Membrane glycoproteomics of fetal lung fibroblasts using LC/MS

Some aberrant N‐glycosylations are being used as tumor markers, and glycoproteomics is expected to provide novel diagnosis markers and targets of drug developments. However, one has trouble in mass spectrometric glycoproteomics of membrane fraction because of lower intensity of glycopeptides in the existence of surfactants. Previously, we developed a glycopeptide enrichment method by acetone precipitation, and it was successfully applied to human serum glycoproteomics. In this study, we confirmed that this method is useful to remove the surfactants and applicable to membrane glycoproteomics. The glycoproteomic approach to the human fetal lung fibroblasts membrane fraction resulted in the identification of over 272 glycoforms on 63 sites of the 44 glycoproteins. According to the existing databases, the structural features on 41 sites are previously unreported. The most frequently occurring forms at N‐glycosylation site were high‐mannose type containing nine mannose residues (M9) and monosialo‐fucosylated biantennary oligosaccharides. Several unexpected N‐glycans, such as fucosylated complex‐type and fucosylated high‐mannose and/or fucosylated pauci‐mannose types were found in ER and lysosome proteins. Our method provides new insights into transport, biosynthesis, and degradation of glycoproteins.     

Design and synthesis of magnetic binary metal oxides nanocomposites through dopamine chemistry for highly selective enrichment of phosphopeptides

In this work, for the first time, magnetic binary metal oxides nanocomposites which integrated Zr and Ti into one entity on an atomic scale on polydopamine coated magnetic graphene (magG/PD/(Zr‐Ti)O₄) was designed and synthesized, and applied to the enrichment of phosphopeptides. The newly prepared magG/PD/(Zr‐Ti)O₄ composites gathered the advantages of large surface area, superparamagnetism, biocompatibility and the enhanced affinity properties to phosphopeptides. MagG/PD/ZrO₂, magG/PD/TiO₂, as well as the simple physical mixture of them were introduced to compare with magG/PD/(Zr‐Ti)O₄ composites. High sensitivity (1 pg/μL or 4.0 × 10^–11 M) and selectivity (weight ratio of β‐casein and BSA reached up to 1:8000) toward phosphopeptides were also presented for magG/PD/(Zr‐Ti)O₄ composites. Additionally, mouse brain tissue was chose as the real samples to further investigate the phosphopeptides enrichment ability of this new material.     

Facile synthesis of magnetic poly(styrene‐co‐4‐vinylbenzene‐boronic acid) microspheres for selective enrichment of glycopeptides

In this work, the composites of magnetic Fe₃O₄@SiO₂@poly (styrene‐co‐4‐vinylbenzene‐boronic acid) microspheres with well‐defined core–shell–shell structure were facilely synthesized and applied to selectively enrich glycopeptides. Due to the relatively large amount of vinyl groups introduced by 3‐methacryloxy‐propyl‐trimethoxysilane on the core‐shell surface, the poly(styrene‐co‐4‐vinylbenzeneboronic acid) (PSV) was coated with high efficiency, resulting in a large amount of boronic acid on the outermost polymer shell of the Fe₃O₄@SiO₂@PSV microspheres, which is of great importance to improve the enrichment efficiency for glycopeptides. The obtained Fe₃O₄@SiO₂@PSV microspheres were successfully applied to the enrichment of glycopeptides with strong specificity and high selectivity, evaluated by capturing glycopeptides from tryptic digestion of model glycoprotein HRP diluted to 0.05 ng/μL (1.25 × 10^?13 mol, 100 μL), tryptic digest of HRP and nonglycosylated BSA up to the ratio of 1:120 w/w and the real complex sample human serum with 103 unique N‐glycosylation peptides of 46 different glycoproteins enriched.     

