Spawn viability in edible mushrooms after freezing in liquid nitrogen without a cryoprotectant

Five strains of edible mushrooms (Lentinula boryana, Lentinula edodes, Pleurotus djamor, Pleurotus pulmonarius, and Volvariella volvacea) were studied. Spawn were prepared from sorghum seeds and then incubated for 14 days under optimum conditions for each species. Once covered by mycelia, the sorghum seeds were placed in polycarbonate vials for freezing in liquid nitrogen. The effect of adding a cryoprotective solution before freezing (either 10% glycerol v/v or 5% dimethylsulfoxide v/v) was evaluated as a function of mycelial growth and percent viability. Three main treatments were undertaken: (1) freezing with a glycerol or dimethylsulfoxide cryoprotectant, (2) freezing with water and (3) freezing without cryoprotectant or water. Samples were maintained frozen for a week, after which time they were thawed (10 min at 30 degrees C) and the seeds placed in Petri dishes with a culture medium. A recovery rate of 96.8% was obtained for the total number of samples summed over all strains and treatments. In contrast, 99.2% of the samples frozen without cryoprotectant were recovered. The recovery of frozen mycelia was delayed with respect to a control group, which was not frozen. However, no difference was observed in percent recovery and mycelial diameter when a new series of spawn was prepared from mycelia that had been previously frozen. Results obtained from this experiment demonstrate that an adequate recovery of mycelia can be obtained without using a cryoprotectant. This capacity might enable large quantities of commercial mushroom strains to be handled at reduced production costs. It is suggested that the mycelia survived freezing without cryoprotectants because they were embedded and protected within the sorghum seeds used to elaborate the spawn.     

Enhanced enzymatic hydrolysis of langostino shell chitin with mixtures of enzymes from bacterial and fungal sources

A combination of enzyme preparations from Trichoderma atroviride and Serratia marcescens was able to completely degrade high concentrations (100 g/L) of chitin from langostino crab shells to N-acetylglucosamine (78%), glucosamine (2%), and chitobiose (10%). The result was achieved at 32 degrees C in 12 days with no pre-treatment (size reduction or swelling) of the substrate and without removal of the inhibitory end-products from the mixture. Enzymatic degradation of three forms of chitin by Serratia/Trichoderma and Streptomyces/Trichoderma blends was carried out according to a simplex-lattice mixture design. Fitted polynomial models indicated that there was synergy between prokaryotic and fungal enzymes for both hydrolysis of crab chitin and reduction of turbidity of colloidal chitin (primarily endo-type activity). Prokaryotic/fungal enzymes were not synergistic in degrading chitosan. Enzymes from prokaryotic sources had much lower activity against chitosan than enzymes from T. atroviride.     

Flow process for electroextraction of intracellular enzymes from the fission yeast, Schizosaccharomyces pombe

Flow treatment of the yeast, Schizosaccharomyces pombe, with high intensity electric field pulses released intracellular enzymes such as glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. Over 70% of the total activity was liberated within 4 h after pulse application. The optimal field intensities were considerably higher than that needed for irreversible plasma membrane permeabilization.     

Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta

Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30 h and adult worms for 4-6 h. Ammonia was the main excretory product, with about 20% urea. For the first 4-6 h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30 h. An initial external ammonia concentration of 600 μM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets.     

The surface structure of a complex substrate revealed by enzyme kinetics and Freundlich constants for α-amylase interaction with the surface of starch

Background

Starch is a main source of carbohydrate in human diets, but differences are observed in postprandial glycaemia following ingestion of different foods containing identical starch contents. Such differences reflect variations in rates at which different starches are digested in the intestine. In seeking explanations for these differences, we have studied the interaction of α-amylase with starch granules. Understanding this key step in digestion should help with a molecular understanding for observed differences in starch digestion rates.

Methods

For enzymes acting upon solid substrates, a Freundlich equation relates reaction rate to enzyme adsorption at the surface. The Freundlich exponent (n) equals 2/3 for a liquid-smooth surface interface, 1/3 for adsorption to exposed edges of ordered structures and 1.0 for solution–solution interfaces. The topography of a number of different starch granules, revealed by Freundlich exponents, was compared with structural data obtained by differential scanning calorimetry and Fourier transform infrared spectroscopy with attenuated total internal reflectance (FTIR-ATR).

