Isolation and characterization of antimicrobial proteins and peptide from chicken liver.

Endogenous antimicrobial peptides and proteins are crucial components of the innate immune system and play an essential role in the defense against infection. Antimicrobial activity was detected in the acid extract of livers harvested from healthy adult White Leghorn hens, Gallus gallus. Two antimicrobial proteins and one antimicrobial polypeptide were isolated from the liver extract by cation-exchange and gel filtration chromatography, followed by two-step reverse-phase high-performance liquid chromatography (RP-HPLC). These antimicrobial components were identified as histones H2A and H2B.V, and histone H2B C-terminal fragment using peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry. The proteins and the peptide identified in the present study, which exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, were thermostable and showed salt-resistant activity. The antimicrobial properties of histones and histone fragment in chicken provide further evidence that histones, in addition to their role in nucleosome formation, may play an important role in innate host defense against intracellular or extracellular microbe invasion in a wide range of animal species.     

Antimicrobial activity of histones from hemocytes of the Pacific white shrimp.

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. The L. vannamei histone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition against Micrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 microm while a mixture of histones H2B and H4 was active at 3 microm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought.     

Antimicrobial proteins in chicken reproductive system

Antimicrobial activity was detected in the ovary and oviduct tissues of healthy mature White Leghorn hens, Gallus gallus. Two antimicrobial proteins were purified to homogeneity using acid extraction followed by multiple steps of chromatography and the pure proteins were further characterized biochemically. Peptide mixtures obtained after enzymatic digestion of the chicken antimicrobial proteins were analyzed using peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry and the proteins were identified as histones H1 and H2B. Chicken histone antimicrobial proteins were active against both Gram-positive and Gram-negative bacteria. The abundance of these proteins in the reproductive tissues and their broad-spectrum antimicrobial nature may indicate their defensive role against pathogens during the follicle development in the ovary and egg formation in the oviduct. The discovery of antimicrobial histones in chicken reproductive system provides further evidence that histones may play a role in innate immunity against microorganisms in a wide range of animal species.     

Pepsin-mediated processing of the cytoplasmic histone H2A to strong antimicrobial peptide buforin I

The intestinal epithelium forms a first line of innate host defense by secretion of proteins with antimicrobial activity against microbial infection. Despite the extensive studies on the antimicrobial host defense in many gastrointestinal tracts, little is known about the antimicrobial defense system of the stomach. The potent antimicrobial peptide buforin I, consisting of 39 aa, was isolated recently from the stomach tissue of an Asian toad, Bufo bufo gargarizans. In this study we examined the mechanism of buforin I production in toad stomach tissue. Buforin I is produced by the action of pepsin isozymes, named pepsin Ca and Cb, cleaving the Tyr39-Ala40 bond of histone H2A. Immunohistochemical analysis revealed that buforin I is present extracellularly on the mucosal surface, and unacetylated histone H2A, a precursor of buforin I, is localized in the cytoplasm of gastric gland cells. Furthermore, Western blot analysis showed that buforin I is also present in the gastric fluids, and immunoelectron microscopy detected localization of the unacetylated histone H2A in the cytoplasmic granules of gastric gland cells. The distinct subcellular distribution of the unacetylated histone H2A and the detection of the unacetylated buforin I both on the mucosal surface and in the lumen suggest that buforin I is produced from the cytoplasmic unacetylated histone H2A secreted into the gastric lumen and subsequently processed by pepsins. Our results indicate that buforin I along with pepsins in the vertebrate stomach may contribute to the innate host defense of the stomach against invading microorganisms.     

Expression and purification of recombinant human histones

Nucleosomes reconstituted from bacterially expressed histones are useful for functional and structural analyses of histone variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human histone H2A, H2B, and H3 genes were expressed well in Escherichia coli cells, but the human histone H4 gene was poorly expressed. Therefore, we designed a new histone H4 gene with codons optimized for the E. coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histones were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6 M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histones in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed, and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B, and H4.     

