Changes in Structural Features of Free N-Glycan and Endoglycosidase Activity during Tomato Fruit Ripening

In developing plants, free N-glycans occur ubiquitously at micromolar concentrations. Such oligosaccharides have been proposed to be signaling molecules in plant development. As a part of a study to elucidate the physiological roles of de-N-glycosylation machinery involved in fruit ripening, we analyzed changes in the amounts and structural features of free N-glycans in tomato fruits at four ripening stages. The amount of high-mannose type free N-glycans increased significantly in accordance with fruit ripening, and the relative amounts of high-molecular size N-glycans, such as Man_8-9GlcNAc₁, became predominant. These observations suggest that the de-N-glycosylation machinery, including endo-β-N-acetylglucosaminidase (ENGase) activity, is stimulated in the later stages of fruit ripening. But contrary to expectation, we found that total ENGase activities in the tomato fruits did not vary significantly with the ripening process, suggesting that ENGase activity must be maintained at a certain level, and that the expression of α-mannosidase involved in the clearance of free N-glycans decreases during tomato fruit ripening.     

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; Le^a: Lewis a (Galβ1-3(Fucα1-4)GlcNAc); PCT: plant complex type; M3FX: Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA; GN2M3FX: GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA; (Le^a)1GN1M3FX: Galβ1-3(Fucα1-4)GlcNAc1-2 Manα1-6(GlcNAcβ1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA or GlcNAc1-2Manα1-6(Galβ1-3(Fucα1-4)GlcNAc1-2Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA.     

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).     

Salt-Adaptation of Tobacco BY2 Cells Induces Change in Glycoform of N-Glycans: Enhancement of Exo- and Endo-Glycosidase Activities by Salt-Adaptation

In this report, we describe that a salt adaptation of plant cells induces glycoform changes in N-glycoproteins. Intracellular and cell-wall glycopeptides were prepared from glycoproteins expressed in wild-type BY2 cells and salt-adapted cells. N-Glycans were liberated from those glycopeptides by hydrazinolysis, and the released oligosaccharides were N-acetylated and pyridylaminated. The structures of pyridylaminated (PA-) N-glycans were analyzed by a combination of two-dimensional sugar-chain mapping, MS analysis, and exoglycosidase digestion. In both wild-type cells and salt-adapted cells, the plant complex type structure was predominant among N-glycans expressed on glycoproteins, but we found that the Man2Xyl1Fuc1GlcNAc2 structure was significantly expressed on intracellular and cell-wall glycoproteins of the salt-adapted cells. Furthermore, enhancement of the specific activities of α-mannosidase and β-N-acetylglucosaminidase was observed in the salt-adapted BY2 cells, suggesting that the glycoform changes are due to changes in glycosidase activities.     

Antibodies that recognize bisected complex N-glycans on cell surface glycoproteins can be made in mice lacking N-acetylglucosaminyltransferase III

The bisecting GlcNAc is transferred to complex or hybrid N-glycans by the action of N-acetylglucosaminyltransferase III (GlcNAc-TIII) encoded by the Mgat3 gene. CHO cells expressing mouse GlcNAc-TIII were shown by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to produce mainly complex N-glycans with the predicted extra (bisecting) GlcNAc. In order to probe biological functions of the bisecting GlcNAc, antibodies that recognize this residue in the context of complex cell surface glycoconjugates were sought. The LEC10 gain-of-function Chinese hamster ovary (CHO) cell mutant that expresses GlcNAc-TIII and complex N-glycans with the bisecting GlcNAc was used to immunize Mgat3 ^+/+ and Mgat3 ^–/– mice. ELISA of whole sera showed that polyclonal antibodies that bound specifically to LEC10 cells were obtained solely from Mgat3 ^–/– mice. Fluorescence-activated cell cytometry of different CHO glycosylation mutants and western blotting after glycosidase treatments were used to show that anti-LEC10 cell antisera from Mgat3 ^–/– mice recognize cellular glycoproteins with complex N-glycans containing both a bisecting GlcNAc and Gal residues. The polyclonal antibody specificity was similar to that of the lectin E-PHA. IgM-depleted serum containing IgG and IgA antibodies retained full binding activity. Therefore Mgat3 ^–/– mice but not wild type mice can be used effectively to produce polyclonal antibodies that specifically recognize glycoproteins bearing complex N-glycans with a bisecting GlcNAc. Published in 2003.     

