Effects of phospholipases,proteases and neuraminidase on γ-hydroxybutyrate binding sites

Summary -Hydroxybutyric acid (GHB) is a natural compound of mammalian brain synthesized from GABA. The characteristics of its synthesis, transport, release, distribution and turnover, in addition to the presence of a high affinity binding site for this substance in brain are in favor of a modulator role for GHB. The effects of hydrolytic enzymes on the specific binding capacity of GHB have been studied in the present work. Phospholipases A₂ and C, neuraminidase and Pronase markedly decrease GHB binding to crude synaptosomal membranes from rat brain. This effect is time and enzyme concentration dependent. Trypsin, under the conditions employed, is less active. The inhibitory effects of phospholipases is correlated with phospholipid hydrolysis. Lysophospholipids, in the absence of bovine fatty acid free serum albumin partially inhibit GHB binding. The action of neuraminidase has been followed by sialic acid release and modifications of the ganglioside profile. The effects of phospholipase C and of neuraminidase are completely different to those on GABA binding sites. These results represent further data concerning the molecular existence of specific GHB binding sites on rat brain membranes.Abbreviations GHB -hydroxybutyrate - LPC L--lysophosphatidylcholine - LPE Lysophosphatidylethanolamine - PC Phosphatidylcholine - PE Phosphatidylethanolamine - BSA Bovine Serum Albumin     

Characterization of human liver (4-methylumbelliferyl-α-d-N-acetylneuraminic acid) neuraminidase activity

• 1.1. The conditions of a linear assay for 4-methylumbelliferyl-α-d-n-acetylneuraminic acid (4-MU-NANA) neuraminidase activity in human liver have been determined and an acidic liver neuraminidase has been characterized with a pH optimum between 4.4 and 4.8 and an apparent Michaelis constant of O.11 ± O.02 mM for 4-MU-NANA. This acidic neuraminidase is labile to freezing, heating and various storage conditions.
• 2.2. The great majority of human liver neuraminidase activity is found in resuspended pellets after homogenization and centrifugation. A small amount of this resuspended pellet activity can be solubilized with the nonionic detergent Triton X-100.
• 3.3. The properties of human liver neuraminidase are compared to those of other mammalian neuraminidases.

     

Adjustment of receptor-binding and neuraminidase substrate specificties in avian–human reassortant influenza viruses

Balanced action of hemagglutinin (HA) and neuraminidase (NA) is an important condition of influenza virus efficient replication, but a role of HA and NA specificities at oligosaccharide level in maintaining such a balance remains poorly studied. Avian virus HA binds exclusively and NA digests efficiently α2–3-sialylated carbohydrate chains, while human virus HA interacts with α2–6 chains and low-active NA cleaves both α2–3- and α2–6-sialosides. Reassortment between viruses leading to appearance of avian virus HA and human virus NA on the virion surface often resulted in decreasing the replicative potential of the formed variants because of disturbance of a functional balance between “alien” HA and NA. A restoration of the reassortant productivity happened due to the appearance of amino acid substitutions in HA and, sometimes, NA. Here, a role of NA and HA oligosaccharide specificities in a restoration of HA–NA functional balance in high-yield passage variants was studied. Postreassortment changes in HA receptor-binding and NA substrate specificities for three reassortant/passage variant virus pairs towards 3′SiaLac, 3′SiaLacNAc, SiaLe^c, SiaLe^a, SiaLe^x, 6′SiaLac, and 6′SiaLacNAc were determined. Selection of the high-yield variants of the human-avian reassortants led either to twofold decrease in the affinity of HA for most α2–3-sialosides and the appearance of affinity for α2–6-sialosides (H3N2 reassortant), or to decreasing the HA affinity for SiaLe^c and SiaLe^a (H3N1 reassortant), or to enhancing the ability of NA to discriminate between α2–3/2–6 substrates (H4N1 reassortant). Thus, all postreassortment changes in oligosaccharide specificities of “alien” HA and NA were directed towards their adjustment to each other, but by different manner.     

