Improved method for preparation and purification of recombinant α-synuclein: high-mannose-type free N-glycan prepared from an edible bean (Vigna angulari, Azuki bean) inhibits α-synuclein aggregation.

Parkinson's disease is characterized by the accumulation of amyloid, which consists of α-synuclein (α-Syn). To screen compounds with amyloid aggregation inhibitory activity, an effective method for the preparation of α-Syn is a prerequisite. We established a simpler method for α-Syn preparation using freeze-thaw treatment of transformed Escherichia coli. Furthermore, we found that the high-mannose type free N-glycans could prevent α-Syn aggregation.

Structural features of free N-glycans in α1,3/4-fucosidase-deficient Arabidopsis thaliana: deletion of α1,3/4-fucosidase activity induced accumulation of plant complex type GN1 free N-glycans.

Deletion of α-1,3/4-fucosidase activity in Arabidopsis thaliana resulted in the accumulation of GN1-type free N-glycans with the Lewis a epitope (GN1-FNG). This suggests that the release of α-fucose residue(s) may trigger rapid degradation of the plant complex-type (PCT) GN1-FNG. The fact that PCT-GN1-FNG has rarely been detected to date is probably due to its easier degradation compared with PCT-GN2-FNG.

Improved assay system for acidic peptide: N-glycanase (aPNGase) activity in plant extracts.

Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography.

Purification and molecular characterization of a truncated-type Ara h 1, a major peanut allergen: oligomer structure, antigenicity, and glycoform.

Peanut allergies are among the most severe food allergies, and several allergenic proteins referred to as Ara h 1-Ara h 17 have been identified from peanut seeds. The molecular characterization of Ara h 1 (63 kDa), a glycosylated allergen, has almost been completed, and the occurrence of two homologous genes (clone 41B and clone P17) has been identified. In this study, we found a new variant of Ara h 1 i.e. 54 kDa, in which the N-terminal amino acid sequence was EGREGEQ-, indicating that the N-terminal domain of 63 kDa Ara h 1 had been removed. This new isoform was obtained from the run-through fraction of hydrophobic interaction chromatography while 63 kDa Ara h 1 was tightly bound to the hydrophobic resins, suggesting that the removal of the N-terminal domain resulted in extreme hydrophilic properties. We found that 63 kDa Ara h 1 occurs as higher order homo-oligomeric conformations such as decamer or nonamer, while 54 kDa Ara h 1 occurs exclusively as a homotrimer, indicating that the N-terminal domain of the 63 kDa molecule may be involved in higher order oligomerization. When antisera from peanut-allergic patients were treated with both the Ara h 1 molecules, the immunoglobulin E (IgE) antibodies in these sera reacted with each Ara h 1 molecule, suggesting that the C-terminal as well as the N-terminal domains of Ara h 1 contribute significantly to the epitope formations of this peanut glycoallergen. Furthermore, the glycoform analyses of N-glycans linked to 63 kDa and 54 kDa Ara h 1 subunits revealed that both typical high-mannose type and ß-xylosylated type N-glycans are linked to the molecules. The cross-reactivity of IgE against Ara h 1 in the serum of one peanut allergy patient was completely lost by de-N-glycosylation, indicating the N-glycan of Ara h 1 was the sole epitope for the Ara h 1- specific IgE in the patient.

Large-scale preparation of sialyl-Tn antigen-containing peptides from mucin-like glycoproteins in boar seminal gel.

Sialyl-Tn antigen, a tumor antigen, is a valuable ligand for the purification of proteins that specifically bind to it. Here, we developed a new method for the preparation of large amounts of sialyl-Tn antigen-containing peptides from an unused resource, boar seminal gel. The glycopeptides were prepared from the actinase E digests by a combination of gel filtration and hydrophilic partitioning.

Direct evidence of cytosolic PNGase activity in Arabidopsis thaliana: in vitro assay system for plant cPNGase activity.

Cytosolic peptide:N-glycanase (cPNGase), which occurs ubiquitously in eukaryotic cells, is involved in the de-N-glycosylation of misfolded glycoproteins in the protein quality control system. In this study, we aimed to provide direct evidence of plant cPNGase activity against a denatured glycoprotein using a crude extract prepared from a mutant line of Arabidopsis thaliana lacking 2 acidic PNGase genes.

Plant complex type free N-glycans occur in tomato xylem sap.