Upregulation in response to infection and antibacterial activity of oyster histone H4

Several histones and histone-derived peptides have been shown to have antimicrobial activity and a potential role in innate immune defenses. A histone H4 sequence was identified in a subtractive suppression library containing genes upregulated in American cupped oysters, Crassostrea virginica, in response to challenge with the protozoan parasite Perkinsus marinus. Oyster histone H4 protein levels significantly increased in hemocyte lysates and cell free hemolymph of oysters experimentally challenged with P. marinus. The complete histone H4 coding sequence of C. virginica was cloned into a Saccharomyces cerevisiae yeast expression system and recombinant expression was confirmed using SDS-PAGE analysis and western blot. Delivery of yeast cells expressing recombinant oyster histone H4 into the gut of brine shrimp, Artemia salinas, challenged with a streptomycin resistant strain of Vibrio anguillarum resulted in a significant and dose-dependent decrease in the load of V. anguillarum. Purified recombinant histone H4 showed antimicrobial activity against V. anguillarum and Escherichia coli at micromolar concentrations, but did not affect the viability of P. marinus in culture. These results support the role of histone H4 in the defense of oysters against bacterial infection and validate the use of a novel oyster antimicrobial H4 in a yeast feed-based delivery system for the treatment of bacterial infections in aquaculture applications.     

Effect of histone acetylation on the formation and removal of B(a)P chromatin adducts.

The modification of core histone proteins in mouse 10T1/2 cells and human lung epitheloid (A549) cells by B(a)PDE in vivo and in vitro was found to be similar. Only histones H2A and H3 were extensively modified. Also other proteins, possibly A24 protein and the minor histone H1 species seem to be binding relatively high levels of this ultimate carcinogen. Butyrate treatment which causes hyperacetylation of the core histones, did not change the specificity of B(a)PDE binding to core histones, nor did it affect the initial level of DNA modification. The acetylated species of histone H3 were all accessible to B(a)PDE, suggesting that these epsilon-amino-groups of the lysine residues are not the targets of the B(a)PDE. The rate of removal of B(a)P-DNA adducts was not affected by butyrate treatment in either normal human or XP fibroblasts. Furthermore the B(a)P-core histones were not preferentially removed from normal human fibroblast chromatin during a 24 h post-treatment incubation.     

Antimicrobial properties of arginine- and lysine-rich histones and involvement of bacterial outer membrane protease T in their differential mode of actions

There is growing evidence of the antimicrobial properties of histones and histone-derived peptides; however, most of them are specific to lysine (Lys)-rich histones (H1, H2A, and H2B). In the present study, we focused on arginine (Arg)-rich histones (H3 and H4) and investigated their antimicrobial properties in comparison with those of histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against the bacterial outer membrane protease T (OmpT) gene-expressing Escherichia coli strain JCM5491 with calculated 50% growth inhibitory concentrations of 3.8, 10, and 12.7 μM, respectively. A lysate prepared from the JCM5491 cells was capable of strongly, moderately, and slightly fragmenting histones H2B, H3, and H4, respectively. While the lysate prepared from the cells of the ompT-deleted E. coli strain BL21(DE3) did not digest these histones, the ompT-transformed BL21(DE3), termed BL21/OmpT⁺, cell lysate digested the histones more strongly than the JCM5491 cell lysate. Laser confocal and scanning electron microscopic analyses demonstrated that while histone H2B penetrated the cell membrane of JCM5491 or BL21/OmpT⁺ cells, histones H3 and H4 remained on the cell surface and subsequently disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. The BL21(DE3) cells treated with each histone showed no bleb formation, but cell integrity was affected and the cell surface was corrugated. Consequently, it is suggested that OmpT is involved in the antimicrobial properties of Arg- and Lys-rich histones and that the modes of antimicrobial action of these histones are different.     

Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man

Human testis was shown to contain a specific histone, TH2B, having the same electrophoretic mobility as rat TH2B. Testicular and ejaculated human sperm still possessed histones at 50% and 15% of the total basic nuclear proteins, respectively. Comparison of the electrophoretic patterns of histones from human testis, testicular sperm and ejaculated sperm implied that the histones may be removed in the order H2A and H1 before H3, H4 and H2B before TH2B. TH2B which is the major histone fraction in ejaculated sperm has no longer a strong affinity to DNA. TH2B in sperm nuclei could be separated from other basic nuclear proteins by Bio-Gel P-10 column chromatography and its amino acid composition is similar to that of rat TH2B, although no cysteine residue was found.     

