A versatile soluble siglec scaffold for sensitive and quantitative detection of glycan ligands.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. The connections between Siglecs and human disease motivate improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands are systematically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs are clarified. For CD33 (Siglec-3), we demonstrate that it recognizes both α2-3 and α2-6 sialosides in solution and on cells, which has implications for its link to Alzheimer's disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.

Mass Spectrometry-Based Shotgun Glycomics for Discovery of Natural Ligands of Glycan-Binding Proteins.

Glycans attached to lipids and membrane-bound and secreted proteins and peptides mediate many important physiological and pathophysiological processes through interactions with glycan-binding proteins (GBPs). However, uncovering functional glycan ligands is challenging due to the large number of naturally occurring glycan structures, the limited availability of glycans in their purified form, the low affinities of GBP-glycan interactions, and limitations in existing binding assays. This work explores the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening libraries of N-glycans derived from natural sources. The assay was tested by screening a small-defined library of complex N-glycans at equimolar concentrations against plant and human GBPs with known specificities for either α2-3- or α2-6-linked sialosides, with affinities in the millimolar to micromolar range. Validation experiments, performed in negative ion mode, revealed that bound N-glycan ligands are readily released, as intact deprotonated ions, from GBPs in the gas phase using collision-induced dissociation. Moreover, the relative abundances of the released ligands closely match their solution affinities. The results obtained for a natural N-glycan library produced from cultured immune cells serve to highlight the ease with which CaR-ESI-MS can screen complex mixtures of N-glycans for interactions. Additionally, scaling the relative abundances of released glycan ligands according to their relative abundances in solution, as determined by hydrophilic interaction-ultrahigh-performance liquid chromatography of the fluorescently labeled library, allows the relative affinities of glycan ligands to be ranked.

How normative multiculturalism relates to immigrant well-being.

OBJECTIVES: In this study we examine the relationship between contextual factors, that is, perceived multicultural norms, and immigrant well-being. Specifically, we test a model whereby each of the three dimensions of normative multiculturalism, perceived Multicultural Ideology, Multicultural Policies and Practices, and Multicultural Contact, positively predicts immigrant well-being both directly and indirectly via belongingness. METHOD: Korean immigrants in New Zealand (N = 306, 56% female) participated in the research. Their average age was 31.17 (SD = 10.46), and the average length of residence was 10.04 years (SD = 7.21). Participants completed a survey that included the Normative Multiculturalism Scale along with measures of belonging and well-being (flourishing, life satisfaction, and positive affect). RESULTS: Structural equation modeling showed that perceived normative Multicultural Policies and Practices exerted a direct positive effect on well-being and an indirect positive effect via belongingness; Multicultural Ideology exerted only an indirect effect; and Multicultural Contact did not significantly relate to belongingness or subjective well-being. IMPLICATIONS: The results are discussed in terms of everyday experiences of intercultural encounters, social norms and the contextual influences of diversity climates, as well as the importance of distinguishing the defining features of multiculturalism in diversity science research. We also propose that multicultural norm setting and norms marketing may lead to positive social and psychological outcomes for immigrants. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Profiles of plant core-fucosylated N-glycans of acid alpha-glucosidases produced in transgenic rice cell suspension cultures treated with eight different conditions.