Free N-glycans (FNGs) are ubiquitous in growing plants. Further, acidic peptide:N-glycanase is believed to be involved in the production of plant complex-type FNGs (PCT-FNGs) during the degradation of dysfunctional glycoproteins. However, the distribution of PCT-FNGs in growing plants has not been analyzed. Here, we report the occurrence of PCT-FNGs in the xylem sap of the stem of the tomato plant. Abbreviations: RP-HPLC: reversed-phase HPLC; SF-HPLC: size-fractionation HPLC; PA-: pyridylamino; PCT: plant complex type; Hex: hexose; HexNAc: N-acetylhexosamine; Pen: pentose; Deoxyhex: deoxyhexose; Man: D-mannose; GlcNAc: N-acetyl-D-glucosamine; Xyl: D-xylose; Fuc: L-fucose; Lea: Lewis a (Galß1-3(Fucα1-4)GlcNAc); PCT: plant complex type; M3FX: Manα1-6(Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA; GN2M3FX: GlcNAcß1-2Manα1-6(GlcNAcß1-2Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA; (Lea)1GN1M3FX: Galß1-3(Fucα1-4)GlcNAc1-2 Manα1-6(GlcNAcß1-2Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA or GlcNAc1-2Manα1-6(Galß1-3(Fucα1-4)GlcNAc1-2Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA.

Degradation pathway of plant complex-type N-glycans: identification and characterization of a key α1,3-fucosidase from glycoside hydrolase family 29.

Plant complex-type N-glycans are characterized by the presence of α1,3-linked fucose towards the proximal N-acetylglucosamine residue and ß1,2-linked xylose towards the ß-mannose residue. These glycans are ultimately degraded by the activity of several glycoside hydrolases. However, the degradation pathway of plant complex-type N-glycans has not been entirely elucidated because the gene encoding α1,3-fucosidase, a glycoside hydrolase acting on plant complex-type N-glycans, has not yet been identified, and its substrate specificity remains to be determined. In the present study, we found that AtFUC1 (an Arabidopsis GH29 α-fucosidase) is an α1,3-fucosidase acting on plant complex-type N-glycans. This fucosidase has been known to act on α1,4-fucoside linkage in the Lewis A epitope of plant complex-type N-glycans. We found that this glycoside hydrolase specifically acted on GlcNAcß1-4(Fucα1-3)GlcNAc, a degradation product of plant complex-type N-glycans, by sequential actions of vacuolar α-mannosidase, ß1,2-xylosidase, and endo-ß-mannosidase. The AtFUC1-deficient mutant showed no distinct phenotypic plant growth features; however, it accumulated GlcNAcß1-4(Fucα1-3)GlcNAc, a substrate of AtFUC1. These results showed that AtFUC1 is an α1,3-fucosidase acting on plant complex-type N-glycans and elucidated the degradation pathway of plant complex-type N-glycans.

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract.

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.

Occurrence of complex type free N-glycans with a single GlcNAc residue at the reducing termini in the fresh-water plant, Egeria densa.

In our previous study, we found unique free N-glycans (FNGs), which carry a single GlcNAc residue (GN1) at the reducing-end side and the Lewis-a epitope at the non-reducing-end side, in the culture broth of rice cells. Based on the FNG structural features and the substrate specificity of plant ENGase, we hypothesized that there might be a novel biosynthetic mechanism responsible for the production of these unique GN1-FNGs, in which high-mannose type (HMT)-GN1-FNGs produced in the cytosol from misfolded glycoproteins by ENGase are transported back into the endoplasmic reticulum and processed to plant complex type (PCT)-GN1-FNGs in the Golgi apparatus. Until now, however, PCT-GN1-FNGs had only been found in the culture broth of rice cultured cells and never in plants, suggesting that the formation of PCT-GN1-FNGs might be generated under special or artificial conditions. In this study, we confirm the presence of PCT-GN1-FNGs, HMT-GN1-FNGs and PCT-GN2-FNGs in the fresh-water plant Egeria densa. These results suggest that a mechanism responsible for the production of PCT-GN1-FNG is present in native plant tissues.

Structural feature of N-glycans of bamboo shoot glycoproteins: useful source of plant antigenic N-glycans.

An effective method to prepare plant complex type (PCT) N-glycans in large amounts has been required to evaluate their immunological activity. In this study, we found that glycoproteins in bamboo shoots predominantly carry PCT N-glycans including the Lewis a epitope-containing ones, suggesting that bamboo shoot is an excellent source for the plant antigenic glycans to synthesize immunoactive neoglycopolymers.

Phenotype-based clustering of glycosylation-related genes by RNAi-mediated gene silencing.

Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, ß1,4-galactosyltransferase I, ß1,3-galactosyltransferase II, and ß1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.

Possible Contribution of Alpha2,6-Sialylation to Luteolysis in Cows by Inhibiting the Luteotropic Effects of Galectin-1.