Glycoform Analysis of N-Glycans Linked to Glycoproteins Expressed in Rice Culture Cells: Predominant Occurrence of Complex Type N-Glycans

Although it has been found that plant endo-β-N-acetylglucosaminidase shows strong activity towards denatured glycoproteins and glycopeptides with high-mannose type N-glycans and free high-mannose type N-glycans bearing the chitobiosyl unit, the endogenous substrates for plant endoglycosidase have not yet been identified. Recently we purified and characterized an endo-β-N-acetylglucosaminidase from rice culture cells and identified the gene encoded (Maeda, M., and Kimura, Y., Trends Glycosci. Glycotech., 17, 205–214 (2005)). Furthermore, we found structural features of free N-glycans in the cells, indicating that high-mannose type species (Man_9-5GlcNAc₁) occur at concentration of several micromolar (μM). Hence, in this study we analyzed glycoform of N-glycans linked to glycoproteins expressed in rice culture cells to see whether endogenous glycoproteinous substrate occurs in reasonable amounts. Structural analysis revealed that more than 95% of total N-glycans linked to glycoproteins in the rice cells had the plant complex type structure, including Lewis a epitope-harboring type, although high-mannose type structures account for less than 5% of total N-glycans.     

Some common properties of lectins from marine algae

Twelve kinds of lectins isolated from four species of marine algae, Boodlea coacta (Chlorophyta) and Hypnea japonica, Carpopeltis flabellata and Solieria robusta (Rhodophyta), were compared for their chemical and biological properties. These lectins were proteins or glycoproteins, similar to terrestrial plant lectins. However, unlike most terrestrial plant lectins, they had a small molecular size (4,200 to 25,000 daltons), were mostly monomeric, and had no affinity for monosaccharides. They strongly agglutinated trypsin-treated rabbit erythrocytes, and their activities commonly were inhibited by glycoproteins bearing N-glycans. From hemagglutination-inhibition tests with various glycoproteins and related compounds, it was found that B. coacta lectins recognize high-mannose N-glycans; H. japonica lectins complex N-glycans, and C. flabellata and S. robusta lectins recognize both types of N-glycans.     