Recombinant human acid alpha-glucosidase (rhGAA) from Chinese hamster ovary cells is the only approved treatment for patients with Pompe disease. In this study, rhGAAs were produced in transgenic rice cell suspension cultures under eight different conditions; untreated, 5 µM of 2-fluoro-l-fucose (2-FF), 50 µM of 2-FF, 100 µM of 2-FF, 100 µM of 2-FF + 0.5% Pluronic F-68 (PF-68), 100 µM of 2-FF + 0.05% Tween 20 (Tw 20), 0.5% PF-68, and 0.05% Tw 20. The N-glycans of eight rhGAAs were analyzed using ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry. The relative quantity (%) of each glycan was obtained from the corresponding UPLC peak area per the sum (100%) of individual UPLC peak area. Fifteen N-glycans, comprising seven core-fucosylated glycans (71.5%, sum of each relative quantities) that have immunogenicity-inducing potential, three de-core-fucosylated glycans (15.4%), and five non-core-fucosylated glycans (13.1%), were characterized with high mass accuracy and glycan-generated fragment ions. The increases or decreases of relative quantities of each glycan from seven rhGAAs were compared with those of untreated control. The percentages of the sum of the relative quantities of core-fucosylated glycans divided by the sums of those of de-core- (core-fucose removed) and non-core-fucosylated glycans were calculated, and the lowest percentage was obtained in 100 µM of 2-FF combined with 0.5% PF-68. These results indicate that the relative quantity of each glycan of rhGAA produced in rice cell suspension cultures is significantly affected by their culture condition. This study performed the comparison of the N-glycan profiles of rice cell-derived rhGAA to identify the core-fucosylated glycans using UPLC and tandem mass spectrometry.

Seventeen O-acetylated N-glycans and six O-acetylation sites of Myozyme identified using liquid chromatography-tandem mass spectrometry.

O-acetylated sialic acid (SA) attached to the N-glycans of therapeutic glycoproteins reportedly inhibit sialidase activity, increase protein half-life, decrease protein antigenicity, and stabilize protein conformation. Recombinant human acid α-glucosidase (Myozyme) is the only drug approved by the United States Food and Drug Administration for the treatment of Pompe disease. In this study, unreported N-glycans containing O-acetylated SA in Myozyme and the relative quantities of total glycans were investigated using liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The 17 N-glycans (6.4% of total glycans) containing mono-, di-, mono/di-, and di/di-O-acetylated N-acetylneuraminic acid (Neu5Ac) were identified with mass accuracy, glycan-generated fragment ions, and the retention time on an LC column. The analysis of peptides containing mono- and/or di-O-acetylated Neu5Ac ions sorted from all peptides using nano-LC-ESI-HCD-MS/MS confirmed six O-acetylation sites (Asn 140, Asn 233, Asn 390, Asn 470, Asn 652, and Asn 882), at least five of which (Asn 140, Asn 233, Asn 390, Asn 470, and Asn 652) could contribute to the drug efficacy or cellular uptake of Myozyme. This is the first study to identify N-glycans containing O-acetylated Neu5Ac and O-acetylation sites in Myozyme.

Production of recombinant human acid ß-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease.

Gaucher disease is an inherited metabolic disease caused by genetic acid ß -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues. In this study, we introduced a gene encoding recombinant human GBA into both wild-type rice (WT) and rice gnt1 calli. Target gene integration and mRNA expression were confirmed by genomic DNA PCR and Northern blotting, respectively. Secreted rhGBAs in culture media from cell lines originating from both WT (WT-GBA) and rice gnt1 (gnt1-GBA) were detected by Western blotting. Each rhGBA was purified by affinity and ion exchange chromatography. In vitro catalytic activity of purified rhGBA was comparable to commercial Chinese hamster ovary cell-derived rhGBA. N-glycans were isolated from WT-GBA and gnt1-GBA and analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The amounts of high mannose-type N-glycans were highly elevated in gnt1-GBA (100%) compared to WT-GBA (1%).

Multipronged ESI-MS Approach for Studying Glycan-Binding Protein Interactions with Glycoproteins.