The corpus luteum (CL) is essential for establishing pregnancy. If pregnancy does not occur during the estrous cycle, luteolysis is induced by prostaglandin (PG) F2alpha secreted from the uterus. Galectin-1, a beta-galactose-binding protein, is expressed in the functional CL of cows and increases the viability of bovine luteal steroidogenic cells (LSCs) by modifying the functions of membrane glycoproteins. The binding of galectin-1 to glycoproteins is blocked by alpha2,6-sialylation of the terminal galactose residues of glycoconjugates, which is catalyzed by a sialyltransferase (ST6Gal-I). However, the physiological role of alpha2,6-sialic acid in bovine CL is unclear. The level of alpha2,6-sialylation of the bovine CL was higher during the regressed-luteal stage than in other luteal stages. Lectin histochemistry revealed that alpha2,6-sialylated glycoconjugates were localized to luteal endothelial cells throughout the estrous cycle. In addition, alpha2,6-sialylated glycoconjugates concentrated to the membrane of LSCs during the regressed-luteal stage. PGF2alpha treatment for 72 h enhanced the expression of ST6Gal-I mRNA and the level of alpha2,6-sialylated glycoproteins in mid-LSCs. The level of alpha2,6-sialylated glycoproteins of late-stage LSCs (Days 15-17 after ovulation) was higher than that of mid-stage LSCs (Days 8-12 after ovulation), and galectin-1 increased the viability of mid-LSCs but not that of late-stage LSCs. Furthermore, galectin-1 increased the viability of late-LSCs when alpha2,6-sialic acid residues were removed by neuraminidase. The overall findings suggest that alpha2,6-sialylation stimulated by PGF2alpha contributes to luteolysis by inhibiting the luteotropic effects of galectin-1 in bovine CL.

Rice α-fucosidase active against plant complex type N-glycans containing Lewis a epitope: purification and characterization.

Rice α-fucosidase (α-fucosidase Os, 58 kDa) that is active for α1-4 fucosyl linkage in Lewis a unit of plant N-glycans was purified to homogeneity. α-fucosidase Os showed activity against α1-3 fucosyl linkage in Lacto-N-fucopentaose III but not α1-3 fucosyl linkage in the core of plant N-glycans. The N-terminal sequence of α-fucosidase Os was identified as A-A-P-T-P-P-P-L-, and this sequence was found in the amino acid sequence of the putative rice α-fucosidase 1 (Os04g0560400).

Identification of ß1,3-galactosyltransferases responsible for biosynthesis of insect complex-type N-glycans containing a T-antigen unit in the honeybee.

Honeybees (Apis mellifera) produce unique complex-type N-glycans bearing a Galß1-3GalNAc (T-antigen) unit, and honeybee-specific N-glycans are linked to royal jelly glycoproteins. In this study, we identified two novel honeybee ß1,3-galactosyltransferase (ß1,3-GalT) genes responsible for biosynthesis of the T-antigen in insect N-glycans. The products of the two putative ß1,3-GalT genes (ß1,3-GalT1 and ß1,3-GalT2), which were expressed in Sf21 insect cells, transferred galactose (Gal) residues to GalNAc2GlcNAc2Man3GlcNAc2-PA to form the Galß1-3GalNAc unit, indicating that the identified genes were involved in biosynthesis of the ß1-3 Gal-containing N-glycan. Therefore, using biochemistry and molecular biology techniques, we revealed a unique N-glycan biosynthesis mechanism in the cephalic region of honeybees, which has not previously been found in other animal or plant cells.

ß-Galactosidase from Ginkgo biloba seeds active against ß-galactose-containing N-glycans: purification and characterization.

In this study, we purified an acidic ß-galactosidase to homogeneity from Ginkgo biloba seeds (ß-Gal'ase Gb-1) with approximately 270-fold purification. A molecular mass of the purified ß-Gal'ase Gb-1 was estimated about 35 kDa by gel filtration and 32 kDa by SDS-PAGE under non-reducing condition, respectively. On the other hand, ß-Gal'ase Gb-1 produced a single band with a molecular mass of 16 kDa by SDS-PAGE under reducing condition. The N-terminal amino acid sequences of 32 kDa and 16 kDa molecules were the same and identified as H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-, suggesting that ß-Gal'ase Gb-1 may function as a homodimeric structure in vivo. When complex-type N-glycans containing ß-galactosyl residues were used as substrates, ß-Gal'ase Gb-1 showed substantial activity for ß1-4 galactosyl residue and modest activity for ß1-3 galactosyl residue with an optimum pH near 5.0. Based on these results, the involvement of ß-Gal'ase Gb-1 in the degradation of plant complex-type N-glycans is discussed.

