Determination Of Monosaccharide Composition Research Articles

Extraction Optimization, Structural Analysis, and Potential Bioactivities of a Novel Polysaccharide from Sporisorium reilianum.

Sporisorium reilianum is an important biotrophic pathogen that causes head smut disease. Polysaccharides extracted from diseased sorghum heads by Sporisorium reilianum exhibit significant medicinal and edible value. However, the structure and biological activities of these novel polysaccharides have not been explored. In this study, a novel polysaccharide (WM-NP'-60) was isolated and purified from the fruit bodies of S. reilianum and aimed to explore the structural characteristics and substantial antioxidant and antitumor properties of WM-NP'-60. Monosaccharide composition determination, periodate oxidation-Smith degradation, 1D/2D-NMR analysis, and methylation analysis revealed that WM-NP'-60 consisted mainly of β-1,6-D-Glcp, β-1,3-D-Glcp, and β-1,3,6-D-Glcp linkages. The antioxidant assays demonstrated that WM-NP'-60 exhibited great activities, including scavenging free radicals, chelating ferrous ions, and eliminating reactive oxygen species (ROS) within cells. The HepG2, SGC7901, and HCT116 cells examined by transmission electron microscopy (TEM) revealed typical apoptotic bodies. Therefore, a novel fungal polysaccharide (WM-NP'-60) was discovered, extracted, and purified in this experiment, with the aim of providing a reference for the development of a new generation of food and nutraceutical products suitable for human consumption.

Exopolysaccharide from Lacticaseibacillus paracasei alleviates gastritis in Helicobacter pylori-infected mice by regulating gastric microbiota.

Many probiotics have the ability to produce extracellular polysaccharides (EPS). EPS derived from these probiotics has been confirmed to regulate the host intestinal microecological balance and alleviate the symptoms of diseases caused by gastrointestinal microecological imbalance. Lactic acid bacteria (LAB) strain with good exopolysaccharide (EPS) producing ability, namely, Lacticaseibacillus paracasei ZFM54 (L. paracasei ZFM54) was screened. The fermentation conditions of L. paracasei ZFM54 for EPS production were optimized. The EPS54 was characterized by chemical component and monosaccharide composition determination, UV, FT-IR and NMR spectra analysis. Cango red, SEM, AFM and XRD analysis were conducted to characterize the structure of EPS54. The EPS54 effectively reduced the colonization of Helicobacter pylori to AGS cells and recovered the cell morphology. EPS54 could also effectively alleviate the gastritis in the H. pylori-infected mice by down-regulating the mRNA expression levels of pro-inflammatory cytokines IL-6, IL-8, IL-1β and TNF-α and up-regulating the mRNA expression of inflammatory cytokine IL-10 in gastric cells. EPS54 was also found to be able to positively regulate the structure of gastric microbiota. The EPS 54 from L. paracasei ZFM54 can alleviate gastritis in H. pylori-infected mice by modulating the gastric microbiota.

Cell wall polysaccharide and glycoprotein content tracks growth-form diversity and an aridity gradient in the leaf-succulent genus Crassula.

Cell wall traits are believed to be a key component of the succulent syndrome, an adaptive syndrome to drought, yet the variability of such traits remains largely unknown. In this study, we surveyed the leaf polysaccharide and glycoprotein composition in a wide sampling of Crassula species that occur naturally along an aridity gradient in southern Africa, and we interpreted its adaptive significance in relation to growth form and arid adaptation. To study the glycomic diversity, we sampled leaf material from 56 Crassula taxa and performed comprehensive microarray polymer profiling to obtain the relative content of cell wall polysaccharides and glycoproteins. This analysis was complemented by the determination of monosaccharide composition and immunolocalization in leaf sections using glycan-targeting antibodies. We found that compact and non-compact Crassula species occupy distinct phenotypic spaces in terms of leaf glycomics, particularly in regard to rhamnogalacturonan I, its arabinan side chains, and arabinogalactan proteins (AGPs). Moreover, these cell wall components also correlated positively with increasing aridity, which suggests that they are likely advantageous in terms of arid adaptation. These differences point to compact Crassula species having more elastic cell walls with plasticizing properties, which can be interpreted as an adaptation toward increased drought resistance. Furthermore, we report an intracellular pool of AGPs associated with oil bodies and calcium oxalate crystals, which could be a peculiarity of Crassula and could be linked to increased drought resistance. Our results indicate that glycomics may be underlying arid adaptation and drought resistance in succulent plants.