A multipronged electrospray ionization mass spectrometry (ESI-MS) approach for investigating glycan-mediated interactions between water-soluble glycan-binding proteins (GBPs) and glycoproteins (GPs) is described. First, the catch-and-release (CaR)-ESI-MS assay, carried with ion mobility separation prior to GBP "release" (i.e., CaRIMS-ESI-MS), is employed to rapidly identify GBP-GP binding in solution. The apparent affinity ( Ka) of the GBP for the GP is then measured using the competitive proxy ligand-ESI-MS binding assay. Finally, N-glycans, enzymatically released as free oligosaccharides from the GP, are screened against the GBP using ESI-MS to identify the glycans that are recognized by the GBP. Measurements performed at multiple GBP concentrations allow for the affinities of released N-glycans (grouped as compositional isomers) to be ranked. Implementation of the approach is illustrated using the known interactions between a C-terminal domain fragment of human galectin-3 (hGal-3C) and three human serum GPs, α-1-acid glycoprotein (AGP), haptoglobin phenotype 1-1 (Hp1-1) and α-2-macroglobulin (α2M). Specific binding of hGal-3C to each GP was successfully detected by CaRIMS-ESI-MS; no binding was detected for a noninteracting reference protein, which served as a negative control. The Ka measured by proxy ligand-ESI-MS for AGP, Hp1-1 and α2M (4 × 105 M-1, 2 × 105 M-1 and 3 × 105 M-1, respectively) are consistent with the reported apparent affinities of hGal-3 for serum GPs and their N-glycans. Screening the N-glycan libraries of each of the GPs against hGal-3C identified ligands corresponding to a total of 20 different saccharide compositions with sialylated bi-, tri-, and tetra-antennary structures. The results of binding measurements performed at two different hGal-3C concentrations revealed that all of the N-glycan ligands exhibit affinities ≥104 M-1.

N-glycan Remodeling Using Mannosidase Inhibitors to Increase High-mannose Glycans on Acid α-Glucosidase in Transgenic Rice Cell Cultures.

Glycoengineering of plant expression systems is a prerequisite for the production of biopharmaceuticals that are compatible with animal-derived glycoproteins. Large amounts of high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9), which can be favorably modified by chemical conjugation of mannose-6-phosphate, are desirable for lysosomal enzyme targeting. This study proposed a rice cell-based glycoengineering strategy using two different mannosidase inhibitors, kifunensine (KIF) and swainsonine (SWA), to increase Man7/8/9 glycoforms of recombinant human acid α-glucosidase (rhGAA), which is a therapeutic enzyme for Pompe disease. Response surface methodology was used to investigate the effects of the mannosidase inhibitors and to evaluate the synergistic effect of glycoengineering on rhGAA. Both inhibitors suppressed formation of plant-specific complex and paucimannose type N-glycans. SWA increased hybrid type glycans while KIF significantly increased Man7/8/9. Interestingly, the combination of KIF and SWA more effectively enhanced synthesis of Man7/8/9, especially Man9, than KIF alone. These changes show that SWA in combination with KIF more efficiently inhibited ER α-mannosidase II, resulting in a synergistic effect on synthesis of Man7/8/9. In conclusion, combined KIF and SWA treatment in rice cell culture media can be an effective method for the production of rhGAA displaying dominantly Man7/8/9 glycoforms without genetic manipulation of glycosylation.

Four unreported types of glycans containing mannose-6-phosphate are heterogeneously attached at three sites (including newly found Asn 233) to recombinant human acid alpha-glucosidase that is the only approved treatment for Pompe disease.

Myozyme is a recombinant human acid alpha-glucosidase (rhGAA) that is currently the only drug approved for treating Pompe disease, and its low efficacy means that a high dose is required. Mannose-6-phosphate (M6P) glycosylation on rhGAA is a key factor influencing lysosomal enzyme targeting and the efficacy of enzyme replacement therapy (ERT); however, its complex structure and relatively small quantity still remain to be characterized. This study investigated M6P glycosylation on rhGAA using liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The glycans released from rhGAA were labeled with procainamide to improve mass ionization efficiency and the sensitivity of MS/MS. The relative quantities (%) of 78 glycans were obtained, and 1.0% of them were glycans containing M6P (M6P glycans). These were categorized according to their structure into 4 types: 3 newly found ones, comprising high-mannose-type M6P glycans capped with N-acetylglucosamine (GlcNAc) (2 variants, 17.5%), hybrid-type M6P glycans (2 variants, 11.2%), and hybrid-type M6P glycans capped with GlcNAc (3 variants, 6.9%), as well as high-mannose-type M6P glycans (3 variants, 64.4%). HCD-MS/MS spectra identified six distinctive M6P-derived oxonium ions. The glycopeptides obtained from protease-digested rhGAA were analyzed using nano-LC-ESI-HCD-MS/MS, and the extracted-ion chromatograms of M6P-derived oxonium ions confirmed three M6P glycosylation sites comprising Asn 140, Asn 233 (newly found), and Asn 470 attached heterogeneously to nine M6P glycans (two types), eight M6P glycans (four types), and seven M6P glycans (two types), respectively. This is the first study of rhGAA to differentiate M6P glycans and identify their attachment sites, despite rhGAA already being an approved drug for Pompe disease.