Role of complex N-glycans in plant stress tolerance

In plant cells, glycans attached to asparagine (N) residues of proteins undergo various modifications in the endoplasmic reticulum and the Golgi apparatus. The N-glycan modifications in the Golgi apparatus result in complex N-glycans attached to membrane proteins, secreted proteins and vacuolar proteins. Recently, we have investigated the role of complex N-glycans in plants using a series of Arabidopsis thaliana mutants affected in complex N-glycan biosynthesis.¹ Several mutant plants including complex glycan 1 (cgl1) displayed a salt-sensitive phenotype during their root growth, which was associated with radial swelling and loss of apical dominance. Among the proteins whose N-glycans are affected by the cgl1 mutation is a membrane anchored β1,4-endoglucanase, KORRIGAN1/RADIALLY SWOLLEN 2 (KOR1/RSW2) involved in cellulose biosynthesis. The cgl1 mutation strongly enhanced the phenotype of a temperature sensitive allele of KOR1/RSW2 (rsw2-1) even at the permissive temperature. This establishes that plant complex N-glycan modification is important for the in vivo function of KOR1/RSW2. Furthermore, rsw2-1 as well as another cellulose biosynthesis mutant rsw1-1 exhibited also a salt-sensitive phenotype at the permissive temperature. Based on these findings, we propose that one of the mechanisms that cause salt-induced root growth arrest is dysfunction of cell wall biosynthesis that induces mitotic arrest in the root apical meristem.Key words: Arabidopsis, salt stress, complex N-glycans, β1,4-endoglucanase, cell wallIn eukaryotic cells, both soluble and membrane proteins that enter the endoplasmic reticulum (ER) system may undergo post-translational modifications called N-glycosylation. N-glycosylation occurs in two phases, namely, core glycosylation in the ER and glycan maturation in the Golgi apparatus.^2,3 The process and roles of core glycosylation in the ER are well established and ubiquitous for eukaryotes. In the ER, pre-assembled core oligosaccharides (Glc₃Man₉GlcNac₂) are transferred to asparagine residues of the Asn-X-Ser/Thr motives in nascent polypeptides by the function of an oligosaccharyltransferase complex (OST). Terminal glucose residues are recognition sites for ER chaperones calnexin and calreticulin, and thus core N-glycans in the ER function in correct folding of newly synthesized proteins.^2,3Greater diversity exists in the N-glycan maturation steps in the Golgi apparatus and conspicuous roles for the resulting complex N-glycans.^2,4 In general, mature N-glycan structures are classified as oligomannosidic type, hybrid or complex type. Glycoprotein precursors that are exported from the ER carry high-mannose type N-glycan intermediates. Numerous enzymes are involved in the conversion of high-mannose type N-glycans to mature complex N-glycans. The functions of N-glycan modifications in the Golgi apparatus are well established in humans, because lack of N-glycan maturation results in Type II Congenital Disorders of Glycosylation.⁵ In Drosophila melanogaster, the Golgi pathway is necessary for development and function of the central nervous system,⁶ whereas in Candida albicans, it is necessary for cell wall integrity and virulence.⁷The first Arabidopsis thaliana mutant lacking complex N-glycans was reported in 1993.⁸ Since then, several mutants and transgenic plants altered in N-glycan maturation in the Golgi apparatus have been reported.^9–12 Plants with altered N-glycan modification pathways that are devoid of potentially immunogenic complex N-glycans are used for the production of pharmaceutical proteins^12,13 and could serve as potential food crops with reduced allergenicity. Until recently, however, plant complex N-glycans have not been associated with essential biological functions in their host plants due to lack of obvious phenotypes of mutant plants defective in complex N-glycan biosynthesis. We recently reported that mutants defective in complex N-glycans show enhanced salt sensitivity, establishing that complex N-glycans are indispensable for certain biological functions.¹Our previous study using an OST subunit mutant stt3a indicated that protein glycosylation could affect salt tolerance and root growth of A. thaliana.¹⁴ Since OST functions upstream of protein folding processes in the ER, stt3a caused an unfolded protein response (UPR), which is a general ER stress response to protein folding defects, as well as accumulation of under-glycosylated proteins. In our recent study, we tried to address whether the salt stress response of the mutant is caused by an activation of UPR, or by a shortage of functional glycoproteins produced by the cells.¹ The cgl1 mutant is defective in N-acetylglucosaminyltransferase in the Golgi apparatus¹⁵ and only able to produce oligomannosidic-type N-glycans but not complex-type N-glycans.⁸ cgl1 mutants exposed to salt stress exhibited root growth arrest and radial swelling similar to stt3a mutants, however, unlike stt3a, the cgl1 mutation did not cause UPR as judged by expression of an UPR marker gene, BiP_pro-GUS. This indicated that salt sensitivity of cgl1 (and likely also of stt3a) is due to lack of mature N-glycans essential for functionality of certain glycoprotein(s).We have determined that a membrane-anchored β1,4-endoglucanase, KORRIGAN1/RADIAL SWELLING2 (KOR1/RSW2), which functions in cellulose biosynthesis, is a target of CGL1 and involved in the salt stress response of A. thaliana.¹ A temperature sensitive rsw2-1 allele¹⁶ showed specific genetic interaction with both cgl1 and stt3a mutations. The corresponding double mutants exhibited spontaneous growth defects at the permissive temperature that were reminiscent of those of rsw2-1 at the restrictive temperature, of cgl1 and stt3a plants treated with salt, and of the rsw1-1 rsw2-1 double mutant that combines two cellulose deficiency mutations. This showed that cgl1 and stt3a enhance cellulose deficiency of rsw2-1, and in turn indicate that the KOR1/RSW2 protein requires complex N-glycans for its function in vivo. Further pyramiding of these mutations resulted in incremental enhancement of growth defects as well as developmental defects of the host plants (Kang et al., (2008), and Fig. 1). Importance of functional cellulose biosynthesis for salt tolerance was further supported by the novel finding of increased salt-sensitivity of rsw2-1 and rsw1-1 single mutants.¹Our previous and current data have implications that affect our view of protein N-glycosylation in plants. First, after all, plant complex N-glycans confer important in vivo functions to secreted/secretory glycoproteins, i.e., protect root growth from salt/osmotic stress. In contrast to core oligosaccharides in the ER, which globally affect protein folding, complex N-glycans appear to function at the individual protein level. Second, one of the targets of salt/osmotic stress is a component of the cellulose biosynthesis machinery, namely KOR1/RSW2 that requires complex N-glycans for its function. KOR1/RSW2 provides a link to how complex N-glycans protect plants from salt/osmotic stress. However, the mechanism by which salt stress triggers the growth arrest via KOR1/RSW2 dysfunction is not yet understood. We have previously shown that the root apical meristem of stt3a exhibits cell cycle arrest under salt stress, but cell differentiation and lateral root formation continued in the same root tip.¹⁴ This implies that plants, in response to salt stress and compromised cell-wall biosynthesis at the root apical meristem, specifically attenuate cell cycle progression at the old meristem and initiate new meristems. A signal transduction pathway that coordinates cell-wall integrity and cell proliferation is well documented in Sacchromyces cerevisiae, where Protein kinase C1 (Pkc1) and a MAP kinase cascade play essential roles.17 Interestingly, both S. cerevisiae Stt3 and Och1 (a mannosyltransferase in the Golgi apparatus) are involved in the cell-wall integrity pathway.¹⁷ In A. thaliana, mutations in the receptor kinase THESEUS1 suppressed hypocotyl elongation defects and ectopic lignification in several cellulose deficient mutants.¹⁸ However, since THE1 is expressed in elongation zones but not in cell division zones of root tips, and the1 did not suppress the kor1-1 phenotype,¹⁸ it is unlikely that THE1 is involved in the regulation of the salt stress response at the root apical meristem. This implies that dividing cells and expanding cells employ distinct mechanism to sense cellulose deficiency. Understanding how complex N-glycans regulate cell-wall biosynthesis and cell proliferation is an exciting task for the coming years.? Open in a separate windowFigure 1Scanning electron micrograph of one-week-old wild type (A and D), rsw2-1 stt3a-2 cgl1-T (B and E) and rsw2-1 rsw1-1 stt3a-2 cgl1-T (C and F) seedlings grown at 18°C. Severe growth defects in mutants are obvious. In shoot apical meristem (D–F), aberrant trichome development is seen in rsw2-1 stt3a-2 cgl1-T (E). In rsw2-1 rsw1-1 stt3a-2 cgl1-T (F), the meristem is transformed into unorganized mass of cells. Bars indicate 0.5 mm.     

