Multiple forms of growth inhibitors secreted from cultured rat liver cells: purification and characterization

It was found that a non-tumorigenic epithelial cell line from the liver of a Buffalo-strain rat (BRL) secreted into the culture medium various inhibitors of the growth of BRL and RSV-BRL (tumorigenic BRL transformed by infection of Rous sarcoma virus). The secreted inhibitors were classified into two types: one inhibited the growth of BRL to a greater extent than that of RSV-BRL (non-tumorigenic BRL growth inhibitor, NGI), and the other, vice versa (tumorigenic BRL growth inhibitor, TGI). Two NGI (NGI-I and NGI-II) and two TGI (TGI-I and TGI-II) were highly purified from the serum-free conditioned medium. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis without 2-mercaptoethanol, NGI-I and II gave protein bands with molecular weights (Mr) of 56,000 and 21,000, respectively. TGI-I and II gave a band that migrated faster than bromophenol blue marker dye, but they did not pass through an ultrafiltration membrane with an Mr cutoff of 5,000. In the presence of a reducing reagent, only NGI-II showed a decrease of Mr, from 21,000 to 11,000. NGI and TGI showed 50% growth inhibition with BRL and RSV-BRL, respectively, at 5-15 ng/ml in the medium containing 10% fetal calf serum. NGI and TGI all were stable to 1 M acetic acid (pH 2.3) and 6 M urea, but labile to 5 mM dithiothreitol or trypsin. Of the eight cell lines tested, NGI-I was most effective on BRL, NGI-II on BRL and HSC-3 (human tongue squamous carcinoma), and both TGI-I and II on RSV-BRL.     

Effect of orally administered 9-oxononanoic acid on lipogenesis in rat liver

9-Oxononanoic acid, which is one of the major products of the autoxidation of linoleic acid, was administered orally to rats and its effect on hepatic lipid metabolism was investigated. The de novo synthesis of fatty acids was strongly reduced 30 h after the administration of 100 mg of 9-oxononanoic acid as compared to that in the saline-administered group. Activity of acetyl-CoA carboxylase decreased by 60% and the activity of carnitine palmitoyltransferase increased by 35% in the test group. The level of triacylglycerols in serum was low and the level of free fatty acids remained unchanged. Thus, the administration of 9-oxononanoic acid decreased hepatic lipogenesis. It is generally believed that the reduction in lipogenesis is facilitated by a decrease in the NADPH level. The ratio of NADPH/NADP in the test group, however, became high as compared to that in the control group, and the activities of glucose 6-phosphate and isocitrate dehydrogenases increased. On the other hand, the levels of CoA derivatives, especially long-chain acyl-CoA, were higher in the test group than in the control. Therefore, the reduction of hepatic lipogenesis in the 9-oxononanoic acid group could be attributed to the inhibition of acetyl-CoA carboxylase by the accumulated long-chain acyl-CoA.     

Relationsnip Between the Nutritive Value of Dietary Protein and Liver Xanthine Oxidase Activity in Young Rats

The relationship between the nutritive value of dietary protein and the activity of lievr xanthine oxidase in growing rats as related to the growth rate and the protein efficiency ratio has been investigated.

The response curve of liver xanthine oxidase plotted against the protein level in the diet was essentially exponential, and the lower portion of this curve was linear. The slope of this straight portion, i.e., the tangent of the curve was observed to reflect the quality of dietary protein from comparison with the growth rate and the protein efficiency ratio.     

Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages

Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.     

DPM1, the catalytic subunit of dolichol-phosphate mannose synthase, is tethered to and stabilized on the endoplasmic reticulum membrane by DPM3

Dolichol-phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.     

TbGPI16 is an essential component of GPI transamidase in Trypanosoma brucei

Glycosylphosphatidylinositol (GPI) is widely used by eukaryotic cell surface proteins for membrane attachment. De novo synthesized GPI precursors are attached to proteins post-translationally by the enzyme complex, GPI transamidase. TbGPI16, a component of the trypanosome transamidase, shares similarity with human PIG-T. Here, we show that TbGPI16 is the orthologue of PIG-T and an essential component of GPI transamidase by creating a TbGPI16 knockout. TbGPI16 forms a disulfide-linked complex with TbGPI8. A cysteine to serine mutant of TbGPI16 was unable to fully restore the surface expression of GPI-anchored proteins upon transfection into the knockout cells, indicating that its disulfide linkage with TbGPI8 is important for the full transamidase activity.     