O-glycans and O-glycosylation sites of recombinant human GM-CSF derived from suspension-cultured rice cells, and their structural role.

Recombinant human GM-CSF (rhGM-CSF) from yeast has been clinically applied to immunosuppressed patients. The production of suspension-cultured rice-cell-derived rhGM-CSF (rrhGM-CSF), which has a longer blood clearance time and the same bioactivity as yeast-derived rhGM-CSF, and the analysis of its N-glycans have been reported recently. However, there are no previous reports of the O-glycosylation of rhGM-CSF from plant cells, and so this study investigated O-glycans, O-glycosylation sites, and their structural role in rrhGM-CSF. Monosaccharide analysis revealed the presence of O-glycans comprising arabinose and galactose. Eight O-glycans comprising four arabinose residues with zero to seven galactose residues along with their relative quantities were analyzed. Analysis of pronase-digested glycopeptides indicated that the O-glycans are partially attached to Ser 5, Ser 7, Ser 9, or Thr 10 residues, and glycan heterogeneity was confirmed at each site. Pro-to-hydroxyproline conversions occurred at Pro 2, Pro 6, and Pro 8 residues. The preparation of deglycosylated rrhGM-CSFs revealed that deglycosylation greatly affects their α-helix structures. These findings indicate that O-glycans of rrhGM-CSF are essential for maintaining its structural stability and result in an extended in vivo half-life, but without affecting its biological function. This is the first report on the O-glycosylation of rhGM-CSF derived from plant cells.

Effects of a Hot-Water Extract of Allium hookeri Roots on Bone Formation in Human Osteoblast-Like MG-63 Cells In Vitro and in Rats In Vivo.

Allium hookeri is a wild herb found mainly in the Himalayas, growing at altitudes of 1400-4200 m. A. hookeri is widely consumed as a vegetable and herbal medicine in Asia, but its effects on bone health have not been reported previously. This study investigated the effects of a hot-water extract of A. hookeri roots on bone formation. The hot-water extract significantly increased the proliferation of in vitro human osteoblast-like MG-63 cells and the stimulatory effects on osteoblast differentiation were noticeably greater for the hot-water extract than for daidzein (a positive control), as reflected by alkaline phosphatase activity, collagen content, and mineral deposition. Expression of the bone-remodeling marker osteocalcin production and bone microstructural parameters were significantly improved in Sprague-Dawley rats in vivo after oral treatment with the hot-water extract compared with their control (saline-administered) counterparts. The chemical compounds of the hot-water extract were characterized by liquid chromatography-mass spectrometry, and alliin, sinapic acid, and ferulic acid, which exert beneficial effects on bone health, were identified. These findings indicate that A. hookeri can be used as a natural resource for increasing bone formation. This is the first report of the anabolic effects of A. hookeri extracts on bone formation in vitro and in vivo.

Significant reduction in allergenicity of ovalbumin from chicken egg white following treatment with ascidian viscera N-acetylglucosaminidase.