β-Galactosidase-neuraminidase deficiency in adults: Deficiency of a freeze-labile neuraminidase in leukocytes and fibroblasts

Summary 4-Methylumbelliferyl neuraminidase activity was studied in fibroblasts, leukocytes, and frozen tissues from adult patients with -galactosidase-neuraminidase deficiency and specific clinical manifestations. This enzyme was almost completely deficient in fibroblasts, but the residual activity was relatively high (20% of the control mean) in the leukocytes from the patients. The frozen liver from one patient showed the enzyme activity as high as controls.This enzyme consisted of two components, freeze-labile and freeze-stable, and it was demonstrated that only the labile enzyme was deficient in fibroblasts and leukocytes. The apparently normal activity of neuraminidase in frozen autopsy tissues of a patient may be explained by the loss of the labile component in control tissues after a long-term freezing. The neuraminidase activity was variable in parents and no definite conclusion was drawn on the hereditary nature of the disease.     

Human β-galactosidase and α-neuraminidase deficient mucolipidosis: Genetic complementation analysis of the neuraminidase deficiency

Summary Human -galactosidase and -neuraminidase deficient mucolipidosis [ML(gal^-neur^-)] is an inherited lysosomal enzymopathy which recently was designated as a sialidosis. We analyzed the neuraminidase deficiency of this disorder with genetic complementation analyses using a heterokaryon enrichment procedure. The genetic defects of two apparent variants of this disorder complemented the defects of the neuraminidase deficiency diseases, sialidosis I and mucolipidosis I, resulting in the restoration of neuraminidase activity in heterokaryons. The neuraminidase deficiency, therefore, may not be the primary defect in ML(gal^-neur^-) and is not an appropriate test for determining carrier status. The clinical and biochemical characteristics of this disorder suggest that a post-translational or processing event for these enzymes may be defective. The defect, however, is different from I-cell disease and pseudo-Hurler polydystrophy, two disorders of post-translational lysosomal enzyme biosynthesis, since complementation studies demonstrated recovery of intracellular -galactosidase and -neuraminidase levels in heterokaryons. The lack of human -galactosidase expression in man-mouse somatic cell hybrids formed from fibroblasts of the infantile onset type disorder suggests that the defect is not corrected by the mouse genome. The ML(gal^-neur^-) disorder therefore appears to be a distinct subtype of the inherited neuraminidase deficiencies in which the defect may occur in a post-translational or regulatory step which coordinately affects the expression of lysosomal -galactosidase and -neuraminidase.     

Expression, purification and characterization of low-glycosylation influenza neuraminidase in α-1,6-mannosyltransferase defective Pichia pastoris

Influenza A viruses expose two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Although N-glycosylation is essential for many glycoproteins, the glycoproteins expressed in yeast are sometimes hyper-glycosylated, which maybe a primary hindrance to the exploitation of therapeutic glycoprotein production because glycoproteins decorated with yeast-specific glycans are immunogenic and show poor pharmacokinetic properties in humans. To elucidate the NA with different glycosylation in interaction with immunogenicity, here we reported the heterologous expression of influenza NA glycoprotein derived from influenza virus A/newCaledonia/20/99(H1N1) in wide-type Pichia pastoris, α-1,6-mannosyltransferase (och1)-defective P. pastoris and Escherichia coli. We also assessed the immunogenicity of hyper-glycosylated NA expressed in the wide-type, low-glycosylated NA expressed in och1-defective P. pastoris strain and non-glycosylated NA produced in E. coli. Recombinant NA was expressed in wide-type P. pastoris as a 59–97 above kDa glycoprotein, 52–57 kDa in the och1 defective strain, and as a 45 kDa non-glycoprotein in E. coli. The antibody titers of Balb/c mice were tested after the mice were immunized three times with 0.2, 1, or 3 μg purified recombinant NA. Our results demonstrated that after the second immunization, the antibody titer elicited with 1 μg low-glycosylated NA was 1:5,500, while it was 1:10 and 1:13 when elicited by 1 μg hyper-glycosylated and non-glycosylated NA. In the 0.2 μg dose groups, a high antibody titer (1:4,900) was only found after third immunization by low-glycosylated NA, respectively. These results suggest that low-glycosylation in och1-defective P. pastoris enhances the immunogenicity of recombinant NA and elicits similar antibody titers with less antigen when compared with hyper- and non-glycosylated NA. Thus, och1-defective P. pastoris may be a better yeast expression system for production of glycoproteins to research immunogenic characterization.     