A protein with antimicrobial activity in the skin of Schlegel's green tree frog Rhacophorus schlegelii (Rhacophoridae) identified as histone H2B

An extract of the skin of Schlegel's green tree frog, Rhacophorus schlegelii (Anura: Rhacophoridae), contained a protein that inhibited the growth of the Gram-negative bacterium Escherichia coli but was inactive against the Gram-positive bacterium Staphylococcus aureus. The protein was purified to near homogeneity by reverse-phase HPLC and amino acid sequence analysis of the products of an endoproteinase Glu-C digest identified the protein as histone H2B. The complete primary structure of the 125 amino acid residue Rhacophorus histone H2B was determined by nucleotide sequence analysis of a cloned cDNA encoding the protein. Mass spectrometry demonstrated that the protein isolated from the skin was not post-translationally modified. Histone fragments with antimicrobial activity were not identified in the Rhacophorus skin extract nor were cationic, alpha-helical antimicrobial peptides of the kind isolated from the skins of several other frog families. The data provide further evidence that histones play a role in the defense against microorganisms.     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).     

The Histone Database: a comprehensive resource for histones and histone fold-containing proteins

The Histone Database is a curated and searchable collection of full-length sequences and structures of histones and nonhistone proteins containing histone-like folds, compiled from major public databases. Several new histone fold-containing proteins have been identified, including the huntingtin-interacting protein HYPM. Additionally, based on the recent crystal structure of the Son of Sevenless protein, an interpretation of the sequence analysis of the histone fold domain is presented. The database contains an updated collection of multiple sequence alignments for the four core histones (H2A, H2B, H3, and H4) and the linker histones (H1/H5) from a total of 975 organisms. The database also contains information on the human histone gene complement and provides links to three-dimensional structures of histone and histone fold-containing proteins. The Histone Database is a comprehensive bioinformatics resource for the study of structure and function of histones and histone fold-containing proteins. The database is available at http://research.nhgri.nih.gov/histones/.     

Differential mode of antimicrobial actions of arginine-rich and lysine-rich histones against Gram-positive Staphylococcus aureus

We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.0, 4.0, and 5.6 μM, respectively. Laser confocal microscopic analyses revealed that both the Arg-rich and Lys-rich histones associated with the surface of S. aureus. However, while the morphology of S. aureus treated with histone H2B appeared intact, those treated with the histones H3 and H4 closely resembled each other, and the cells were blurred. Electrophoretic mobility shift assay results revealed these histones have binding affinity to lipoteichoic acid (LTA), one of major cell surface components of Gram-positive bacteria. Scanning electron microscopic analyses demonstrated that while histone H2B elicited no obvious changes in cell morphology, histones H3 and H4 disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. Consequently, our results suggest that bacterial cell surface LTA initially attracts both the Arg- and Lys-rich histones, but the modes of antimicrobial action of these histones are different; the former involves cell membrane disruption and the latter involves the cell integrity disruption.     

Enzyme histochemically detectable NAD(P)H oxidase in human placental trophoblasts: normal, preeclamptic, and fetal growth restriction-complicated pregnancy

The purpose of the present study was to determine the subcellular localization of NAD(P)H oxidase, a reactive oxygen species (ROS)-producing enzyme, in the human placenta at various gestational ages. Ultrastructural enzyme histochemistry for NAD(P)H oxidase, using cerium as a capturing agent, was carried out. Placentas from patients with severe preeclampsia and patients who delivered infants with fetal growth restriction (FGR) were also studied. Electron-dense precipitates indicating NAD(P)H oxidase activity were visible in the microvillous membranes of the placentas, especially on the surface plasma membrane of the syncytiotrophoblast microvilli, after 25 weeks of gestation. The distribution pattern and enzyme intensities were apparently the same among normal, preeclamptic, and FGR placentas. Cytochemical control experiments ensured the specific detection of NAD(P)H oxidase activity. These observations indicated that syncytiotrophoblasts possessed NAD(P)H oxidase activity, and thus ROS-generating activity. Placental NAD(P)H oxidase may play a role in placental lipid peroxidation and the placental defense mechanism.     

Histones: a novel class of lipopolysaccharide-binding molecules

Unlike soluble and membrane forms of lipopolysaccharide (LPS)-binding proteins, intracellular LPS-binding molecules are poorly documented. We looked for such molecules in a murine lung epithelial cell line. Two proteins with LPS-binding activity were isolated and unambiguously identified as histones H2A.1 and H4 by mass spectrometry. Synthetic peptides representing partial structures indicated that the LPS binding site is located in the C-terminal moiety of the histones. Extending the study, we found that histones H1, H2A, H2B, H3, and H4 from calf thymus are all able to bind LPS. Bindings were specific, and affinities, determined by isothermal titration calorimetry, were (except for H4) higher than that of the LPS-binding antibiotic polymyxin B. In the presence of H2A the binding of LPS to the macrophage cell line RAW 264.7, and the LPS-induced production of TNF-alpha and nitric oxide by these cells, were markedly reduced. Histones may thus represent a new class of intracellular and extracellular LPS sensors.     