Ovalbumin (OA) is the most abundant ingredient of chicken egg-white allergenic proteins. In the present study we investigated the possibility of reducing OA allergenicity by treatment with a natural protein exhibiting N-acetylglucosaminidase (NA) activity. Ascidian is cultivated as a food resource in northeast Asia. The ascidian viscera NA (AVNA) with almost no other exoglycosidases or proteolytic enzymes was isolated by applying size-exclusion chromatography to a protein precipitate of ascidian viscera. Intact OA was mixed with AVNA containing 0.2, 1.0, and 5.0 Units of NA. Anion-exchange chromatography was then used to isolate OA from AVNA-treated OA. The electrophoretic patterns and N-glycans of each isolated OA from AVNA-treated OA (iOA) were analyzed, and the terminal N-acetylglucosamines of iOA were selectively cleaved with no other degradation occurring. A competitive indirect enzyme-linked immunosorbent assay using rabbit anti-OA sera was performed to investigate the allergenicity of iOA, which was found to be significantly reduced depending on the increased NA activity compared to that of intact OA. These results indicate that OA allergenicity was reduced using a simple and mild treatment process with AVNA, and suggest that ascidian NA is an efficient natural protein for reducing the allergenicity of OA without requiring the use of harsh physical treatments or chemical conjugation.

Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspension-cultured rice cells with coexpression of human ß1,4-galactosyltransferase and human CTLA4Ig.

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4Ig(P)) reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4Ig(M)). However, limitations of hCTLA4Ig(P) include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing (ß1-2)-xylose and α1,3-fucose, which cause immunogenic reactions in humans. In the present study, human ß1,4-galactose-extended hCTLA4Ig(P) (hCTLA4Ig(P)-Gal) was expressed through the coexpression of human ß1,4-galactosyltransferase (hGalT) and hCTLA4Ig in an attempt to overcome these unfavorable effects. The results indicated that both encoding hGalT and hCTLA4Ig were successfully coexpressed, and the analysis of N-glycan and its relative abundance in purified hCTLA4Ig(P)-Gal indicated that not only were the two glycans containing (ß1-4)-galactose newly extended, but also glycans containing both ß1,2-xylose and α1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4Ig(P), respectively. Unlike hCTLA4Ig(P), hCTLA4Ig(P)-Gal was effective as an acceptor via (ß1-4)-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4Ig(P)-Gal in Sprague-Dawley rats was 1.9 times longer than that of hCTLA4Ig(P), and the clearance pattern of hCTLA4Ig(P)-Gal was close to that for hCTLA4Ig(M). These results indicate that the coexpression with hGalT and hCTLA4Ig(P) is useful for both reducing glycan immunogens and increasing in vivo stability. This is the first report of hCTLA4Ig as an effective therapeutics candidate in glycoengineered rice cells.

Effects of selective cleavage of high-mannose-type glycans of Maackia amurensis leukoagglutinin on sialic acid-binding activity.

BACKGROUND: Maackia amurensis leukoagglutinin (MAL) is a glycoprotein and sialic acid-binding lectin that is used widely in the detection and characterization of sialoglycoconjugates and human cancer cells. However, its N-linked glycan structure and role have yet to be determined. METHODS: The N-linked glycans were analyzed using high-performance liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the secondary structure was investigated using circular dichroism analysis. A hemagglutination assay was performed. Furthermore, surface plasmon resonance analysis, and fluorescence microscopy and fluorescence-activated cell-sorting analysis were conducted to assess the sialoglycoprotein-binding ability and its usefulness in the detection of human breast cancer MCF-7 cells, respectively. RESULTS: Analysis of the N-linked glycan structure of MAL confirmed the presence of eight glycans, comprising two α1,3-fucosylated paucimannosidic-type and six high-mannose-type glycans. Glycan analysis of MAL that had been treated with peptide N-glycosidase F (de-M-MAL) revealed that while the two α1,3-fucosylated paucimannosidic glycans remained attached following the treatment, the six high-mannose-type glycans had been completely cleaved from the original MAL. There were almost no secondary structural changes between MAL and de-M-MAL; however, the lectin activities exhibited by MAL, such as hemagglutination and binding to a sialoglycoprotein, were completely absent in de-M-MAL, and the ability to detect human breast cancer MCF-7 cells was 77% lower in de-M-MAL than in MAL. CONCLUSION: The high-mannose-type glycans in intact MAL are closely associated with its lectin activities. GENERAL SIGNIFICANCE: This is the first report of the N-linked glycan structure of MAL and the effect of high-mannose-type glycans on lectin activities.