Concanavalin A‐immobilized magnetic nanoparticles for selective enrichment of glycoproteins and application to glycoproteomics in hepatocelluar carcinoma cell line

Protein glycosylation is one of the most important PTMs in biological organism. Lectins such as concanavalin A (Con A) have been widely applied to N‐glycosylated protein investigation. In this study, we developed Con A‐immobilized magnetic nanoparticles for selective separation of glycoproteins. At first, a facile immobilization of Con A on aminophenylboronic acid‐functionalized magnetic nanoparticles was performed by forming boronic acid‐sugar‐Con A bond in sandwich structure using methyl α‐D ‐mannopyranoside as an intermedium. The selective capture ability of Con A‐modified magnetic nanoparticles for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7703. In total 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Also, the regeneration of the protein‐immobilized nanoparticles can easily be performed taking advantage of the reversible binding mechanism between boronic acid and sugar chain. The experiment results demonstrated that Con A‐modified magnetic nanoparticles by the facile and low‐cost synthesis provided a convenient and efficient enrichment approach for glycoproteins, and are promising candidates for large‐scale glycoproteomic research in complicated biological samples.     

Functional dual hydrophilic dendrimer‐modified metal‐organic framework for the selective enrichment of N‐glycopeptides

Analysis of protein glycosylation remains a significant challenge due to the low abundance of glycoproteins or N‐glycopeptides. Here we have synthesized an amino‐functionalized metal‐organic framework (MOF) MIL‐101(Cr)‐NH₂ whose surface is grafted with a hydrophilic dendrimer poly(amidoamine) (PAMAM) for N‐glycopeptide enrichment based on the hydrophilic interactions. The selected substrate MOF MIL‐101(Cr) owns high surface area which provides nice support for peptide adsorption. In addition, the MOF displayed a good hydrophilic property after being modified with amino groups. Most importantly, the grafted hydrophilic dendrimer PAMAM was firstly applied in the postsynthetic modification of MOFs. And this functionalization route using macromolecular dendrimer opens a new perspective in MOFs design. Owing to its long dendritic chains and abundant amino groups, our material displayed dual hydrophilic property. In the enrichment of standard glycoprotein HRP digestion, the functional MOF material was shown to have low detection limit (1 fmol/μL) and good selectivity when the concentration of nonglycopeptides was 100 fold higher than the target N‐glycopeptides. All the results proved that MIL‐101(Cr)‐NH₂@PAMAM has great potential in the glycoproteome analysis.     

Tailored Electron Transfer Pathways in Aucore/Ptshell–Graphene Nanocatalysts for Fuel Cells

Au_core/Pt_shell–graphene catalysts (G‐Cys‐Au@Pt) are prepared through chemical and surface chemical reactions. Au–Pt core–shell nanoparticles (Au@Pt NPs) covalently immobilized on graphene (G) are efficient electrocatalysts in low‐temperature polymer electrolyte membrane fuel cells. The 9.5 ± 2 nm Au@Pt NPs with atomically thin Pt shells are attached on graphene via l ‐cysteine (Cys), which serves as linkers controlling NP loading and dispersion, enhancing the Au@Pt NP stability, and facilitating interfacial electron transfer. The increased activity of G‐Cys‐Au@Pt, compared to non‐chemically immobilized G‐Au@Pt and commercial platinum NPs catalyst (C‐Pt), is a result of (1) the tailored electron transfer pathways of covalent bonds integrating Au@Pt NPs into the graphene framework, and (2) synergetic electronic effects of atomically thin Pt shells on Au cores. Enhanced electrocatalytic oxidation of formic acid, methanol, and ethanol is observed as higher specific currents and increased stability of G‐Cys‐Au@Pt compared to G‐Au@Pt and C–Pt. Oxygen reduction on G‐Cys‐Au@Pt occurs at 25 mV lower potential and 43 A g_Pt^?1 higher current (at 0.9 V vs reversible hydrogen electrode) than for C–Pt. Functional tests in direct fomic acid, methanol and ethanol fuel cells exhibit 95%, 53%, and 107% increased power densities for G‐Cys‐Au@Pt over C–Pt, respectively.     

Selective isolation and analysis of glycoprotein fractions and their glycomes from hepatocellular carcinoma sera

As one of the most important post‐translational modifications, the discovery, isolation, and identification of glycoproteins are becoming increasingly important. In this study, a Con A‐magnetic particle conjugate‐based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and hepatocellular carcinoma (HCC) case sera. The isolated glycoproteins and their N‐linked glycans were identified by LC‐ESI‐MS/MS and MALDI‐TOF/TOF‐MS, respectively. A total of 93 glycoproteins from the healthy donors and 85 glycoproteins from the HCC cases were identified. There were 34 different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34). Twenty‐eight glycans from the healthy donors and 30 glycans from the HCC cases were detected and there were 22 different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Among these glycoproteins, 50 were known to be N‐linked glycoproteins and three novel glycopeptides from two predicted potential glycoproteins were discovered. Moreover, lectin blotting, Western blotting and lectin/glyco‐antibody microarrays were applied to definitely elucidate the change of selective protein expressions and their glycosylation levels, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.     