Molecular Characterization of a Novel Peroxidase from the Cyanobacterium Anabaena sp. Strain PCC 7120

The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase (Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35°C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzyme efficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, Reactive Blue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256 μM·min⁻¹, respectively. The apparent K_m and k_cat/K_m values for Reactive Blue 5 were 3.6 μM and 1.2 × 10⁷ M⁻¹ s⁻¹, respectively, while the apparent K_m and k_cat/K_m values for H₂O₂ were 5.8 μM and 6.6 × 10⁶ M⁻¹ s⁻¹, respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2- to ∼50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes.In textile, food, and dyestuff industries, reactive dyes such as azo and anthraquinone (AQ) and pthalocyanine-based dyes constitute one of the extensively used classes of synthetic dyes. However, it has been estimated that approximately 50% of the applied reactive dye is wasted because of hydrolysis during the dyeing process (26, 35). This results in a great effluent problem for the industries because of the recalcitrant nature of these dyes. With increased public concern and ecological awareness, in addition to stricter legislative control of wastewater discharge in recent years, there is an increased interest in various methods of dye decolorization. Dye decolorization using physicochemical processes such as coagulation, adsorption, and oxidation with ozone has proved to be effective. However, these processes are usually expensive, generate large volumes of sludge, and require the addition of environmentally hazardous chemical additives (26). There are several reports of microorganisms capable of decolorizing synthetic dyes. This has been attributed to their growth and production of enzymes such as laccase (1, 9, 40), azoreductases (3), and peroxidases, for example, lignin peroxidase (12, 25, 36), manganese peroxidase (10, 38), and versatile peroxidase (16). However, most of the synthetic dyes are xenobiotic compounds that are poorly degraded using the typical biological aerobic treatments. Furthermore, microbial anaerobic reductions of synthetic dyes are known to generate compounds such as aromatic amines that are generally more toxic than the dyes themselves (3). Therefore, for environmental safety, the use of enzymes instead of enzyme-producing microorganisms presents several advantages such as increased enzyme production, enhanced stability and/or activity, and lower costs by using recombinant DNA technology.Peroxidases are heme-containing enzymes that use hydrogen peroxide (H₂O₂) as the electron acceptor to catalyze numerous oxidative reactions. They are found widely in nature, both in prokaryotes and eukaryotes, and are largely grouped into plant and animal superfamilies. They are one of the most studied enzymes because of their inherent spectroscopic properties and potential use in both diagnostic and bioindustrial applications. In particular, their ability to degrade a wide range of substrates has recently stimulated interest in their potential application in environmental bioremediation of recalcitrant and xenobiotic wastes (10, 25, 26).Recently, a novel family of heme peroxidases characterized by broad dye decolorization activity has been identified in various fungal species such as Thanatephorus cucumeris Dec1 (18), Termitomyces albuminosus (15), Polyporaceae sp. (15), Pleurotus ostreatus (13), and Marasmius scorodonius (27). Because of their broad substrate specificity, low pH optima, lack of a conserved active site distal histidine, and structural divergence from classical plant and animal peroxidases (32), these proteins have been proposed to belong to the novel DyP peroxidase family. Over 400 proteins of prokaryotic and eukaryotic origins have been grouped in the DyP peroxidase family, Pfam 04261 (http://pfam.sanger.ac.uk/), and it is apparent from genome databases that many species possess DyP. The ability of these proteins to effectively degrade hydroxyl-free AQ and azo dyes as well as the specificity for typical peroxidase substrates illustrates their potential use in the bioremediation of wastewater contaminated with synthetic dyes. However, with the exception of a DyP from the plant pathogenic fungus T. cucumeris Dec1 (an anamorph of Rhizoctonia solani, a very common fungal plant pathogen), which has been characterized extensively (18, 28, 30-32, 34), little information is available on other members of the DyP family. In particular, studies on bacterial DyPs have been limited to only the automatically translated sequence or structural data (41, 42). Within the context of further understanding the structure-function and potential applicability of these novel types of enzymes in general, we have taken an interest in DyP-type enzymes, particularly, the less known bacterial groups.Cyanobacteria (blue-green algae) represent the most primitive, oxygenic, plant-type photosynthetic organisms and are thought to be involved in greater than 20 to 30% of the global photosynthetic primary production of biomass, accompanied by the cycling of oxygen. Anabaena sp. strain PCC 7120 is a filamentous, heterocyst-forming cyanobacterium capable of nitrogen fixation and has long been used as a model organism to study the prokaryotic genetics and physiology of cellular differentiation, pattern formation, and nitrogen fixation (14). This strain''s genome sequence is complete and annotated (17). From bioinformatics analysis of the Anabaena sp. strain PCC 7120 genome, we identified an open reading frame (ORF), alr1585, encoding a putative heme-dependent peroxidase exhibiting homology to T. cucumeris Dec1, DyP. Here, we report on the characterization of this novel bacterial DyP, designated AnaPX (for Anabaena peroxidase), from the cyanobacterium Anabaena sp. strain PCC 7120, with broad specificity for both aromatic compounds and synthetic dyes such as AQ dyes.     

Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background

An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.

Methods

We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.

Results

Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.

Conclusions

Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.

General significance

Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.