Characterization of two novel bacterial type A <Emphasis Type="Italic">exo</Emphasis>-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-d-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N′-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-d-glucosaminide (1 → 4)-β-linkages and are thus “exo-chitobiose hydrolases.” In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.     

New aspects of biosynthesis and metabolism of <Emphasis Type="Italic">N</Emphasis>-acyldopamines in rat tissues

Possible biosynthetic pathways of N-acyldopamines in rat tissues were compared. It was shown that an insignificant amount of the conjugation products was formed during the incubation of arachidonic acid and dopamine, whereas the substitution of tyrosine for dopamine resulted in the productive biosynthesis of N-arachidonoyldopamine. The biosynthesis presumably involves several closely conjugated enzymatic stages, and free fatty acids rather than their CoA esters served as the starting substrates. The decarboxylation stage probably precedes the stage of catechol system formation, because N-acetyltyramine (a probable intermediate) was easily oxidized by monophenol monooxygenase to N-acyldopamine, whereas N-acyltyrosine is hydrolyzed under these conditions. Biosynthesis of N-acyldopamines in a cell-free medium was accompanied by their methylation. The possibility of oxidative metabolism of N-acyldopamines, which could serve as co-substrates or inhibitors of different oxidoreductases, was shown for the first time.     

Extracellular hydrolases of strain <Emphasis Type="Italic">Bacillus</Emphasis> sp. 739 and their involvement in the lysis of micromycete cell walls

The mycolytic bacterial strain Bacillus sp. 739 produces extracellular enzymes which degrade in vitro the cell walls of a number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process correlated with the levels of the β-1,3-glucanase and protease activity. The comparative characteristic of Bacillus sp. 739 enzymatic preparations showed that efficient hydrolysis of the fungus cell walls was the result of the action of the complex of enzymes produced by the strain when grown on chitin-containing media. Among the enzymes of this complex, chitinases and β-1,3-glucanases hydrolyzed most actively the disintegrated cell walls of B. sorokiniana. However, only β-1,3-glucanases were able to degrade the cell walls of native fungal mycelium in the absence of other hydrolases, which is indicative of their key role in the mycolytic activity of Bacillus sp. 739.     

Isolation and properties of fungal β-glucosidases

Using chromatography on different matrixes, three β-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) β-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (β-glucan from barley and laminarin); ii) β-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).     

Mode of action and specificity of a chitinase from unicellular microalgae, <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Key message

Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds. However, chitin-mediated defense system intensively studied in higher plants remains to be investigated in this microalga. Recently, Taira et al. (Biosci Biotechnol Biochem 82:1090–1100, 2018) isolated a unique chitinase gene, comprising two catalytic domains almost homologous to each other (Cat1 and Cat2) and two chitin-binding domains (CBD1 and CBD2), from E. gracilis. We herein examined the mode of action and the specificity of the recombinant Cat2 by size exclusion chromatography and NMR spectroscopy. Both Cat1 and Cat2 appeared to act toward chitin substrate with non-processive/endo-splitting mode, recognizing two contiguous N-acetylglucosamine units at subsites ??2 and ??1. This is the first report on a chitinase having two endo-splitting catalytic domains. A cooperative action of two different endo-splitting domains may be advantageous for defensive action of the E. gracilis chitinase.

Abstract

The unicellular alga, E. gracilis, produces a chitinase consisting of two GH18 catalytic domains (Cat1 and Cat2) and two CBM18 chitin-binding domains (CBD1 and CBD2). Here, we produced a recombinant protein of the Cat2 domain to examine its mode of action as well as specificity. Cat2 hydrolyzed N-acetylglucosamine (A) oligomers (A_n, n?=?4, 5, and 6) and partially N-acetylated chitosans with a non-processive/endo-splitting mode of action. NMR analysis of the product mixture from the enzymatic digestion of chitosan revealed that the reducing ends were exclusively A-unit, and the nearest neighbors of the reducing ends were mostly A-unit but not exclusively. Both A-unit and D-unit were found at the non-reducing ends and the nearest neighbors. These results indicated strong and absolute specificities for subsites ??2 and ??1, respectively, and no preference for A-unit at subsites +?1 and +?2. The same results were obtained from sugar sequence analysis of the individual enzymatic products from the chitosans. The subsite specificities of Cat2 are similar to those of GH18 human chitotriosidase, but differ from those of plant GH18 chitinases. Since the structures of Cat1 and Cat2 resemble to each other (99% similarity in amino acid sequences), Cat1 may hydrolyze the substrate with the same mode of action. Thus, the E. gracilis chitinase appears to act toward chitin polysaccharide chain through a cooperative action of the two endo-splitting catalytic domains, recognizing two contiguous A-units at subsites ??2 and ??1.