N‐glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm

Using cell surface capture technology, the cell surface N‐glycoproteome of human‐induced pluripotent stem cell derived hepatic endoderm cells was assessed. Altogether, 395 cell surface N‐glycoproteins were identified, represented by 1273 N‐glycopeptides. This study identified N‐glycoproteins that are not predicted to be localized to the cell surface and provides experimental data that assist in resolving ambiguous or incorrectly annotated transmembrane topology annotations. In a proof‐of‐concept analysis, combining these data with other cell surface proteome datasets is useful for identifying potentially cell type and lineage restricted markers and drug targets to advance the use of stem cell technologies for mechanistic developmental studies, disease modeling, drug discovery, and regenerative medicine.     

Electrochromatographic enantioseparation of amino acids using polybutylmethacrylate-based chiral monolithic column by capillary electrochromatography

This article describes the development of a polybutylmethacrylate‐based monolithic capillary column as a chiral stationary phase. The chiral monolithic column was prepared by polymerization of butyl methacrylate (BMA), ethylene dimethacrylate (EDMA), and N‐methacryloyl‐l ‐glutamic acid (MAGA) in the presence of porogens. The porogen mixture included N,N‐dimethyl formamide and phosphate buffer. MAGA was used as a chiral selector. The effect of MAGA content was investigated on electrochromatographic enantioseparation of d,l ‐histidine, d,l ‐tyrosine, d,l ‐phenyl alanine, and d,l ‐glutamic acid. The effect of acetonitrile (ACN) content in mobile phase on electro‐osmotic flow was also investigated. It was demonstrated that the poly(BMA‐EDMA‐MAGA) monolithic chiral column can be used for the electrochromatographic enantioseparation of amino acids by capillary electrochromatography (CEC). The mobile phase was ACN/10 mM phosphate buffer (45:55%) adjusted to pH 2.7. It was observed that l ‐enantiomers of the amino acids migrated before d ‐enantiomers. The separation mechanism of electrochromatographic enantioseparation of amino acids in CEC is discussed. Chirality 24:606–609, 2012. © 2012 Wiley Periodicals, Inc.     

GMDP: unusual physico‐chemical and biological properties of the anomeriс forms

Disaccharide containing unit of peptidoglycan from bacterial cell wall, N‐acetyl‐d ‐glucosaminyl‐N‐acetylmuramyl‐l ‐alanyl‐d ‐glutaminamide (gluсosaminyl‐muramyl‐dipeptide) registered in Russia as an immunomodulatory drug, is shown to participate in slow equilibrium of α and β anomeric forms. Data of NMR spectra and molecular dynamics indicate that the α‐anomer predominantly acquires a folded conformation stabilized by intramolecular hydrogen bond between the alanyl carbonyl and muramyl NH proton. The β‐form displays a considerable fraction of extended, non‐hydrogen bonded structures. In the standard immunoadjuvant test system, the α‐form is practically inactive, and the activity of the equilibrium mixture with α : β = 68 : 32 ratio is due to the presence of β‐anomer. Such unique α–β selectivity of biological action must be considered at the design of related immunoactive glycopeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.     