Regulatory Effects Mediated by Enteromorpha prolifera Polysaccharide and Its Zn(II) Complex on Hypoglycemic Activity in High-Sugar High-Fat Diet-Fed Mice.

In order to investigate and develop functional foods of marine origin with hypoglycemic activity, Enteromorpha prolifera polysaccharide-Zn(II) (EZ) complex was first prepared by marine resourced E. prolifera polysaccharide (EP) and ZnSO4 and their anti-diabetes activities against high-sugar and high-fat-induced diabetic mice were evaluated. The detailed structural characterization of EZ was elucidated by UV-Vis spectroscopy, infrared spectroscopy, and monosaccharide composition determination. The pharmacological research suggests that EZ has a potent hypoglycemic effect on high-sugar and high-fat-induced diabetic mice by inhibiting insulin resistance, improving dyslipidemia, decreasing inflammatory status, repairing pancreas damage, as well as activating the IRS/PI3K/AKT signaling pathway and regulating GLUT2 gene expression. At the same time, microbiota analysis indicates that a high dose of EZ could enhance the abundance of dominant species, such as Staphylococcaceae, Planococcaceae, Muribaculaceae, Aerococcaceae, and Lacrobacillaceae, in intestinal microbiota distribution. Thus, EZ could be considered as a potential candidate for developing an ingredient of functional foods for Zn(II) supplements with hypoglycemic activity.

Structural characterizations and α-glucosidase inhibitory activities of four Lepidium meyenii polysaccharides with different molecular weights

Four polysaccharides (MCPa, MCPb, MCPc, MCPd) were obtained from Lepidium meyenii Walp. Their structures were characterized by chemical and instrumental methods including total sugar, uronic acid and protein content determination, UV, IR and NMR spectroscopy, as well as monosaccharide composition determination and methylation analyses. Four polysaccharides were a group of glucans with different molecular weights ranging from 3.12 to 14.4 kDa, and shared a similar backbone chain consisting of (1→4)-glucose linkages with branches attached to C-3 and C-6. Furthermore, bioactivity assay showed that MCPs had concentration-dependent inhibitory activity on α-glucosidase. MCPb (Mw = 10.1 kDa) and MCPc (Mw = 5.62 kDa) with moderate molecular weights exhibited higher inhibitory activity compared with MCPa and MCPd.Graphical

Determination of monosaccharide composition in human serum by an improved HPLC method and its application as candidate biomarkers for endometrial cancer

Altered glycan levels in serum have been associated with increased risk of cancer. In this study, we have developed and validated a HPLC-based method to analyze monosaccharide composition (D-mannose, Glucosamine, Galactosamine, Glucuronic acid, D-glucose, D-galactose, D-xylose, L-fucose) in human serum, with L-rhamnose, being used as internal standard. Monosaccharides obtained from hydrolyzed serum samples were derivatized by 1-Phenyl-3-methyl-5-pyrazolone. A ZORBAX XDB-C18 column(150×4.6mm) was used for chromatographic separation with 100 mM ammonium acetate buffer (NH4Ac-HAc, PH=5.5, solvent A), acetonitrile (ACN, solvent B) as a mobile phase. The calibration standard curves for the eight monosaccharides showed good linearity over the range of 2.5-500μg/mL with R2 > 0.995. The relative standard deviation values for intra-day and inter-day precision were ≤ 5.49%. Recovery was 69.01-108.96%. We observed that this column exhibited high specificity and selectivity to separate monosaccharides from serum. This method was then applied to quantitatively analyze the serum monosaccharide levels in 30 patients with endometrial cancer and 30 matched healthy controls. Statistical analysis indicated that the serum monosaccharide levels were significantly higher in patients compared with healthy controls (P value< 0.0001). Overall, we report here a simple, reliable, low-cost, and reproducible HPLC method for the separation and quantification monosaccharides in the human serum, which has potential value to serve as a screening marker for endometrial cancer.

The Effect of Sugarcane Straw Aging in the Field on Cell Wall Composition.