     

Neuro-and psychotropic activity of <Emphasis Type="Italic">N</Emphasis>-uronoylamino acids and <Emphasis Type="Italic">N</Emphasis>-uronoylpeptides

Peculiarities of the rat behavior were studied in a series of experimental stress models after a systemic administration of new N-uronoyl derivatives of amino acids. The psychotropic effect was shown to be determined by the nature of the amino acid fragment. N-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyraneuronoyl)-glycylglycine exhibited an anxiolytic effect more pronounced than that of pyracetam, whereas N-(1,2:3,4-di-O-isopropilidene-α-D-galactopyranuronoyl)-glycylglutamic acid has antidepressant action stronger than that of amitriptyline. Mechanisms for the psychotropic effects of the examined derivatives are discussed.     

Mutational analysis of substrate specificity in a <Emphasis Type="Italic">Citrus paradisi</Emphasis> flavonol 3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis vinifera anthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.     

The ammonia-catalyzed release of glycoprotein <Emphasis Type="Italic">N</Emphasis>-glycans

Despite the great significance of release and analysis of glycans from glycoproteins, the existing N-glycan release methods are undermined by some limitations and deficiencies. The traditional enzymatic protocols feature high N-glycan release specificity but are generally costly and inefficient for some types of N-glycans. The existing chemical methods require harsh reaction conditions or are accompanied by the remarkable formation of by-products. Herein, we describe a versatile chemical method for the release and analysis of N-glycans from glycoproteins. This method differs from the existing methods as only aqueous ammonia is used to catalyze the N-glycan release reactions. Optimization of reaction conditions was performed using RNase B as a model glycoprotein and the obtained results indicated a highest N-glycan yield in ammonia at 60 °C for 16 h. Comparison of this method with traditional enzymatic protocols and recently reported NaClO methods confirmed the good reliability and efficiency of the novel approach. We also successfully applied this method to some complex biological samples, such as Ginkgo seed protein, fetal bovine serum (FBS) and hen egg white, and demonstrated its great compatibility with various neutral N-glycans, core α-1,3-fucosylated N-glycans and sialylated N-glycans. This method is very simple and cost-effective, enabling convenient analysis and large-scale preparation of released reducing N-glycans from various biological samples for structural and functional glycomics studies.     

Expressing accessory proteins in cellulolytic <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to improve the conversion yield of recalcitrant cellulose

Background

A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate.

Results

The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively.

Conclusions

This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.

     

Kinetic Analyses of the Substrate Inhibition of <Emphasis Type="Italic">Paramecium</Emphasis> Arginine Kinase

Paramecium tetraurelia expresses four types of arginine kinase (AK1–AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis–Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3–15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R², AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, V_max^SES?=?30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, V_max^ES?=?241.7. The dissociation constant for the SES complex (K_i^SES: 0.34 mM) was two times smaller than that of the ES complex (K_s^ES: 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower V_max^SES. The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the K_i^SES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, V_max^ES and V_max^SES values for the mutants decreased by one-third, except for the V_max^SES of the S79A mutant, which had a value that was comparable with the value for the wild-type.     