Effects of Glucosinolates from Turnip (Brassica rapa L.) Root on Bone Formation by Human Osteoblast-Like MG-63 Cells and in Normal Young Rats.

Turnip (Brassica rapa L.) root ethanol extract (TRE) was prepared, and its chemical constituents were characterized by ultra-performance liquid chromatography and mass spectrometry. Thirteen glucosinolates (GSLs) were identified, comprising eight aliphatic, four indolic, and one aromatic compounds. The effects of these GSLs on bone formation were investigated in vitro by incubating human osteoblast-like MG-63 cells with TRE and then analyzing their viability, alkaline phosphatase (ALP) activity, collagen content, and mineralization and in vivo by administering TRE orally to normal young rats (500 mg/kg/day) and assessing subsequent changes in serum osteocalcin and bone microstructure in these animals. No TRE-related toxicity was found, and the levels of cell viability, ALP activity, collagen synthesis, and mineralization were significantly increased relative to the negative control. In particular, stimulatory effects on the differentiation of MG-63 cells were strongly enhanced as compared with a positive control (daidzein). Serum osteocalcin was also significantly increased, and some important bone microstructural parameters were improved in TRE-administered rats compared with their saline-administered counterparts. GSLs therefore appear to have a stimulatory effect on bone formation in both MG-63 cells and normal young rats. This is the first report on the usefulness of turnip root and its GSL compounds for bone formation.

Effects of Watercress Containing Rutin and Rutin Alone on the Proliferation and Osteogenic Differentiation of Human Osteoblast-like MG-63 Cells.

Most known osteoporosis medicines are effective for bone resorption, and so there is an increasing demand for medicines that stimulate bone formation. Watercress (N. officinale R. Br.) is widely used as a salad green and herbal remedy. This study analyzed a watercress extract using ultra-performance liquid chromatography/mass spectrometry, and identified a rutin as one of its major constituents. Osteogenic-related assays were used to compare the effects of watercress containing rutin (WCR) and rutin alone on the proliferation and differentiation of human osteoblast-like MG-63 cells. The reported data are expressed as percentages relative to the control value (medium alone; assigned as 100%). WCR increased cell proliferation to 125.0±4.0% (mean±SD), as assessed using a cell viability assay, and increased the activity of alkaline phosphatase, an early differentiation marker, to 222.3±33.8%. In addition, WCR increased the expression of collagen type I, another early differentiation marker, to 149.2±2.8%, and increased the degree of mineralization, a marker of the late process of differentiation, to 122.9±3.9%. Rutin alone also increased the activity of ALP (to 154.4±12.2%), the expression of collagen type I (to 126.6±6.2%), and the degree of mineralization (to 112.3±5.0%). Daidzein, which is reported to stimulate bone formation, was used as a positive control; the effects of WCR on proliferation and differentiation were significantly greater than those of daidzein. These results indicate that WCR and rutin can both induce bone formation via the differentiation of MG-63 cells. This is the first study demonstrating the effectiveness of either WCR or rutin as an osteoblast stimulant.

Cleavage of the terminal N-acetylglucosamine of egg-white ovalbumin N-glycans significantly reduces IgE production and Th2 cytokine secretion.