Cellulosic ethanol is an alternative for increasing the amount of bioethanol production in the world. In Brazil, sugarcane leads the bioethanol production, and to improve its yield, besides bagasse, sugarcane straw is a possible feedstock. However, the process that leads to cell wall disassembly under field conditions is unknown, and understanding how this happens can improve sugarcane biorefinery and soil quality. In the present work, we aimed at studying how sugarcane straw is degraded in the field after 3, 6, 9, and 12 months. Non-structural and structural carbohydrates, lignin content, ash, and cellulose crystallinity were analyzed. The cell wall composition was determined by cell wall fractionation and determination of monosaccharide composition. Non-structural carbohydrates degraded quickly during the first 3 months in the field. Pectins and lignin remained in the plant waste for up to 12 months, while the hemicelluloses and cellulose decreased 7.4 and 12.4%, respectively. Changes in monosaccharide compositions indicated solubilization of arabinoxylan (xylose and arabinose) and β-glucans (β-1,3 1,4 glucan; after 3 months) followed by degradation of cellulose (after 6 months). Despite cellulose reduction, the xylose:glucose ratio increased, suggesting that glucose is consumed faster than xylose. The degradation and solubilization of the cell wall polysaccharides concomitantly increased the level of compounds related to recalcitrance, which led to a reduction in saccharification and an increase in minerals and ash contents. Cellulose crystallinity changed little, with evidence of silica at the latter stages, indicating mineralization of the material. Our data suggest that for better soil mineralization, sugarcane straw must stay in the field for over 1 year. Alternatively, for bioenergy purposes, straw should be used in less than 3 months.

Determination of monosaccharides in Lycium barbarum fruit polysaccharide by an efficient UHPLC-QTRAP-MS/MS method.

Pre-column derivatisation using 1-phenyl-3-methyl-5-pyrazolone (PMP) is a common method for monosaccharide determination. Herein, a specific ultra-high-performance liquid chromatography quadrupole trap tandem mass spectrometry (UHPLC-QTRAP-MS/MS) method was developed and used for the determination of monosaccharide composition of Lycium barbarum polysaccharide (LBP). Exploration of a new efficient method for monosaccharide determination. In our study, hydrochloric acid was used to hydrolyse the polysaccharide obtained from L. barbarum fruits. Principal component analysis (PCA) and technique for order of preference by similarity to ideal solution (TOPSIS) analysis were used for comprehensive evaluation of the samples. The results showed that LBP was composed of seven monosaccharides: galactose, arabinose, mannose, rhamnose, xylose, ribose, and glucose. The linear relationship of the seven monosaccharides was optimum within a certain concentration range. Quantitative recoveries of the seven monosaccharides from the samples ranged from 94.76% to 102.11%. A rapid quantitative detection method was established, in which the hydrolysis time was reduced from 12h to 2h. By using LBP as one of the indexes to evaluate the quality of L. barbarum, L. barbarum from Zhongning County, Ningxia Province, was identified as having the best quality among the varieties tested. The UHPLC-QTRAP-MS/MS pre-column derivatisation method used in this study was simple, accurate, and sensitive, with good repeatability, and can be used for quality evaluation and origin distinction of L. barbarum and other medicinal plants.

Determination of monosaccharide composition of kefiran using HPLC

Kefiran is an exopolysaccharide (EPS) produced by specific microorganisms in the kefir grain. In this research, kefir grain was activated in various carbon sources (glucose, galactose or lactose) containing medium. The monosaccharides composition of the formed kefiran were determined by a high performance liquid chromatography (HPLC). HPLC with refractive index (RI) detector was used in order to make qualitative and quantitative analysis of sucrose, glucose, galactose, arabinose, xylose, and ribose present in kefiran. Also, the physical properties of differently kefiran formed were determined. The amount of kefiran in the control group and in the sample from the galactose containing medium were determined to be 511.00 and 610.55 mg/L, respectively. The kefiran form obtained from the medium containing galactose was more positively affected than in other media. The apparent viscosity, at a shear rate of 66.0 s-1of the kefiran produced in glucose containing medium, was determined to be 29.2 mPas. The surface morphologies of different kefirans were analysed and roughness parameters were determined by atomic force microscopy (AFM); the maximum height of lumps ranged from 16.80 to 22.10 nm. In this study, of sucrose, glucose, galactose, arabinose, xylose, and ribose were found in sugar composition in all kefiran samples.

