Innate defense against influenza A virus: activity of human neutrophil defensins and interactions of defensins with surfactant protein D

Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some instances when relatively SP-D-resistant strains were treated with SP-D and HNPs. HNPs were found to bind to the neck and/or carbohydrate recognition domain of SP-D. This binding was specific because no, or minimal, binding to other collectins was found. HNPs precipitated SP-D from bronchoalveolar lavage fluid and reduced the antiviral activity of bronchoalveolar lavage fluid. HNP-1 and -2 differed somewhat in their independent antiviral activity and their binding to SP-D. These results are relevant to the early phase of host defense against IAV, and suggest a complex interplay between SP-D and HNPs at sites of active inflammation.     

Isolation and partial characterization of the entire human pro alpha 1(II) collagen gene.

Using a cDNA probe specific for the bovine Type II procollagen, a series of overlapping genomic clones containing 45 kb of contiguous human DNA have been isolated. Sequencing of a 54 bp exon, number 29, provided direct evidence that the recombinant clones bear human Type II collagen sequences. Localization of the 5' and 3' ends of the gene indicated that the human Type II collagen gene is 30 kb in size. This value is significantly higher than that of the homologous avian gene. The segregation of a polymorphic restriction site in informative families conclusively demonstrated that the Type II gene is found in a single copy in the human haploid genome. Finally, sequencing of a triple helical domain exon has confirmed that a rearrangement leading to the fusion of two exons occurred in the pro alpha 1(I) gene, following the divergence of the fibrillar collagens.     

Impact of pro segments on the folding and function of human neutrophil alpha-defensins

Human neutrophil alpha-defensins (HNPs) are synthesized in vivo as inactive precursor proteins, i.e. preproHNPs. A series of sequential proteolytic events excise the N-terminal inhibitory pro peptide, leading to defensin maturation and storage in azurophilic granules. The anionic pro peptide, required for correct sub-cellular trafficking and sorting of proHNPs, inhibits the antimicrobial activity of cationic defensins, either inter or intra-molecularly, presumably through charge neutralization. To better understand the role of the pro peptide in the folding and functioning of alpha-defensins and/or pro alpha-defensins, we chemically attached the proHNP1 pro peptide or (wt)pro peptide and the following artificial pro segments to the N terminus of HNP1: polyethylene glycol (PEG), Arg(10) (polyR), Ser(10) (polyS), and (cr)pro peptide, a charge-reversing mutant of the pro peptide where Arg/Lys residues were changed to Asp, and Asp/Glu residues to Lys. Comparative in vitro folding suggested that while all artificial pro segments chaperoned defensin folding, with PEG being the most efficient, the pro peptide catalyzed the folding of proHNPs likely through two independent mechanisms: solubilization of and interaction with the C-terminal defensin domain. Further, the N-terminal artificial pro segments dramatically altered the bactericidal activity of HNP1 against both Escherichia coli and Staphylococcus aureus. Surprisingly, (cr)pro peptide and (wt)pro peptide showed similar properties with respect to intra-molecular and inter-molecular catalysis of defensin folding as well as alpha-defensin binding, although their binding modes appeared different. Our findings identify a dual chaperone activity of the pro peptide and may shed light on the molecular mechanisms by which pro alpha-defensins fold in vivo.     

Prepro-endothelin-1 mRNA and its mature peptide in human appendix

Because the precise immunopathological events occurring in appendicitis are not completely understood, possible local production of endothelin-1 (ET-1) in human appendix was investigated. We used immunohistochemistry and in situ hybridization to detect the presence, distribution, and phenotype of ET-1-positive cells and prepro-ET-1 (pp-ET-1) mRNA-expressing cells. ET-1-positive stromal cells and pp-ET-1 mRNA-expressing cells were detected with different distributions and relative frequencies in normal control appendix, histologically normal appendix, and inflamed appendix. Six of 20 histologically normal appendixes from patients with a clinical diagnosis of acute appendicitis had many ET-1-positive stromal cells and high pp-ET-1 mRNA expression, similar to inflamed appendix. Forty percent of the pp-ET-1 mRNA-expressing cells were neutrophils, and the other positive cells were mast cells and macrophages. We suggest that local production of ET-1 by neutrophils and other inflammatory cells could be a molecular sign of focal inflammation in histologically normal appendixes and that ET-1 could be implicated, with other cytokines, in the pathogenesis of appendicitis by inducing appendiceal ischemia through vasoconstriction.     