Predictors of hypofibrinogenemia in blunt trauma patients on admission.

PURPOSE: Massive bleeding usually leads to critically low levels of clotting factors, including fibrinogen. Although reduced fibrinogen levels correlate with increased mortality, predictors of hypofibrinogenemia have remained poorly understood. We investigated whether findings available on admission can be used as predictors of hypofibrinogenemia. METHODS: We retrospectively reviewed serum fibrinogen levels tested on arrival in 290 blunt trauma patients transported to a level I trauma center during a 3-year period. The primary outcome was prehospital predictors for hypofibrinogenemia. Covariates included age, sex, prehospital fluid therapy, prehospital anatomical and physiological scores, time from injury, base excess, and lactate on arrival. All variables with values of p < 0.10 in univariate analysis were included in a multivariate logistic regression model. The relationships between the variables and the 7-day mortality rate were evaluated in a Cox proportional hazards model. RESULTS: Patient's age [odds ratio (OR): 0.97, p < 0.001], Triage Revised Trauma Score (T-RTS) (OR: 0.81, p = 0.003), and prehospital fluid therapy (OR: 2.54, p = 0.01) were detected as independent predictors for hypofibrinogenemia in multivariate logistic regression analysis. Serum fibrinogen level [hazard ratio (HR): 0.99, p = 0.01] and T-RTS (HR: 0.77, p < 0.01) were associated with the 7-day mortality rate. CONCLUSION: T-RTS is considered to play an important role in predicting hypofibrinogenemia and 7-day mortality in blunt trauma patients.

Oral vaccination with a liposome-encapsulated influenza DNA vaccine protects mice against respiratory challenge infection.

It is well accepted that vaccination by oral administration has many advantages over injected parenteral immunization. The present study focuses on whether oral vaccination with a DNA vaccine could induce protective immunity against respiratory challenge infection. The M1 gene of influenza A virus was used to construct DNA vaccine using pcDNA 3.1(+) plasmid, a eukaryotic expression vector. The cationic liposomes were used to deliver the constructed DNA vaccine. In vitro and in vivo expression of M1 gene was observed in the cell line and in the intestine of orally vaccinated C57BL/6 mice, respectively. It became clear that this type of oral DNA vaccination was capable of inducing both humoral and cellular immune responses, together with an augmentation of IFN-γ production. In addition, oral vaccination with liposome-encapsulated DNA vaccine could protect the mice against respiratory challenge infection. These results suggest that gastrointestinal tract, a constituent member of the common mucosal immune system, is a potent candidate applicable as a DNA vaccine route against virus respiratory diseases.

Infection with respiratory syncytial virus influences FasL-mediated apoptosis of pulmonary γδ T cells in a murine model of allergen sensitization.

BACKGROUND: It has been reported that adoptive transfer of γδ T cells increases the cellular infiltration, especially eosinophils, in the lungs of allergic mice, suggesting that γδ T cells may play a proinflammatory role in allergic airway inflammation. Respiratory syncytial virus (RSV) infection can decrease the number of Th2-type γδ T cells. However, the underlying mechanisms remain unknown. METHODS: BALB/c mice were inoculated intranasally with RSV before or after sensitization to OVA. The amounts of Th1/Th2 cytokines as well as the levels of specific antibodies were determined by ELISA. The apoptotic death of pulmonary γδ T cells was analyzed by flow cytometry. RESULTS: Adoptive transfer of γδ T cells increased the production of Th2 cytokines in the lungs and allergy-related antibodies in the serum, further confirming that γδ T cells act as pro-inflammatory cells or a promoter for the development of allergic asthma. RSV infection before sensitization to OVA enhanced apoptotic death of pulmonary γδ T cells. The percentage and absolute number of FasL-expressing γδ T cells in the lungs of allergic mice were elicited significantly by prior RSV infection. Blocking FasL with monoclonal antibody diminished apoptotic death of γδ T cells, suggesting that FasL is important for RSV-induced apoptosis of pulmonary γδ T cells. CONCLUSIONS: This work provides evidence that RSV infection suppresses the subsequent development of OVA-induced allergic responses partly by enhancing FasL-mediated apoptosis of pulmonary γδ T cells.

Large-scale preparation of glycopeptides harboring the TF-antigen unit from royal jelly.

We have reported that new N-glycans carrying the TF-antigen occurred on a major royal jelly glycoprotein, and we have identified the glycosylation site to which the antigenic N-glycan is linked, but an appropriate procedure has not been established to prepare non-labeled immunoreactive glycopeptides in large amounts for functional analysis. In this study, we developed an effective method of preparing Asn-glycopeptide bearing TF-antigen.