Extraction of a novel water-soluble gum from nettle (Urtica dioica) seeds: Optimization and characterization

Nettle seed gum (NSG) which is a novel potential source of hydrocolloid was characterized in terms of yield, physicochemical, rheological, functional and thermal properties. According to Response Surface Methodology, the maximum extraction yield was determined as 6.17% on dry basis. In order to clarify the structural information of the gum, the FT-IR analysis and monosaccharide composition determination were performed. In the results, the NSG had 8.48% moisture, 10.81% total ash, 2.89% total protein, 1.15% total oil and 76.67% total carbohydrate. The FT-IR analysis revealed the existence of carboxyl groups, which gives the gum the capability of ion binding. Furthermore, Mannose/Galactose ratio was calculated as 1.07, indicating the water solubility of gum. DSC analysis showed that the NSG was highly stable in thermal process. Intrinsic viscosity, average viscosity and molecular weight values were determined as 8.56 ± 0.4 dL/g and 1.89 ± 0.1 × 106 g/mol, respectively. The emulsification capacity, emulsion stability and foam formation capacity of the NSG were 48%, 40% and 4%, respectively. The NSG exhibited shear thinning behavior while dynamic rheological analysis indicated the NSG had viscoelastic properties and G′ (the storage modulus) was higher than G″ (the loss modulus). In conclusion, the present study supplied novel knowledge on the NSG, which will be inestimable for clarifying its matchless functional properties.

Variation of Chemical and Morphological Properties of Different Parts of Prunus Amygdalus L. and Their Effects on Pulping

Chemical and morphological of Prunus amygdalus L. characterization (shells and stems) were studied in order to assess their potentiality for chemical pulp. In this work, a simple, rapid and sensitive analytical method for the determination of monosaccharide composition in almond shells and stems polysaccharide was developed and validated. The soda-anthraquinone process was also applied in order to evaluate pulp properties. The effects of delignification and bleaching process were statistically investigated by employing response surface methodology. The isolated fibers were investigated and characterized using several techniques such as gel permeation chromatography, X-ray diffraction, morfi analyzer and scanning electron microscope. The obtained results can be considered as a potential source of cellulose for the production of cellulose derivatives, cellulose pulp for papermaking applications and natural fibres reinforced polymer composite.

Characterization of nutrient status of Halamphora luciae (Bacillariophyceae) using matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)

ABSTRACTThe effects of N and P depletion on the production and structural characterization of the cellular carbohydrate polymers of the estuarine diatom Halamphora luciae in batch culture were examined using matrix-assisted laser desorption-ionization time-of flight mass spectrometry (MALDI-TOF MS) complemented with monosaccharide composition determination and structural analyses by methylation of aqueous extracted product. The MALDI MS analysis of the cells showed a similar profile in control and N- and P-depleted media, with a displacement to higher molecular weight for cells grown in depleted media. In the monosaccharide analyses, both nutrient depletion and culture ageing led to an increase in glucose content, indicating that MALDI-TOF MS in whole cells was detecting the changes in chrysolaminarin. The maxima for the ions from f/2-P and to a lesser extent in f/2-N were displaced to higher m/z values indicating a higher degree of polymerization (DP). Methylation analysis confirmed the presence of chrysolaminarin, a (1→3)-β-d-glucan with branching in C2 and C6, where the glucan backbone had a substitution every four glucose residues. The (1→3)-β-d-glucan was also detected in the cingule by fluorescence with aniline blue.

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics.

The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes.

Ion-exchange chromatography combined with direct current amperometric detection at CuNPs/reduced graphene oxide–chitosan composite film modified electrode for determination of monosaccharide composition of polysaccharides from Phellinus igniarius

A novel Cu nanoparticles/reduced graphene oxide-chitosan (CuNPs/r-GO-chitosan) composite film modified glassy carbon electrode (GCE) was fabricated by dispersing CuNPs uniformly on a stable r-GO-chitosan thin film through electrodeposition process. The modified electrode was characterized by cyclic voltammetry, scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS), and exhibited efficiently electrocatalytic oxidation toward monosaccharides with high stability. The good electrocatalytic activity of this modified electrode might be attributed to the synergistic effect of r-GO and CuNPs, and the stability might be attributed to the r-GO and chitosan thin matrix film. When the CuNPs/r-GO-chitosan/GCE was used as an electrochemical sensor in high performance anion exchange chromatography-direct current amperometric detection (HPAEC-DC) flowing system for the determination of monosaccharides under constant working potential of +0.55 V, the detection limits (S/N=3) ranged from 0.006 to 0.02 mg L(-1) for the analyzed sugars, and the dynamic linear ranges spanned from 0.02 to 500 mg L(-1). The proposed method has been applied for the determination of monosaccharide composition of crude polysaccharides from phellinus igniarius real samples, and the results were satisfactory.