Preparation and characterization of the extracellular domain of human Sid-1

In C. elegans, the cell surface protein Sid-1 imports extracellular dsRNA into the cytosol of most non-neuronal cells, enabling systemic spread of RNA interference (RNAi) throughout the worm. Sid-1 homologs are found in many other animals, although for most a function for the protein has not yet been established. Sid-1 proteins are composed of an N-terminal extracellular domain (ECD) followed by 9-12 predicted transmembrane regions. We developed a baculovirus system to express and purify the ECD of the human Sid-1 protein SidT1. Recombinant SidT1 ECD is glycosylated and spontaneously assembles into a stable and discrete tetrameric structure. Electron microscopy (EM) and small angle x-ray scattering (SAXS) studies reveal that the SidT1 ECD tetramer is a compact, puck-shaped globular particle, which we hypothesize may control access of dsRNA to the transmembrane pore. These characterizations provide inroads towards understanding the mechanism of this unique RNA transport system from structural prospective.     

The pro domain of pre-pro-transforming growth factor beta 1 when independently expressed is a functional binding protein for the mature growth factor

Transforming growth factor beta 1 (TGF-beta 1) is proteolytically derived from the carboxyl terminus of a 390 amino acid precursor molecule termed pre-pro-TGF-beta 1. Previous studies have suggested that the pro piece of pre-pro-TGF-beta 1 may play an important role in the formation of an inactive, latent complex. These latent forms are thought to be important in the regulation of TGF-beta 1 activity. To understand this latent complex in more detail, we have expressed the pro domain of pre-pro-TGF-beta 1 in tissue culture cells independent of the mature growth factor. A stop codon was genetically engineered into the cDNA of pre-pro-TGF-beta 1 by changing the Arg-278 codon from CGA to the STOP codon TGA. The resulting protein is truncated just prior to the amino-terminal Ala residue of the mature growth factor. Transient expression studies and immunoblotting indicate that this pro piece is readily made and secreted by the COS-1 cells; the major form of the expressed pro piece, when analyzed by SDS-polyacrylamide gel electrophoresis, behaves as a disulfide-linked dimer (Mr 80,000). Bioassays, using mink lung indicator cells, reveal that the pro domain forms an inactive complex with exogenously added mature TGF-beta 1. Treatment of this complex with heat or acid results in the release of active TGF-beta 1, indicating an in vitro structure similar to natural, latent TGF-beta 1 complexes. The pro piece from TGF-beta 1 was also found to form latent structures with two closely related family members, TGF-beta 1.2 and TGF-beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and characterization of five overlapping cDNAs specific for the human pro alpha 1(I) collagen chain.

We report the cloning of five overlapping cDNAs bearing sequences specific for the human pro alpha 1(I) collagen chain. Poly-A RNA enriched for collagen sequences was purified from normal human fibroblasts and used as template to synthesize double stranded cDNA. The cDNA was inserted into the Eco RI site of pBR 322 by blunt-ending and dG:dC tailing. The clones were screened by colony hybridization using the original RNA population and the resulting five positive clones subjected to restriction endonuclease mapping analysis and DNA sequencing. These overlapping clones cover from residue 247 in the alpha chain to part of the 3' end untranslated region of the pro alpha 1(I) mRNA for a total of 3400 nucleotides.     

Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.     

Endothelin-1 stimulates its own synthesis in human endothelial cells.

We studied whether endothelin-1 (ET-1) would affect its own synthesis. Human umbilical cord vein endothelial cells in methionine-poor culture medium containing [35S] methionine were treated with synthetic ET-1 or ET-3. Immunoprecipitation of 35 S-labeled ET-1 was performed with rabbit ET-1 antiserum. ET-1 caused an 40 +/- 4% (mean +/- SEM) increase of immunoprecipitable 35 S-labeled ET-1 as confirmed by its elution point in reversed phase high power liquid chromatography (HPLC). ET-3 caused a 23 +/- 2% increase in ET-1 concentration. Amplification of cDNA by PCR showed both ET-1 and ETB receptor mRNAs in human cord vein endothelial cells. We conclude that ET-1 increases its own synthesis in endothelial cells. This suggests a positive autocrine feed-back action of ET-1 on its own synthesis, an effect which is probably mediated by non-specific ETB receptors.     