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.     

Small-scale, high-throughput method for plant N-glycan preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis

A simple, small-scale, and high-throughput method for preparation of plant N-glycans for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) is described. This method entailed the extraction of soluble proteins, pepsin digestion, release of N-glycans by glycopeptidase A, and a three-step chromatographic purification process using cation exchange, anion exchange, and graphitized carbon. Homemade minicolumns using commercially available filter unit devices were used for N-glycan purification steps. All purification steps were designed to be easy. Using this method, N-glycans from 10-mg leaf samples of different plant species and only 2 μg of pure horseradish peroxidase were successfully purified.     

Expression of complex-type N-glycans in developmental periods of zebrafish embryo

As a first step to elucidate a role of N-glycans in development of vertebrates, we analyzed structures of the glycans expressed in early stages of zebrafish embryo. N-glycans were prepared from zebrafish embryos at several developmental stages followed by tagging with a fluorophore, 2-aminopyridine. The labeled glycans were analyzed by two modes of HPLCs. The comparison of the elution profiles of HPLCs unveil the change of the oligosaccharide structure during the development. These peaks were merely detected during 4–7 h after fertilization, however, increased from 12 h, and at 15 h a fairly amount of them was appeared. Structure analysis revealed that they were bianntenary complex-type N-glycans with or without fucose and/or bisecting N-acetylglucosamine residues. These results suggest that the complex-type N-glycans are concerned in some developmental event from segmentation period downward in zebrafish. Published in 2005.     

Inhibitory and Thermodynamic Studies on the Hydrolysis of Cyclodextrins Catalyzed by Taka-amylase A

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz ¹H-NMR spectrometry.

The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing β1-2 xylosyl and/or α1-3 fucosyl residue(s) and occurrence of β1-4GlcNAc residue in the insect glycoproteins.