Results

Enzyme binding rate and FTIR-ATR peak ratio were directly proportional to n and Δ_gelH was inversely related to n. Amylase binds fastest to solubilised starch and to granules possessing smooth surfaces at the solid–liquid interface and slowest to granules possessing ordered crystalline surfaces.

Conclusions

Freundlich exponents provide information about surface blocklet structures of starch that supplements knowledge obtained from physical methods.

General Significance

Nanoscale structures at the surface of starch granules influence hydrolysis by α-amylase. This can be important in understanding how dietary starch is digested with relevance to diabetes, cardiovascular health and cancer.     

超声波对铜绿微囊藻超微结构和生理特性的影响

为了研究超声波对蓝藻细胞的影响,利用超声波（40W）处理200 mL铜绿微囊藻（Microcystis aeruginosa） 悬浮液20min,之后继续培养并于不同时间取样检测。检测悬浮藻细胞生物量发现其3d降低了97.84%;分别观察1、3、5d时沉降藻细胞超微结构变化,发现13d时细胞内脂质颗粒和藻青素颗粒增多、类囊体片层断裂、藻胆体脱落,5d时拟核区萎缩消失、细胞基础结构解体、胞质出现空洞、胞内结构颗粒降解;检测藻细胞光合放氧速率、叶绿素a （Chl.a）、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性、膜透性以及跨膜ATP酶活性,发现光合放氧速率3d下降24.83%,Chl.a含量5d下降23.75%,超声组细胞SOD活性变化幅度比较大,但总体上活性降低,而CAT活性则表现为先增后减,活性始终大于对照组,同时胞内有机物渗出量增大,三种跨膜ATP酶活性（Na+/K+-ATPase、Mg2+-ATPase 和Ca2+-ATPase）均先升后降,并与膜透性变化相关。以上结果表明,超声波使铜绿微囊藻细胞沉降,并对其造成了胁迫,使部分藻细胞光合作用减弱,光合色素遭到损伤,细胞膜透性增大,甚至引起藻细胞程序性死亡。SOD活力的快速降低表明超声波使藻细胞内超氧离子（O2-）过量累积,从而对藻细胞造成氧化损伤,除此之外,超声波使藻细胞基础结构破坏、细胞内结构颗粒降解、细胞膜透性增大,这些都可能是致使部分铜绿微囊藻细胞死亡的重要原因。铜绿微囊藻细胞CAT以及跨膜ATP酶活性增大,表明藻细胞增强抗氧化酶活性以及离子调控和能量活动以抵御超声波的胁迫,而当胁迫随着时间减小后,细胞开始恢复生长和代谢,酶活力开始降低。       

Schisandra fructus extract ameliorates doxorubicin-induce cytotoxicity in cardiomyocytes: altered gene expression for detoxification enzymes

The effect of Schisandra fructus extract (SFE) on doxorubicin (Dox)-induced cardiotoxicity was investigated in H9c2 cardiomyocytes. Dox, which is an antineoplastic drug known to induce cardiomyopathy possibly through production of reactive oxygen species, induced significant cytotoxicity, intracellular reactive oxygen species (ROS), and lipid peroxidation. SFE treatment significantly increased cell survival up to 25%, inhibited intracellular ROS production in a time- and dose-dependent manner, and inhibited lipid peroxidation induced by Dox. In addition, SFE treatment induced expression of cellular glutathione S-transferases (GSTs), which function in the detoxification of xenobiotics, and endogenous toxicants including lipid peoxides. Analyses of 31,100 genes using Affymetrix cDNA microarrays showed that SFE treatment up-regulated expression of genes involved in glutathione metabolism and detoxification [GST theta 1, mu 1, and alpha type 2, heme oxygenase 1 (HO-1), and microsomal epoxide hydrolase (mEH)] and energy metabolism [carnitine palmitoyltransferase-1 (CPT-1), transaldolase, and transketolase]. These data indicated that SFE might increase the resistance to cardiac cell injury by Dox, at least partly, together with altering gene expression, especially induction of phase II detoxification enzymes.     