5-Brom-3-indolyl-α-ketoside of 5-N-acetyl-D-neuraminic acid a new substrate for the light and electron microscopic demonstration of mammalian neuraminidase

Summary By means of the 5-brom-3-indolyl--ketoside of 5-N-acetyl-D-neuraminic acid mammalian neuraminidase can be localized for the first time on the cellular and subcellular level in rat and mouse organs. The results depend on the pretreatment of the tissue and the auxiliary reagents used.     

Low expression of Neu2 sialidase in the thymus of SM/J mice—existence of neuraminidase positive cells “Neu-medullocyte” in the murine thymus

We have already reported that the homogenate of the A/J mouse thymus shows a high sialidase activity at the neutral pH region and that in both soluble and membrane fractions optimal pH was 6.5–7 (Kijimoto-Ochiai et al., Glycoconj. J., 20:375–384, 2004). In the present study, we investigated the level of sialidase activities in the thymus of the SM/J mouse, a mouse strain that we know to have a Neu1^a allele that reveals a low level of sialidase activity in the liver. We found that while in the A/J thymus the soluble sialidase activity at pH 6.5 was high, the SM/J thymus lacked all such activity. A QTL analysis of SMXA recombinant inbred strains showed that soluble sialidase activity correlated well with the D1Mit8/9 marker on chromosome 1. The murine whole DNA-sequence data and the results of our FISH analysis (Kotani et al., Biochem. Biophys. Res. Comm., 286:250–258, 2001) showed that this location is consistent with the position of Neu2 gene. We confirmed that it is hard to detect the Neu2 enzyme of the SM/J mouse thymus by an anti-Neu2 antibody using a Western blot analysis. We also found that while the mRNA expression of Neu2 was quite normal in the SM/J mouse liver, it was very low in the SM/J mouse thymus. We therefore conclude that the lack of soluble sialidase activity in the SM/J mouse thymus is due to the thymus-specific low expression level of the Neu2 gene. We have previously shown that the sialidase positive cell which contains the Mac-1 and immunoglobulin, and which is located sparsely in the corticomedullar region or medullary region of the A/J mouse thymus (Kijimoto-Ochiai et al., Glycoconj. J., 20:375–384, 2004). We showed now in this paper that the detection of this cell in the SM/J mouse thymus at pH 7.0 was difficult. We propose, therefore, to name the cell “Neu-medullocyte”.     

Bacterial neuraminidase increases IL-8 production in lung epithelial cells via NF-κB-dependent pathway

Bacterial neuraminidase, a sialic acid-degrading enzyme, is one of the virulent factors produced in pathogenic bacteria like as other bacterial components. However little is known about whether bacterial neuraminidase can initiate or modify a cellular response, such as cytokine production, in epithelial cells at infection and inflammation. We demonstrate here that bacterial neuraminidase, but not heat-inactivated neuraminidase, up-regulates expression of interleukin-8 (IL-8) mRNA and protein in lung epithelial A549 and NCI-H292 cells. We also show that bacterial neuraminidase significantly up-regulates IL-8 promoter activity as well as nuclear factor-kappaB (NF-κB) reporter activity. Moreover, inhibition of NF-κB signaling suppressed IL-8 mRNA expression induced by bacterial neuraminidase. Taken together, desialylation-induced IL-8 production in lung epithelial cells may play an important role in infection-associated inflammatory events.     

Transforming growth factor-β: activation by neuraminidase and role in highly pathogenic H5N1 influenza pathogenesis

Transforming growth factor-beta (TGF-β), a multifunctional cytokine regulating several immunologic processes, is expressed by virtually all cells as a biologically inactive molecule termed latent TGF-β (LTGF-β). We have previously shown that TGF-β activity increases during influenza virus infection in mice and suggested that the neuraminidase (NA) protein mediates this activation. In the current study, we determined the mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 by mobility shift and enzyme inhibition assays. We also investigated whether exogenous TGF-β administered via a replication-deficient adenovirus vector provides protection from H5N1 influenza pathogenesis and whether depletion of TGF-β during virus infection increases morbidity in mice. We found that both the influenza and bacterial NA activate LTGF-β by removing sialic acid motifs from LTGF-β, each NA being specific for the sialic acid linkages cleaved. Further, NA likely activates LTGF-β primarily via its enzymatic activity, but proteases might also play a role in this process. Several influenza A virus subtypes (H1N1, H1N2, H3N2, H5N9, H6N1, and H7N3) except the highly pathogenic H5N1 strains activated LTGF-β in vitro and in vivo. Addition of exogenous TGF-β to H5N1 influenza virus-infected mice delayed mortality and reduced viral titers whereas neutralization of TGF-β during H5N1 and pandemic 2009 H1N1 infection increased morbidity. Together, these data show that microbe-associated NAs can directly activate LTGF-β and that TGF-β plays a pivotal role protecting the host from influenza pathogenesis.     