Analysis of monosaccharide composition of Cyclocarya paliurus polysaccharide with anion exchange chromatography

A simple, rapid and sensitive analytical method for the determination of monosaccharide composition in Cyclocarya paliurus polysaccharide was developed and validated. This method was based on hydrolysis of the polysaccharides followed by high-performance anion-exchange chromatography analysis. The effects of sodium hydroxide concentration and column temperature on retention and separation of the monosaccharide were investigated with Carbo PAC™ PA10 analytical column. The established method was validated and the results showed that this method had good linearity (R2, 0.9987–0.9999), adequate accuracy (98.53–102.13% recovery), high precision (relative standard deviation <3.8%) and sensitive detection limits (2.57–7.86nM), with a simple preparation of the samples (no need to derivatize the samples) and short run time (20min). The results showed that the C. paliurus polysaccharide consisted of rhamnose, arabinose, galactose, glucose, mannose and xylose in the molar ratio of 1.00:1.85:3.26:3.12:0.85:0.29.

Development and application of a rapid and efficient CZE method coupled with correction factors for determination of monosaccharide composition of acidic hetero‐polysaccharides from Ephedra sinica

Ephedrine alkaloids cannot account for all the effects of Ephedra sinica and the polysaccharides are also demonstrated to be one of the main bioactive constituents of E. sinica. However, no work has been reported on the analysis of monosaccharide composition of purified polysaccharides isolated from the stem of E. sinica. To develop a rapid and efficient capillary zone electrophoresis (CZE) method based on pre-column derivatisation with 1-phenyl-3-methyl-5-pyrazolone for the simultaneous determination of neutral and acidic sugars of purified polysaccharides from E. sinica. Three polysaccharides (ESP-A3, ESP-A4 and ESP-B4) were isolated and purified by ion exchange and gel-filtration chromatography from the stem of E. sinica. The effects of background electrolyte pH and concentration, applied voltage and temperature on the separation were investigated. Meanwhile, factors affecting the hydrolysis of ESP-B4 with sulphuric acid were investigated by changing the hydrolysis time, acid concentration and hydrolytic temperature to achieve complete hydrolysis. The standard curves coupled with correction factors were used to calculate molar ratios. The optimal CZE method coupled with correction factors was successfully applied to the determination of molar ratios of three purified polysaccharides and their corresponding partial acid hydrolysis products. ESP-A3, ESP-A4 and ESP-B4 were all typical acidic hetero-polysaccharides and consisted of xylose, arabinose, glucose, rhamnose, mannose, galactose, glucuronic acid and galacturonic acid, and their corresponding molar ratios were 6.8:7.5:1.0:14.0:13.7:22.3:10.2:3.8 for ESP-A3, 1.2:4.1:1.0:5.1:1.6:17.3:3.1:2.2 for ESP-A4, and 1.0:4.5:1.0:2.0:1.0:5.5:1.5:50.0 for ESP-B4. The results provided scientific evidence for the further study of the structure and bioactivity of complex acidic E. sinica polysaccharides.

O-polysaccharide structure in serogroup I azospirilla

Lipopolysaccharides (LPS) and O-specific polysaccharides (OPS) were obtained from the outer membrane of four Azospirillum strains previously assigned to serogroup I based on the serological affinity revealed by the antibodies (AB) to the LPS of A. brasilense Sp245. Investigation, including determination of monosaccharide composition, methylation analysis, and one- and two-dimensional NMR spectroscopy, was carried out to determine the OPS structure. The OPSs of A. brasilense Sp107 and S27 and of A. lipoferum RG20a were found to have an identical structure of repeating units represented by a linear penta-D-rhamnan, as was previously described for the OPSs of A. brasilense Sp245 and SR75. The OPS of A. brasilense SR15 was found to consist of tetrasaccharide repeating units of the following structure: → 2)-α-D-Rhap-(1 → 2)-β-D-Rhap-(1 → 3)-α-D-Rhap-(1 → 2)-α-D-Rhap-(1 →. An opine compound, Nδ-(1-carboxyethyl)-ornithine, closely associated with the LPS of A. brasilense SR15, was identified in azospirilla for the first time. The presence of a 6-deoxisugar (D-rhamnose) in the OPS structure was shown to be the chemical basis of the serological similarity and the reason for classification of these strains within the serogroup I.