In silico restriction landmark genome scanning analysis of Xanthomonas oryzae pathovar oryzae MAFF 311018

We have developed a restriction landmark genome scanning (RLGS) system in silico, involving two-dimensional electrophoretic analysis of DNA by computer simulation that is based on the availability of whole-genome sequences for specific organisms. We applied the technique to the analysis of the Xanthomonas oryzae pathovar oryzae (Xoo) MAFF 311018, which causes bacterial blight in rice. The coverage that was found to be achievable using RLGS in silico, as a percentage of the genomic regions that could be detected, ranged from 44.5% to 72.7% per image. However, this reached a value of 96.7% using four images that were obtained with different combinations of landmark restriction enzymes. Interestingly, the signal intensity of some of the specific spots obtained was significantly lower than that of other surrounding spots when MboI, which cleaves unmethylated 5'-GATC-3' sites, was used. DNA gel blot analysis with both DNA adenine methylase (Dam)-sensitive and -insensitive isoschizomers (MboI and Sau3AI) revealed that Dam-mediated DNA adenine methylation had indeed occurred at these particular sites. These results suggest that a significant portion of the 5'-GATC-3' sites within the Xoo genome is stably methylated by Dam.     

Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE

The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.     

Effect of a formulation of Bacillus firmus on root-knot nematode Meloidogyne incognita infestation and the growth of tomato plants in the greenhouse and nursery

Bacillus firmus, commercial WP formulation (BioNem) was evaluated against the root-knot nematode Meloidogyne incognita in a laboratory, greenhouse and under field conditions on tomato plants. In the laboratory tests, an aqueous suspension of BioNem at 0.5%, 1%, 1.5% and 2% concentration reduced egg hatching from 98% to 100%, 24-days after treatment. Treatment of second-stage juveniles with 2.5% and 3% concentration of BioNem, caused 100% inhibition of mobility, 24 h after treatment. In the green house trials, BioNem applied at 8 g/pot (1200 cc soil) planted with a tomato seedlings reduced gall formation by 91%, final nematode populations by 76% and the number of eggs by 45%. Consequently, plant height and biomass was increased by 71% and 50%, respectively, compared to the untreated control, 50-days after treatment application. Application of BioNem at 16 g/pot was phytotoxic to plants. In the field trails, BioNem applied at 200 and 400 kg ha⁻¹ was effective in reducing the number of galls (75-84%), and increased shoot height (29-31%) and weight (20-24%) over the untreated control, 45-days after treatment. Our results indicate that B. firmus is a promising microorganism for the biological control of M. incognita in tomato pots.     

Influence of organic co-solvents on the activity and substrate specificity of feruloyl esterases

Organic co-solvents can expand the use of enzymes in lignocellulose deconstruction through making substrates more soluble and thus more accessible. In choosing the most adequate co-solvent for feruloyl esterases, hydrolysis of methyl p-hydroxycinnamates by three pure enzymes (and a multi-enzyme preparation) was evaluated. Low concentrations of dimethylsulfoxide (DMSO) enhanced hydrolysis by two of the enzymes while at levels >20%, activity was reduced. DMSO also enhanced acetyl esterase-type activity of the enzymes. The co-solvent effect was different for each enzyme-substrate couple, indicating that other factors are also involved. Kinetic studies with a Talaromyces stipitatus feruloyl esterase showed low concentrations of dimethylsulfoxide enhanced the hydrolytic rate while K_m also increased. Moreover, long-term incubation (96 h) of an Aspergillus niger feruloyl esterase in dimethylsulfoxide:water provided to the enzyme the ability to hydrolyze methyl p-coumarate, suggesting an active-site re-arrangement. Dimethylsulfoxide (10-30%) is proposed as an adequate co-solvent for feruloyl esterase treatment of water-insoluble substrates.     

Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

Background

Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°C.

Results

A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50–70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in T_m) and improved half-life at 60°C (t_1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t_1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.

Conclusion

Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0118-z) contains supplementary material, which is available to authorized users.     

Intracellular delivery of glutathione S-transferase into mammalian cells

Protein transduction domains (PTDs) derived from human immunodeficiency virus Tat protein and herpes simplex virus VP22 protein are useful for the delivery of non-membrane-permeating polar or large molecules into living cells. In the course of our study aiming at evaluating PTD, we unexpectedly found that the fluorescent-dye-labeled glutathione S-transferase (GST) from Schistosoma japonicum without known PTDs was delivered into COS7 cells. The intracellular transduction of GST was also observed in HeLa, NIH3T3, and PC12 cells, as well as in hippocampal primary neurons, indicating that a wide range of cell types is permissive for GST transduction. Furthermore, we showed that the immunosuppressive peptide VIVIT fused with GST successfully inhibits NFAT activation. These results suggest that GST is a novel PTD which may be useful in the intracellular delivery of biologically active molecules, such as small-molecule drugs, bioactive peptides, or proteins.     