Prospecting Anticancer Compounds in Actinomycetes Recovered from the Sediments of Saint Peter and Saint Paul's Archipelago,Brazil

Saint Peter and Saint Paul's Archipelago is a collection of 15 islets and rocks remotely located in the equatorial Atlantic Ocean. In this particular site, the present project intended to assess the biodiversity and biotechnological potential of bacteria from the actinomycete group. This study presents the first results of this assessment. From 21 sediment samples, 268 strains were isolated and codified as BRA followed by three numbers. Of those, 94 strains were grown in liquid media and submitted to chemical extractions with AcOEt (A), BuOH (B), and MeOH (M). A total of 224 extracts were screened for their cytotoxic activity and 41 were significantly active against HCT‐116 cancer cells. The obtained IC₅₀ values ranged from 0.04 to 31.55 μg/ml. The HR‐LC/MS dereplication analysis of the active extracts showed the occurrence of several known anticancer compounds. Individual compounds, identified using HR‐MS combined with analysis of the AntiMarin database, included saliniketals A and B, piericidins A and C and glucopiericidin A, staurosporine, N‐methylstaurosporine, hydroxydimethyl‐staurosporine and N‐carbamoylstaurosporine, salinisporamycin A, and rifamycins S and B. BRA‐199, identified as Streptomyces sp., was submitted to bioassay‐guided fractionation, leading to isolation of the bioactive piericidins A and C, glucopiericidin, and three known diketopiperazines, cyclo(l ‐Phe‐trans‐4‐OH‐l ‐Pro), cyclo(l ‐Phe‐l ‐Pro), and cyclo(l ‐Trp‐l ‐Pro).     

Subcellular and anatomical distribution in rat brain of glycoproteins that contain mannose-rich heteropolysaccharide chains

Mannose-rich glycopeptides derived from brain glycoproteins were obtained by proteolysis of bovine brain tissue or subcellular fractions derived from rat brain tissue. The dialyzable mannose-rich glycopeptides were isolated by colum electrophoresis and gel flitration. These glycopeptides contained, on the average, six mannose and two N-acetylglucosamine residues with variable amounts of fucose and galactose. Over 50% of the mannose-rich glycopeptides of rat brain were localized in the microsomal and synaptosomal fractions; myelin and the soluble fraction contained lesser amounts. None was recovered from the mitochondria. The amount, per mg protein, of mannose-rich oligosaccharide chains in the myelin exceeded the concentration found in the microsomal and synaptosomal fractions. The concentration of mannose-rich glycopeptides derived from glycoproteins was 50% higher in white matter than in gray. On the other hand, the non-dialyzable and acidic sialoglycopeptides showed a three-fold enrichment in gray matter compared to white. The relatively lower ratio of sialoglycopeptides to mannose-rich glycopeptides observed in white matter (2.5) compared to gray matter (6.9) is reflected in the lower value for the ratio in myelin (1.1) compared to synpatosomes (2.1). Although glycoproteins that contain mannose-rich oligosaccharide chains are present in the nerve cell and its terminals, these glycoproteins appear to be relatively enriched in myelin and/or glial membranes.     

Distribution, developmental and stress responses of antioxidant metabolism in Malus

A comprehensive study was carried out to examine the interactions between the two major hydrophilic antioxidants l ‐ascorbate (vitamin C, l ‐AA), and glutathione (γ‐glutamyl cysteinylglycine, GSH), and other antioxidant pools in tissues of Malus, to identify factors affecting steady‐state cellular concentrations. We show that in Malus, each tissue type has a characteristic and different l ‐AA/GSH ratio and that in fruit, exocarp (epidermal) tissue acclimated to high light has higher l ‐AA levels but lower GSH levels than shaded (green) areas. Maturing seeds were characterized by the highest concentrations of GSH and a highly oxidized l ‐AA pool. It is demonstrated that fruit seeds are capable of l ‐AA biosynthesis, but that this occurs exclusively by means of the Smirnoff–Wheeler pathway. By contrast, foliar tissue was also able to synthesize l ‐AA using uronic acid substrates. Unlike the fruit of some other plant species however, the remaining fruit tissues are incapable of de novol ‐AA biosynthesis. The observed differences in the steady‐state concentrations of l ‐AA and GSH and the capacity to withstand stress in fruit, were also independent of the rates of uptake of photosynthate or of l ‐AA, but were correlated with the protective effect provided by phenolic compounds in these tissues. During development and maturation, l ‐AA and GSH levels in apple fruit declined steadily while foliar levels remained essentially constant throughout. However there was no apparent relationship between the free sugar contents of the fruit and antioxidant concentrations.     