Development of schemes of induced mutagenesis for improving the productivity of <Emphasis Type="Italic">Aspergillus</Emphasis> strains producing amylolytic enzymes

In order to improve the biosynthesis of amylolytic enzymes by industrial Aspergillus strains, the efficiency of stepwise application of the following methods of induced mutagenesis was studied: ultraviolet (UV) irradiation, gamma irradiation, and treatment with N-methyl-N-nitro-N-nitrosoguanidine (NG). It was found that at the early stages of mutagenesis of the glucoamylase-producing A. awamori strain UV and NG, used either alone or in combination, were efficient enough as mutagenic agents, providing an increase of glucoamylase activity by 30–35 and 50–60%, respectively. At the later stages, serial UV mutagenesis and gamma-irradiation showed high efficiency for both A. awamori, and A. oryzae strains. Gamma-mutagenesis of Aspergillus strains using a cobalt source provided the most stable and highly active strains retaining 90–95% of their activity after five transfers on agar medium. The experiments resulted in significant improvement of the studied industrial strains, more than doubling activity of the target enzymes.     

The parasitism of the mouldPenicillium purpurogenum onAspergillus niger

The enzymatic composition of thePenicillium purpurogenum preparation cultivated on Czapek medium (P_Cz), and on the medium prepared from the mycelium of the mouldAspergillus niger (P_An), has been followed, causing the lysis ofAspergillus niger cell walls.Penicillium purpurogenum forms cellulolytic enzymes, enabling the utilization of cellulose as a sole carbon source.In vitro experiments with enzymatic preparations (P_An) indicated a rapid decrease inviscosity of carboxymethylcellulose at a negligible increase in reduction. The enzymatic preparations (P_Cz), causing also cell wall lysis inAspergillus niger, were however cellulolytically inactive, their presence in the systems causing their lysis is therefore not assumed.Penicillium purpurogenum also forms amylases, in cell walls ofAspergillus niger however no corresponding substrate is present. Proteases are synthetized only adaptively, and the results indicate that even lipases, also present in the substrate, are not a part of the lytic system. Chitinases are of the constitutive type, and are presumably a part of the enzymatic complex causing cell lysis. The main factor is anyhow an enzyme, or an enzymatic system, which is subjected to further investigation.     

Comparison of L-lactate dehydrogenases of different origins produced in the cells of yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The expression of L-lactate dehydrogenase genes ldh1 (Bos taurus), ldhA (Homo sapiens), ldhA (Rhizopus oryzae), ldh1 (Lactobacillus plantarum), and ldh1 (Lactobacillus pentosus) in the cells of yeast Schizosaccharomyces pombe VKPM U-3106 has been investigated. The catalytic characteristics of the enzymes encoded by these genes have been compared, and the intensity of lactic acid synthesis by the recombinant strains obtained has been evaluated. The enzymatic activity of L-lactate dehydrogenases from L. plantarum and L. pentosus was the highest (approximately 2 to 2.5 times higher than that of the mammalian enzymes), and these enzymes therefore appear to have the highest potential for the development of lactic-acid producing strains of yeast S. pombe.     

Degradation and polymerization of monolignols by <Emphasis Type="Italic">Abortiporus biennis</Emphasis>, and induction of its degradation with a reducing agent

This study was carried out to better understand the characteristic modification mechanisms of monolignols by enzyme system of Abortiporus biennis and to induce the degradation of monolignols. Degradation and polymerization of monolignols were simultaneously induced by A. biennis. Whole cells of A. biennis degraded coniferyl alcohol to vanillin and coniferyl aldehyde, and degraded sinapyl alcohol to 2,6-dimethoxybenzene- 1,4-diol, with the production of dimers. The molecular weight of monolignols treated with A. biennis increased drastically. The activities of lignin degrading enzymes were monitored for 24 h to determine whether there was any correlation between monolignol biomodification and ligninolytic enzymes. We concluded that complex enzyme systems were involved in the degradation and polymerization of monolignols. To degrade monolignols, ascorbic acid was added to the culture medium as a reducing agent. In the presence of ascorbic acid, the molecular weight was less increased in the case of coniferyl alcohol, while that of sinapyl alcohol was similar to that of the control. Furthermore, the addition of ascorbic acid led to the production of various degraded compounds: syringaldehyde and acid compounds. Accordingly, these results demonstrated that ascorbic acid prevented the rapid polymerization of monolignols, thus stabilizing radicals generated by enzymes of A. biennis. Thereafter, A. biennis catalyzed the oxidation of stable monolignols. As a result, ascorbic acid facilitated predominantly monolignols degradation by A. biennis through the stabilization of radicals. These findings showed outstanding ability of A. biennis to modify the lignin compounds rapidly and usefully.     