The antimicrobial action of histones in the reproductive tract of cow

An infection of any part of female reproductive tract can severely interfere with fertility and reproduction. The fluids and epithelium from the lumen of the female reproductive tract (uterus, oviduct and ovarian follicle) are a known source of antimicrobial action in several species. In this study, we compared the antimicrobial properties of fluids from the reproductive tract of a cow. After removal of small molecules, we demonstrated that there is an antimicrobial activity connected with a fraction of compounds with a molecular mass range between 3500 and 30,000. The most probable candidates responsible for the observed antimicrobial effect were subsequently identified by mass spectroscopy as histones H2A type 2-C, H2B type 1-K, H3.3, and H4. The antimicrobial role of histone H2B was further confirmed by using an antibody against this histone.     

Antimicrobial peptides in the first line defence of human colon mucosa

Antimicrobial peptides and proteins are effector molecules in the protection of epithelial surfaces. We have evaluated the presence of antimicrobial peptides/proteins that can participate in human colonic defence against microbes. A peptide/protein extract of normal human colon mucosa was found to be active against Gram-positive bacteria, Gram-negative bacteria, and fungi. Four polypeptides with antimicrobial activity were isolated from this material and they were identified by N-terminal amino acid sequence analysis as ubiquicidin, histone H2B, eosinophil cationic protein, and phospholipase A(2) (PLA(2)). Using immunodetection and mass spectrometry, LL-37, HNP1-3, and HBD-1 were also identified. Combined, these results indicate that the colon mucosa is protected by a complex mixture of polypeptides, able to kill invading microbes and working in synergy as a barrier against bacterial invasion.     

The heparin-binding lectin from ovine placenta: Purification and identification as histone H4

The heparin-binding lectin complex from ovine placental cotyledons was purified by affinity chromatography on heparin-agarose column. It showed three protein bands, which had molecular weights of 13 000, 15 000 and 17 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the presence of DNA by agarose gel electrophoresis. The protein components of the complex were separated by reverse-phase HPLC. The minimum inhibitory concentrations of glycosaminoglycans were significantly different for the lectin complex and the separated proteins, suggesting affinity changes upon DNA binding. The haemagglutinating activity specificity allowed the characterization of the fraction with a molecular weight of 13 000 as the heparin-binding lectin. This protein was identified as histone H4 by internal sequencing, thus showing that this is the histone responsible for the heparin-binding property of the complex. The accompanying proteins were tentatively identified as histones H2A and H2B. This revised version was published online in November 2006 with corrections to the Cover Date.     

Prostaglandin E(2) 9-keto reductase activity in bovine retained and not retained placenta

Prostaglandin E(2) 9-keto reductase (9-KPR) activity shifts reversibly PGE(2) into PGF(2 alpha) and may be responsible for the control of prostaglandins (PGs) levels in, among others, placental tissues. The retention of fetal membranes in cows is the postpartum disorder where the disturbances in PGs metabolism have been reported. It has been argued whether these disturbances are due to alterations in 9-KPR activity. In this study, the activity of the enzyme was determined in maternal and fetal bovine placental tissues which were divided into 6 groups as follows: (A) caesarian section before term without retained fetal membranes (n=10), (B) caesarian section before term with retained fetal membranes (n=10), (C) caesarian section at term without retained fetal membranes (n=12), (D) caesarian section at term with retained fetal membranes (n=12), (E) spontaneous delivery at term without retained fetal membranes (n=12), (F) spontaneous delivery at term with retained fetal membranes (n=12). The enzyme activity was measured spectrophotometrically and expressed in nanokatals (nkat) per protein content. The activity increased towards parturition and was significantly higher in maternal than in fetal part of placenta in all groups examined. The significantly higher values in retained than in not retained placental tissues were observed in the samples examined. The present results indicate that the disturbances in 9-KPR activity in bovine retained placenta exist but their reasons still require further experiments.