Effect of pharynx epithelial cells surface desialylation on receptor‐mediated adherence of Staphylococcus aureus

Aims: To characterize the interaction between cell surface carbohydrates and Staphylococcus aureus. Methods and Results: In the present study, in vitro adherence of S. aureus to Detroit 562 cells, amount of cell surface desialylation and effect of subterminal monosaccharides on desialylated glycoproteins on adherence was studied with colony counting, HPLC, fluorescence microscopy and fluorometric techniques. According to our findings, S. aureus adherence to pharynx cells was enhanced (40%) after neuraminidase treatment, and neuraminidase also cleave great amount of Detroit 562 cells surface sialic acid (39–60%). Adherence assay with various monosaccharides‐pretreated bacteria, and lectin competitive inhibition, showed that the residual subterminal galactose, fucose and N‐acetyl‐d ‐glucosamine remaining on desialylated Detroit 562 cell surface glycoproteins responsible for this binding. Conclusion: The results are the first to show that galactose, fucose and N‐acetyl‐d ‐glucosamine remaining on desialylated pharynx cell surface glycoproteins serve as the adhesine receptors for S. aureus. Significance and Impact of the Study: This study may explain the predisposition of severe S. aureus pneumonia complication in respiratory viral infections.     

Cysteine inhibits amyloid fibrillation of lysozyme and directs the formation of small worm‐like aggregates through non‐covalent interactions

In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l ‐cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l ‐arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm‐like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non‐covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:470–478, 2014     

Screening for glucose‐triggered modifications of glutathione

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. These structures may undergo further Amadori rearrangement and free radical‐mediated oxidation to finally generate irreversible advanced glycation end products (AGEs). One of the factors known to modulate the glycation of proteins is glutathione, the most abundant nonprotein thiol tripeptide with the γ‐linkage, H‐Glu(Cys‐Gly‐OH)‐OH (GSH). Screening for products formed by GSH with D ‐glucose is an essential step in understanding the participation of GSH in glycation (the Maillard) reaction. Under the conditions used in these studies we observed N‐(1‐deoxy‐D ‐fructos‐1‐yl)‐pyroglutamic acid as the major glycation product formed in the mixtures of GSH and glucose in vitro. A RP HPLC/MS and tandem MS analyses of the GSH/glucose mixtures revealed that cleavage of the N‐terminal glutamic acid and the formation of pyroglutamic acid‐related Amadori product were accompanied by generation of Cys‐Gly‐derived Amadori and thiazolidine compounds. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of the peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) fromSilene alba cells

The peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992)Glycoconjugate J 9:191–97] was partially purified from culturedSilene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of unconjugated N-glycans in a suspension medium of culturedSilene alba cells.Abbreviations GlcNAc N-acetylglucosamine - PNGase peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase - BSA bovine serum albumin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TLC thin layer chromatography - HPAEC-PAD High pH anion exchange chromatography-pulsed amperometric detection     

Involvement of Nitric Oxide in UVB‐Induced Pigmentation in Guinea Pig Skin

Ultraviolet (UV) B irradiation evokes erythema and delayed pigmentation in skin, where a variety of toxic and modulating events are known to be involved. Nitric oxide (NO) is generated from l ‐arginine by NO synthases (NOS). Production of NO is enhanced in response to UVB‐stimulation and has an important role in the development of erythema. NO has recently been demonstrated as a melanogen which stimulates melanocytes in vitro, however, no known in vivo data has been reported to support this finding. In this study, we investigated the contribution of NO with UV‐induced pigmentation in an animal model using an NOS inhibitor. UVB‐induced erythema in guinea pig skin was reduced when an NOS inhibitor, l ‐NAME (N‐nitro‐ l ‐arginine methylester hydrochloride), was topically applied to the skin daily, beginning 3 days before UVB‐irradiation. Delayed pigmentation and an increased number of DOPA‐positive melanocytes in the skin were markedly suppressed by sequential daily treatment with l ‐NAME. Furthermore, melanin content 13 days after UVB‐irradiation was significantly lower in skin treated with l ‐NAME than in the controls. In contrast, d ‐NAME (N‐nitro‐ d ‐arginine methylester hydrochloride), an ineffective isomer of l ‐NAME, demonstrated no effect on these UV‐induced skin responses. These results suggest that NO production may contribute to the regulation of UVB‐induced pigmentation.     

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.