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.     

Expression profiles of hydrophobic surfactant proteins in children with diffuse chronic lung disease

Background

Abnormalities of the intracellular metabolism of the hydrophobic surfactant proteins SP-B and SP-C and their precursors may be causally linked to chronic childhood diffuse lung diseases. The profile of these proteins in the alveolar space is unknown in such subjects.

Methods

We analyzed bronchoalveolar lavage fluid by Western blotting for SP-B, SP-C and their proforms in children with pulmonary alveolar proteinosis (PAP, n = 15), children with no SP-B (n = 6), children with chronic respiratory distress of unknown cause (cRD, n = 7), in comparison to children without lung disease (n = 15) or chronic obstructive bronchitis (n = 19).

Results

Pro-SP-B of 25–26 kD was commonly abundant in all groups of subjects, suggesting that their presence is not of diagnostic value for processing defects. In contrast, pro-SP-B peptides cleaved off during intracellular processing of SP-B and smaller than 19–21 kD, were exclusively found in PAP and cRD. In 4 of 6 children with no SP-B, mutations of SFTPB or SPTPC genes were found. Pro-SP-C forms were identified at very low frequency. Their presence was clearly, but not exclusively associated with mutations of the SFTPB and SPTPC genes, impeding their usage as candidates for diagnostic screening.

Conclusion

Immuno-analysis of the hydrophobic surfactant proteins and their precursor forms in bronchoalveolar lavage is minimally invasive and can give valuable clues for the involvement of processing abnormalities in pediatric pulmonary disorders.     

Enhancement of transglycosylation activity by construction of chimeras between mesophilic and thermophilic beta-glucosidase

The family 3 beta-glucosidase from Thermotoga maritima is a highly thermostable enzyme (85 degrees C) that displays transglycosylation activity. In contrast, the beta-glucosidase from Cellvibrio gilvus is mesophilic (35 degrees C) and displays no such transglycosylation activity. Both enzymes consist of two domains, an N-terminal and a C-terminal domain, and the amino acid identities between the two enzymes in these domains are 32.4 and 36.4%, respectively. In an attempt to identify the molecular basis underpinning the display of transglycosylation activity and the requirements for thermal stability, eight chimeric genes were constructed by shuffling the two parental beta-glucosidase genes at four selected borders, two in the N-terminal domain and two in the C-terminal domain. Of the eight chimeric genes constructed, only two chimeric enzymes (Tm578/606Cg and Tm638/666Cg) gave catalytically active forms and these were the ones shuffled in the C-terminal domain. For these active chimeric enzymes, 80% (Tm578/606Cg) and 88% (Tm638/666Cg) of their amino acid sequences originated from T. maritima. With regard to their thermal profiles, the two active chimeric enzymes, Tm578/606Cg and Tm638/666Cg, displayed profiles intermediate to those of the two parental enzymes as they were optimally active at 65 and 70 degrees C, respectively. These two chimeric enzymes were optimally active at pH 4.1 and 3.9, which is closer to that observed for the T. maritima enzyme (pH 3.2-3.5) than that for the C. gilvus enzyme (pH 6.2-6.5). Kinetic parameters for the chimeric enzymes were investigated with five different substrates including pNP-beta-D-glucopyranoside. The kinetic parameters obtained for the chimeric enzymes were closer to those of the T. maritima enzyme than to those of the C. gilvus enzyme. Transglycosylation activity was observed for both chimeric enzymes and the activity of the Tm578/606Cg chimera was at a level twice that observed with the T. maritima enzyme. This study is an effective demonstration of the usefulness of chimeric enzymes in altering the characteristics of an enzyme.     

