granule-associated phosphoproteins upon exocytosis, neutrophils were pretreated with a TAT-fusion protein containing a SNARE domain from SNAP-23 (TAT-SNAP-23), which inhibited fMLF-stimulated CD66b-containing granule exocytosis by 100±10%. Following TAT-SNAP-23 pretreatment, neutrophils were stimulated with the chemotactic peptide fMLF for 0 min, 1 min, and 2 min. Granules were isolated by gradient centrifugation and subjected to proteolytic digestion with trypsin or chymotrypsin to obtain peptides from the outer surface of the granule. Phosphopeptides were enriched by gallium or TiO2 affinity chromatography, and phosphopeptides and phosphorylation sites were identified by reversed phase high performance liquid chromatography-electrospray ionization-tandem MS. This resulted in the identification of 243 unique phosphopeptides corresponding to 235 proteins, including known regulators of vesicle trafficking. The analysis identified 79 phosphoproteins from resting neutrophils, 81 following 1 min of fMLF stimulation, and 118 following 2 min of stimulation. Bioinformatic analysis identified a potential Src tyrosine kinase motif from a phosphopeptide corresponding to G protein coupled receptor kinase 5 (GRK5). Phosphorylation of GRK5 by Src was confirmed by an in vitro kinase reaction and by precursor ion scanning for phospho-tyrosine specific immonium ions containing Tyr251 and Tyr253. Immunoprecipitation of phosphorylated GRK5 from intact cells was reduced by a Src inhibitor. In conclusion, targets of signal transduction pathways were identified that are candidates to regulate neutrophil granule exocytosis.     

Peroxynitrite regulates exocytosis of neutrophil granules

Peroxynitrite is formed in the organism by activated neutrophils as a result of the enhanced production of nitrogen monoxide and superoxide anion radical in the inflammation foci. Since peroxynitrite modifies the structure of macromolecules, including the elements of actin cytoskeleton, it can influence signal transduction pathways that regulate intracellular granule exocytosis. In this paper we explore a dual effect of peroxynitrite on the processes of neutrophil degranulation by the methods of flow cytometry, light microscopy, and atomic force microscopy. We showed that peroxynitrite at concentrations less than 300 μM activated graded exocytosis of neutrophil intracellular granules, which resulted in the enhancement of neutrophil adhesion to the substrate, cell spreading on the substrate, and activation of neutrophil ability to kill microorganisms. Peroxynitrite at higher concentrations inhibited exocytosis of neutrophil granules and hindered cell adhesion to the substrate. The character of influence of the specific agents, such as colchicine and cytochalasin that selectively disrupt cytoskeletal structures, on peroxynitrite-induced changes in neutrophil morphology indicates an important role of actin cytoskeleton in the regulation of intracellular granule exocytosis induced by peroxynitrite. Our results support the hypothesis suggesting that peroxynitrite is a natural regulator of neutrophil effector functions.     

Collagenase is a component of the specific granules of human neutrophil leucocytes.

Azurophil and specific granules were isolated from human polymorphonuclear neutrophil leucocytes. Collagenase was almost exclusively a component of the specific granules. This finding is in contrast with the distribution of other proteolytic enzymes, which are localized in the azurophil (or lysosomal) granules.     

Ligand preference inferred from the structure of neutrophil gelatinase associated lipocalin

Neutrophil gelatinase associated lipocalin (NGAL), a constituent of neutrophil granules, is a member of the lipocalin family of binding proteins. NGAL can also be highly induced in epithelial cells in both inflammatory and neoplastic colorectal disease. NGAL is proposed to mediate inflammatory responses by sequestering neutrophil chemoattractants, particularly N-formylated tripeptides and possibly leukotriene B(4) and platelet activating factor. The crystal structures of NGAL display a typical lipocalin fold, albeit with an unusually large and atypically polar binding site, or calyx. The fold of NGAL is most similar to the epididymal retinoic acid-binding protein, another lipocalin, though the overall architecture of the calyces are very different. The crystal structures also reveal either sulfate ions or an adventitiously copurified fatty acid bound in the binding site. Neither ligand is displaced by added N-formylated tripeptides. The size, shape, and character of the NGAL calyx, as well as the low relative affinity for N-formylated tripeptides, suggest that neither the copurified fatty acid nor any of the proposed ligands are likely to be the preferred ligand of this protein. Comparisons between the crystal structures and the recently reported solution structure of NGAL reveal significant differences, in terms of both the details of the structure and the overall flexibility of the fold.     

Isolation and characterization of a human neutrophil aggregation defective mutant of Fusobacterium nucleatum

Fusobacterium nucleatum is known to adhere to human polymorphonuclear neutrophils (PMNs) and cause them to aggregate. In this study, we isolated a spontaneously occurring aggregation defective (AGG(-)) mutant and this mutant will be used for future study of the interactions between this bacterium and human PMN. Genomic DNA fingerprinting by random-primed polymerase chain reaction method revealed a difference between the parent strain and the AGG(-) mutant. This mutant also showed an altered phenotype in both microbicidal and phagocytic assays, suggesting that the bacterial factor involved in the aggregation may also be very important for the phagocytosis and, subsequently, the killing by human PMNs. Further study of this mutant may help to clarify the molecular mechanisms of the interaction between this pathogen and human PMNs.