The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man_9~4GlcNAc₂) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc₂Man₃GlcNAc₂) 8.4%, and hybrid type structure (GlcNAc₁Man₄GlcNAc₂) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc₃Hex₃HexNAc₂: 3.0%), D2 (HexNAc₂Hex₅HexNAc₂: 4.5%), and D3 (HexNAc₃Hex₄HexNAc₂: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of β-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a β1-4 linkage N-acetylglucosaminyl residue.     

A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans

Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVN^Lec1. We found that Sf-RVN and Sf-RVN^Lec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVN^Lec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVN^Lec1 cells were Endo H-cleavable Man₅GlcNAc₂ structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.     

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

In this study, we purified an acidic β-galactosidase to homogeneity from Ginkgo biloba seeds (β-Gal’ase Gb-1) with approximately 270-fold purification. A molecular mass of the purified β-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, β-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 β-Gal’ase Gb-1 may function as a homodimeric structure in vivo. When complex-type N-glycans containing β-galactosyl residues were used as substrates, β-Gal’ase Gb-1 showed substantial activity for β1-4 galactosyl residue and modest activity for β1-3 galactosyl residue with an optimum pH near 5.0. Based on these results, the involvement of β-Gal’ase Gb-1 in the degradation of plant complex-type N-glycans is discussed.     

Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers

We have analyzed the structures of glycosphingolipids and intracellular free glycans in human cancers. In our previous study, trace amounts of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans with a single GlcNAc at each reducing terminus (Gn1 type) was found to accumulate intracellularly in colorectal cancers, but were undetectable in most normal colorectal epithelial cells. Here, we used cancer glycomic analyses to reveal that substantial amounts of free Neu5Ac-containing complex-type N-glycans, almost all of which were α2,6-Neu5Ac-linked, accumulated in the pancreatic cancer cells from three out of five patients, but were undetectable in normal pancreatic cells from all five cases. These molecular species were mostly composed of five kinds of glycans having a sequence Neu5Ac-Gal-GlcNAc-Man-Man-GlcNAc and one with the following sequence Neu5Ac-Gal-GlcNAc-Man-(Man-)Man-GlcNAc. The most abundant glycan was Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc, followed by Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal evidence for the occurrence of free Neu5Ac-linked N-glycans in human cancer tissues. Our findings suggest that free Neu5Ac-linked glycans may serve as a useful tumor marker.     

Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products

N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear.     

Purification and Characterization of a Co(II)-Sensitive α-Mannosidase from Ginkgo biloba Seeds

An α-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified α-mannosidase was estimated to be 120 kDa by SDS–PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo α-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala–Phe–Met–Lys–Tyr–X–Thr–Thr–Gly–Gly–Pro–Val–Ala–Gly–Lys–Ile–Asn–Val–His–Leu–. The α-mannosidase activity for Man₅GlcNAc₁ was enhanced by the addition of Co²⁺, but the addition of Zn²⁺, Ca²⁺, or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man₆GlcNAc₁ was significantly faster than that for pyridylaminated Man₆GlcNAc₂, suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013–1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an α-mannosidase, which is activated by Co²⁺ and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.     

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.     

Changes in N-Linked Oligosaccharides during Seed Development of Ginkgo biloba

Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-β-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253-261), which should be involved in the production of high-mannose type free N-glycans.

The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc_2~0Man₃Xyl₁Fuc_1~0GlcNAc₂) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc₂Man₃Xyl₁Fuc₁GlcNAc₂ and GlcNAc₁Man₃Xyl₁Fuc₁GlcNAc₂ decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages.

Concerning the endogenous substrates for plant endo-β-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized glycoproteins.     

In vivo activity of recombinant human lewis fucosyltransferase III in leaves of Nicotiana tabacum L.

Fucosylation in plants occurs in glycoproteins and polysaccharides but the function of fucosylation is largely unknown. We aimed to analyze the effects of over-expression of human fucosyltransferase III (hFucT III) on in vivo N-glycan accumulation in tobacco plant leaves and focused on comparing the amount of Lewis a (Le^a)-epitope accumulation in transgenic and in wild-type plants. Fucosyltransferase assays, Western blot and mass spectrometry were used to identify, quantify and analyse Le^a N-glycans. We found that constitutive overexpression of hFucT III activity had no effect on Le^a complex type N-glycans accumulation. Our results suggest that tobacco recombinant hFucT III acts more as a hydrolase than as a transferase.