Feedback mechanisms regulate retinoic acid production and degradation in the zebrafish embryo

Retinoic acid (RA) signaling in vertebrate embryos occurs in a distinct physical and temporal pattern. Regulating this spatial distribution is crucial to the development of the embryo, as RA in excess or in inappropriate tissues is teratogenic. In order to understand how RA availability is determined in zebrafish we have investigated the expression of cyp26a1, an enzyme that inactivates RA, and its relationship to raldh2, one of the enzymes that produce RA from retinal. cyp26a1 expression follows three phases: in presumptive anterior neurectoderm and in a circumblastoporal ring during gastrulation, in the tailbud throughout somitogenesis, and in multiple specific tissue types beginning at mid-somitogenesis and continuing through 48 h postfertilization (hpf). This expression was either adjacent or opposite to those tissues expressing raldh2. We then investigated how RA production might regulate these relationships. Endogenous RA produced by raldhs did not play a role in setting cyp26a1 expression in most tissues. However, exogenous RA regulates expression of both enzymes. cyp26a1 is up regulated in the embryo in a time, concentration, and tissue-dependent manner. Conversely, raldh2 expression is reduced with RA treatment. Tests of the raldh2 promoter in cell transfections proved that RA directly represses its activity. These data demonstrate that the feedback mechanisms regulating production and degradation of RA must be considered in any experiments altering levels of RA in the developing vertebrate embryo.     

Traps of carnivorous pitcher plants as a habitat: composition of the fluid, biodiversity and mutualistic activities

BACKGROUND: Carnivorous pitcher plants (CPPs) use cone-shaped leaves to trap animals for nutrient supply but are not able to kill all intruders of their traps. Numerous species, ranging from bacteria to vertrebrates, survive and propagate in the otherwise deadly traps. This paper reviews the literature on phytotelmata of CPPs. PITCHER: Fluid as a Habitat The volumes of pitchers range from 0·2 mL to 1·5 L. In Nepenthes and Cephalotus, the fluid is secreted by the trap; the other genera collect rain water. The fluid is usually acidic, rich in O(2) and contains digestive enzymes. In some taxa, toxins or detergents are found, or the fluid is extremely viscous. In Heliamphora or Sarracenia, the fluid differs little from pure water. INQUILINE: Diversity Pitcher inquilines comprise bacteria, protozoa, algae, fungi, rotifers, crustaceans, arachnids, insects and amphibia. The dominant groups are protists and Dipteran larvae. The various species of CPPs host different sets of inquilines. Sarracenia purpurea hosts up to 165 species of inquilines, followed by Nepenthes ampullaria with 59 species, compared with only three species from Brocchinia reducta. Reasons for these differences include size, the life span of the pitcher as well as its fluid. MUTUALISTIC: Activities Inquilines closely interact with their host. Some live as parasites, but the vast majority are mutualists. Beneficial activities include secretion of enzymes, feeding on the plant's prey and successive excretion of inorganic nutrients, mechanical break up of the prey, removal of excessive prey and assimilation of atmospheric N(2). CONCLUSIONS: There is strong evidence that CPPs influence their phytotelm. Two strategies can be distinguished: (1) Nepenthes and Cephalotus produce acidic, toxic or digestive fluids and host a limited diversity of inquilines. (2) Genera without efficient enzymes such as Sarracenia or Heliamphora host diverse organisms and depend to a large extent on their symbionts for prey utilization.     

Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway

Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2) is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing β-N-acetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. N-acetylglucosaminyltransferase I (GNT-I) contributes to branch point intermediate production and can potentially reverse the FDL trimming reaction. However, there has been no concerted effort to evaluate the relationships among these three enzymes in any single insect system. Hence, we extended our previous studies on Spodoptera frugiperda (Sf) FDL to include GNT-I and -II. Sf-GNT-I and -II cDNAs were isolated, the predicted protein sequences were analyzed, and both gene products were expressed and their acceptor substrate specificities and intracellular localizations were determined. Sf-GNT-I transferred N-acetylglucosamine to Man(5)GlcNAc(2), Man(3)GlcNAc(2), and GlcNAc(β1-2)Man(α1-6)[Man(α1-3)]ManGlcNAc(2), demonstrating its role in branch point intermediate production and its ability to reverse FDL trimming. Sf-GNT-II only transferred N-acetylglucosamine to Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2), demonstrating that it initiates complex N-glycan production, but cannot use Man(3)GlcNAc(2) to produce hybrid or complex structures. Fluorescently tagged Sf-GNT-I and -II co-localized with an endogenous Sf Golgi marker and Sf-FDL co-localized with Sf-GNT-I and -II, indicating that all three enzymes are Golgi resident proteins. Unexpectedly, fluorescently tagged Drosophila melanogaster FDL also co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. Thus, the substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the net outcome of the insect cell N-glycosylation pathway.