Evaluation of oligosaccharide methods for carbohydrate analysis in a fully human monoclonal antibody and comparison of the results to the monosaccharide composition determination by a novel calculation

Carbohydrates can change a drug's properties including solubility, affinity towards antigen, pharmacokinetics and pharmacodynamics. Due to this importance, carbohydrate composition is utilized as a parameter to evaluate a drug candidate's quality. In this study, the compositional monosaccharides of a drug candidate are measured by HPAEC-PAD, while the oligosaccharides are studied by HPAEC-PAD, CE-LIF and LC–MS. The advantages and limitations of these various approaches for oligosaccharide analysis are reviewed in this work. While the methods used for oligosaccharide analysis are well established we have devised a new and novel calculation for determining monosaccharide content using the relative percentages of the N-glycans. This calculation was used to evaluate the accuracy of the oligosaccharide determination methods by comparison of the N-glycan data to the experimental monosaccharide data. The results obtained from this novel calculation demonstrate that the relative abundance of carbohydrates as determined from these various approaches are consistent.

Determination of monosaccharide composition in plant fiber materials by capillary zone electrophoresis

The neutral sugar composition of acid hydrolyzed extracts of cellulose fiber samples, i.e. oat spelt, wheat straw, thermomechanica pulp (TMP) made of spruce, aspen stemwood, and bleached birch kraft pulp, was determined by a new capillary zone electrophoresis (CZE) method employing an alkaline background electrolyte. The method relies on in-capillary reaction and direct UV detection at wavelength 270 nm. Neutral carbohydrates d-(+)-galactose, d-(+)-glucose, l-rhamnose, d-(+)-mannose, d-(−)-arabinose, and d-(+)-xylose were simultaneously separated. The calibration plots were linear over a range from 10 to 150 mg/L for d-(+)-galactose, l-rhamnose, d-(+)-mannose, and d-(−)-arabinose and from 50 to 400 mg/L for d-(+)-glucose and d-(+)-xylose. Relative standard deviations (RSDs) of peak areas during a 5-day analysis period varied from 3.3% for galactose to 11.8% for rhamnose. RSDs of migration times varied between 0.3 and 0.7%. The detection limit (at S/N 3) was 5 mg/L for each monosaccharide. The results obtained by CZE agreed well with results obtained by high-performance anion-exchange chromatography. Glucose and xylose were the two predominant monosaccharides in the plants, except in the spruce TMP sample where glucose and mannose dominated.

Nucleolin Undergoes Partial N- and O-Glycosylations in the Extranuclear Cell Compartment

Nucleolin is an ubiquitous, nonhistone nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation, and growth. Nucleolin was primarily found in the nucleus, but it was also proposed as a possible shuttle between the nucleus, cytoplasm, and cell membrane. We report here that part of the extranuclear nucleolin undergoes complex N- and O-glycosylations. A band with higher molecular mass (113 kDa) than the 105-kDa classical major nucleolin band was detected on SDS-PAGE gel that cross-reacted with specific anti-nucleolin antibodies and was identified as a nucleolin isoform by mass spectrometry. The presence of N-glycans was first suggested by sensibility of the 113-kDa nucleolin isoform to tunicamycin treatment. Determination of monosaccharide composition by heptafluorobutyrate derivation followed by gas-chromatography mass spectrometry indicated the presence of N- and O-glycans. The structures of N- and O-glycans were first investigated using specificity of binding to lectins. This approach allowed a partial characterization of N-glycan structures and revealed O-glycan structures that could otherwise go unnoticed. Further study of N-glycans by mass spectrometry using direct exoglycosidase treatment on MALDI-TOF target allowed the complete definition of their structures. Finally, the use of peptide mass fingerprinting with sinapinic acid allowed identification of N317 and N492 as the two N-glycosylation sites. N317 and N492 belong to RNA-binding domains 1 and 3 of nucleolin, respectively, that suggests a role of glycosylation in regulating the function of the protein.