Isolation and characterization of Neurospora crassa mutants resistant to antifungal plant defensins

Twenty-five Neurospora crassa mutants obtained by chemical mutagenesis were screened for increased resistance to various antifungal plant defensins. Plant defensin-resistant N. crassa mutants were further tested for their cross-resistance towards other families of structurally different antimicrobial peptides. Two N. crassa mutants, termed MUT16 and MUT24, displaying resistance towards all plant defensins tested but not to structurally different antimicrobial peptides were selected for further characterization. MUT16 and MUT24 were more resistant towards plant defensin-induced membrane permeabilization as compared to the N. crassa wild-type. Based on the previously demonstrated key role of fungal sphingolipids in the mechanism of growth inhibition by plant defensins, membrane sphingolipids of MUT16 and MUT24 were analysed. Membranes of these mutants contained structurally different glucosylceramides, novel glycosylinositolphosphorylceramides, and an altered level of steryl glucosides. Evidence is provided to link these clear differences in sphingolipid profiles of N. crassa mutants with their resistance towards different plant defensins.     

Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1

MICALs form a conserved multidomain protein family essential for cytoskeletal rearrangements. To complement structural information available, we produced the FAD-containing monooxygenase-like domain of human MICAL-1 (MICAL-MO) in forms differing for the presence and location of a His-tag, which only influences the protein yields. The K(m) for NADPH of the NADPH oxidase reaction is sensitive to ionic strength and type of ions. The apparent k(cat) (pH 7) is limited by enzyme reduction by NADPH, which occurs without detectable intermediates, as established by anaerobic rapid reaction experiments. The sensitivity to ionic strength and type of ions and the pH dependence of the steady-state kinetic parameters extend MICAL-MO similarity with enzymes of the p-hydroxybenzoate hydroxylase class at the functional level. The reaction is also sensitive to solvent viscosity, providing a tool to monitor the conformational changes predicted to occur during turnover. Finally, it was confirmed that MICAL-MO promotes actin depolymerization, and it was shown that F-actin, but not G-actin, stimulates NADPH oxidation by increasing k(cat) and k(cat)/K(NADPH) (≈5 and ≈200-fold, respectively) with an apparent K(m) for actin of 4.7μM, under conditions that stabilize F-actin. The time-course of NADPH oxidation shows substrate recycling, indicating the possible reversibility of MICAL effect.     

Solution structure of the Reps1 EH domain and characterization of its binding to NPF target sequences

The recently described EH domain recognizes proteins containing Asn-Pro-Phe (NPF) sequences. Using nuclear magnetic resonance (NMR) data, we determined the solution structure of the EH domain from the Reps1 protein and characterized its binding to linear and cyclic peptides derived from a novel targeting protein. The structure calculation included 1143 distance restraints and 122 angle restraints and resulted in structures with a root-mean-square deviation of 0.40 +/- 0.05 A for backbone atoms of superimposed secondary structural elements. The structure comprises two helix-loop-helix motifs characteristic of EF-hand domains. Titration data with NPF-containing peptides showed evidence of intermediate exchange on the NMR chemical shift time scale, which required an analysis that includes curve fitting to obtain accurate equilibrium constants and dissociation rate constants. The cyclic and linear peptides bound with similar affinities (Kd = 65 +/- 17 and 46 +/- 14 microM, respectively) and to the same hydrophobic pocket formed between helices B and C. The cyclic peptide formed a complex that dissociated more slowly (k(off) = 440 +/- 110 s(-1)) than the linear peptide (k(off) = 1800 +/- 250 s(-1)), but had little change in affinity because of the slower rate of association of the cyclic peptide. In addition, we characterized binding to a peptide containing a DPF sequence (Kd = 0.5 +/- 0.2 mM). The characterization of binding between the Reps1 EH domain and its target proteins provides information about their role in endocytosis.     

Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide

Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)