Ovalbumin (OA) is one of the most abundant of the glycoprotein allergens, and induces a T-helper type 2 immune response that results in an IgE-mediated hypersensitivity. In this study, the terminal carbohydrates of N-glycans from intact OA were cleaved with the exoglycosidases galactosidase, mannosidase, and N-acetylglucosaminidase to generate degalactosylated-OA, demannosylated-OA, and de-N-acetylglucosaminylated-OA, respectively, in order to evaluate their role in allergenicity. The exoglycosidase digestion procedure did not result in either degradation or contamination of the three deglycosylated sample, and the digestion efficiency was confirmed by comparing the results of glycan analysis of the three exoglycosidase-treated OAs with that of glycans of intact OA. Mice were immunized with either intact or exoglycosidase-treated OAs, and their respective allergic reactions were compared. IgE production in the de-N-acetylglucosaminylated-OA group was reduced to 58.8% of that in the intact OA group. In addition, the production levels of the cytokines interleukin-4 and interleukin-5 were significantly reduced in the de-N-acetylglucosaminylated-OA group to 53.4% and 45.8% of the levels in the intact OA group, respectively. However, there were almost no changes (or only slight reductions) in the degalactosylated-OA and demannosylated-OA groups, respectively. These results indicate that cleavage of the terminal carbohydrate, and particularly N-acetylglucosamine, reduces the allergenicity of OA. This is the first report of the effect of cleavage of the terminal carbohydrate on glycoprotein allergenicity.

Type and branched pattern of N-glycans and their structural effect on the chicken egg allergen ovotransferrin: a comparison with ovomucoid.

Ovotransferrin (OT), a multifunctional glycoprotein with defensive and protective activities, accounts for approximately 13% of chicken egg white proteins and is known as a major egg-associated allergen along with ovomucoid (OM). In contrast to the well-characterized N-glycans of OM, the N-glycan structure of OT has not been reported. Here, using HPLC equipped with a fluorescence detector and mass spectrometric analysis in combination with exoglycosidase digestion, we investigated the N-glycan type and branched pattern of OT, and compared them with those of OM. The HPLC peak area was used to calculate the relative quantity (%) of each glycan. Seventeen N-glycans, including 11 glycans (1 core structure and 10 complex-type oligosaccharides), that commonly exist in ovotransferrin and ovomucoid were identified. Six characteristic glycans (2 truncated structures, 1 complex-type, and 3 hybrid-type oligosaccharides) in OT and eight characteristic glycans in OM were classified. OT contains the following branched complex-type structures: mono-(13.2%), bi-(23.9%), tri-(9.0%), tetra-(2.7%), and penta-(2.8%) antennary oligosaccharides. However, OM contained mostly tri-(33.5%) and penta-(31.2%) antennary oligosaccharides. The N-glycan-containing bisecting N-acetylglucosamine comprised 43.4% and 79.8% of the total glycans in OT and OM, respectively. Moreover, using circular dichroism analysis, we observed that the secondary structure of the deglycosylated OT is quite different from that of the intact protein. To our knowledge, this is the first study to analyze N-glycans in OT in comparison with those of OM.

Binding Specificity of Philyra pisum Lectin to Pathogen-Associated Molecular Patterns, and Its Secondary Structure.

We recently reported a Philyra pisum lectin (PPL) that exerts mitogenic effects on human lymphocytes, and its molecular characterization. The present study provides a more detailed characterization of PPL based on the results from a monosaccharide analysis indicating that PPL is a glycoprotein, and circular dichroism spectra revealing its estimated α-helix, ß-sheet, ß-turn, and random coil contents to be 14.0%, 39.6%, 15.8%, and 30.6%, respectively. These contents are quite similar to those of deglycosylated PPL, indicating that glycans do not affect its intact structure. The binding properties to different pathogen-associated molecular patterns were investigated with hemagglutination inhibition assays using lipoteichoic acid from Gram-positive bacteria, lipopolysaccharide from Gram-negative bacteria, and both mannan and ß-1,3-glucan from fungi. PPL binds to lipoteichoic acids and mannan, but not to lipopolysaccharides or ß-1,3-glucan. PPL exerted no significant antiproliferative effects against human breast or bladder cancer cells. These results indicate that PPL is a glycoprotein with a lipoteichoic acid or mannan-binding specificity and which contains low and high proportions of α-helix and ß-structures, respectively. These properties are inherent to the innate immune system of P. pisum and indicate that PPL could be involved in signal transmission into Gram-positive bacteria or fungi.