Phenol Oxidase Activity of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 Mutants with Modified Motility and <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 Phase Variants with Different Plasmid Composition

Herein, we reveal the alteration in phenol oxidase enzymes complex production from Azospirillum brasilense Sp245 omegon mutants with polar and lateral flagella dysfunction and from A. brasilense Sp7 phase variants with different plasmid composition. The enzymatic activities for various laccases, tyrosinases, Mnperoxidases, and lignin peroxidases as well as the isomorphic composition of intracellular laccases and tyrosinases were estimated for the studied variants and the parent strains. It was noted that various genetic events correlating with phenotypic heterogeneity in A. brasilense populations affect their phenol oxidase activity level.     

Effective to census population size ratios in two Near Threatened Mediterranean amphibians: <Emphasis Type="Italic">Pleurodeles waltl</Emphasis> and <Emphasis Type="Italic">Pelobates cultripes</Emphasis>

Efforts to mitigate amphibian declines are hindered by a lack of information about basic aspects of their biology and demography. The effective to census population size ratio (N _e /N _c ) is one of the most important parameters for the management of wildlife populations because it combines information on population abundance and genetic diversity and helps predict population viability in the long term. Few studies have calculated this ratio in amphibians, which sometimes show low ratios, associated with a higher extinction risk. Here we integrate field-based (capture-mark-recapture studies, egg string counts) and molecular approaches (estimation of the effective number of breeders (N _b ) and the effective population size (N _e ) based on genotypes from larval cohorts and candidate parents) to produce the first estimates of the N _e /N _c and N _b /N _c ratios in two amphibians, the Iberian ribbed newt Pleurodeles waltl and the western spadefoot Pelobates cultripes. Additionally, we investigate sex-biased dispersal in both species based on direct (field observations) and indirect (genetic) evidence. Both species showed similar ratios, slightly lower in Pleurodeles (0.21–0.24) than in Pelobates (0.25–0.30). Observed displacement rates were low in both species (P. waltl?=?0.51%; P. cultripes?=?1.23%). We found no evidence for sex-biased dispersal in P. cultripes, but both direct and indirect evidences suggest a tendency for female-biased dispersal in P. waltl. We discuss differences in the genetic estimates of N _e and N _b provided by three inference methods and the implications of our findings for the management of these species, characteristic of Mediterranean wetlands in the Iberian Peninsula and listed as Near Threatened.     

Insight into the molecular mechanism of the sulfur oxidation process by reverse sulfite reductase (rSiR) from sulfur oxidizer <Emphasis Type="Italic">Allochromatium vinosum</Emphasis>

Sulfur metabolism is one of the oldest known biochemical processes. Chemotrophic or phototrophic proteobacteria, through the dissimilatory pathway, use sulfate, sulfide, sulfite, thiosulfate or elementary sulfur by either reductive or oxidative mechanisms. During anoxygenic photosynthesis, anaerobic sulfur oxidizer Allochromatium vinosum forms sulfur globules that are further oxidized by dsr operon. One of the key redox enzymes in reductive or oxidative sulfur metabolic pathways is the DsrAB protein complex. However, there are practically no reports to elucidate the molecular mechanism of the sulfur oxidation process by the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum. In the present context, we tried to analyze the structural details of the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum by molecular dynamics simulations. The molecular dynamics simulation results revealed the various types of molecular interactions between DsrA and DsrB proteins during the formation of DsrAB protein complex. We, for the first time, predicted the mode of binding interactions between the co-factor and DsrAB protein complex from Allochromatium vinosum. We also compared the binding interfaces of DsrAB from sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris. This study is the first to provide a comparative aspect of binding modes of sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

Alteration of a recombinant protein <Emphasis Type="Italic">N</Emphasis>-glycan structure in silkworms by partial suppression of <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase gene expression

Objective

To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae.

Results

Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man₃GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins.

Conclusions

Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

     

Recombinant human heterodimeric IL-15 complex displays extensive and reproducible <Emphasis Type="Italic">N</Emphasis>- and <Emphasis Type="Italic">O</Emphasis>-linked glycosylation

Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and β-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.