Reinvestigation of the synthesis of 4-methylcoumarin-7-yl 5-acetamido-3,5-dideoxy-α-d-glycero-d-galacto-2-nonulopyranosidonic acid,a fluorogenic substrate for neuraminidase

A crystalline tetrabutylammonium salt of 7-hydroxy-4-methylcoumarin was prepared and shown to contain two coumarin residues for each ammonium group. Condensation of this salt with the glycosyl chloride of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-β-d-glycero-d-galacto-2-nonulopyranosonate in dry acetonitrile at room temperature gave the corresponding α-glycoside in higher yield and purity than previously reported methods. Removal of the acetyl and methyl ester blocking-groups gave the free glycoside, which was shown to have the α configuration by n.m.r. spectroscopy. In contrast, the reaction of the free coumarin derivative with the chloro sugar in refluxing, dry toluene in the presence of cadmium carbonate as acid acceptor gave none of the above glycoside, but gave the corresponding glycal in good yield.     

155 3D QSAR and protein–protein interaction studies on neuraminidase against Clostridium perfringens: An approach toward target identification using structure-based drug designing

The rapid onset of resistance to new drugs and emergence of antibiotic resistant bacteria has led to resurgence in life-threatening bacterial infections. These problems have revitalized interest in antibiotics and lead to new research. To gain further insight between structural and biological activity of Clostridium perfringens, a gram-positive anaerobe responsible for food poisoning, mynecrosis in wound infections, and enterotoxemia in humans. We have considered various in silico approaches for developing new drug leads, based on small ligand structure. The importance of neuraminidases in the virulence of C. perfringens makes it a potent target for the studies of drug designing against this microbe. Natural products or their direct derivatives play crucial roles in many diseases. In the present study, 3D QSAR analysis using kNN-MFA method was performed on a series of pterocarpan derivatives as Clostridial neuraminidase inhibitors. Twenty-five compounds using random selection and sphere exclusion method for the division of dataset into training and test set were chosen. kNN-MFA methodology with stepwise, simulated annealing and genetic algorithm was used for model building and four predictive models have been generated. The most significant model has a high internal predictivity of 64.80% (q ²?=?0.6480) and an external predictivity of 95.46% (r ²?=?0.9546). Model showed that electrostatic and steric interactions play important role in determining neuraminidase inhibitory activity. The kNN-MFA plots provide further understanding of the relationship between structural features of substituted pterocarpan derivatives and their activities which were applied for designing new compounds as inhibitors. The drug likeliness of these compounds was checked through ADME property prediction and their interaction with the neuraminidase was checked by molecular docking studies. Moreover, analysis of protein–protein interaction network of NanI in C. perfringens was done.     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Selection of optimal variants of Gō-like models of proteins through studies of stretching

The Gō-like models of proteins are constructed based on the knowledge of the native conformation. However, there are many possible choices of a Hamiltonian for which the ground state coincides with the native state. Here, we propose to use experimental data on protein stretching to determine what choices are most adequate physically. This criterion is motivated by the fact that stretching processes usually start with the native structure, in the vicinity of which the Gō-like models should work the best. Our selection procedure is applied to 62 different versions of the Gō model and is based on 28 proteins. We consider different potentials, contact maps, local stiffness energies, and energy scales—uniform and nonuniform. In the latter case, the strength of the nonuniformity was governed either by specificity or by properties related to positioning of the side groups. Among them is the simplest variant: uniform couplings with no i, i + 2 contacts. This choice also leads to good folding properties in most cases. We elucidate relationship between the local stiffness described by a potential which involves local chirality and the one which involves dihedral and bond angles. The latter stiffness improves folding but there is little difference between them when it comes to stretching.