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-beta-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-beta-N-acetylglucosaminidase from rice culture cells and identified the gene encoded. Furthermore, we found structural features of free N-glycans in the cells, indicating that high-mannose type species (Man(9-5)GlcNAc(1)) occur at concentration of several micromolar (microM). 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.     

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen*

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing alpha1-3 fucose and/or beta1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).     

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans

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 beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc 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 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 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 beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.     

Structure of O-glycosidically linked oligosaccharides from glycoproteins of Trypanosoma cruzi CL-Brener strain: evidence for the presence of O-linked sialyl-oligosaccharides

Glycoproteins on the cell surface of Trypanosoma cruzi are known to play important roles in the interaction of the parasite with the host cells. We previously determined the structures of the O-glycan chains from the sialoglycoproteins (mucin-like molecules) of the G- and Y-strains and observed significant differences between them. We now report the structures of the sialylated and nonsialylated O-linked oligosaccharides isolated from the cell surface glycoproteins of the myotropic CL-Brener strain grown in the presence of fetal calf serum. The structures of the O-linked oligosaccharide alditols obtained by reductive beta-elimination of the sialoglycoprotein were determined by a combination of methylation analysis, fast atom bombardment-mass spectrometry and nuclear magnetic resonance spectroscopy. The presence of a beta-galactopyranose substituent on the N-acetylglucosamine O-4 position shows that these O-linked oligosaccharides from CL-Brener strain belong to the same family as those isolated from mucins expressed by T. cruzi Y strain, a reticulotropic strain. In addition, novel O-glycans, including alpha2-3 mono-sialylated species are described.     

A micromethod for the estimation of oligosaccharides containing glycosidically linked sialic acid or hexoses, or both, in glycoproteins

The peeling reaction, the process by which oligosaccharides are degraded in alkali, was used as the basis for an assay to provide structural information about glycosidically linked oligosaccharides in glycoproteins. Glycoproteins were treated with 0.05 M NaOH at 50 degrees to induce release, and subsequent degradation ("peeling"), of glycosidically linked, but not of N-glycosydically linked, oligosaccharides. Among the degradation products generated from O-linked chains were three 3-deoxy sugar acids whose formation was correlated with certain structural features of the oligosaccharides. N-Acetylneuraminic acid was released from terminal positions in the oligosaccharides, and iso- and meta-saccharinic acids were derived from the degradation of 4-O- and 3-O-substituted hexoses, respectively. All of these sugar acids were detected colorimetrically by periodate oxidation and reaction of the product with 2-thiobarbituric acid. The ability of the method to generate 3-deoxy sugar acids was tested in 8 alkali-treated glycoproteins. 3-Deoxy sugar acids were detected only in those glycoproteins whose glycosidically linked carbohydrates contained N-acetylneuraminic acid, or 3-O- or 4-O-substituted hexoses, or both. As little as 0.12 microgram of 3-deoxy sugar acid produced from 5 micrograms of human chorionic gonadotropin was sufficient for detection. This method is novel in its ability to distinguish sialylation of glycosidically linked carbohydrates. Furthermore, it combines the specificity of beta-elimination with the sensitivity of the 2-thiobarbituric acid assay in targeting degradation products of the peeling reaction as candidates for an assay method.     

Poly(N-acetyllactosaminyl) oligosaccharides in glycoproteins of PC12 pheochromocytoma cells and sympathetic neurons

Endo-beta-galactosidase treatment of glycopeptides derived from the trypsinate and membranes of PC12 pheochromocytoma cells and cultured sympathetic neurons demonstrated the presence of poly(N-acetyllactosaminyl) units on tri- and tetraantennary oligosaccharides, some of which have a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Nerve growth factor induced differentiation of the PC12 cells led to a small but significant decrease in the proportion of these oligosaccharides. Poly(N-acetyllactosaminyl) oligosaccharides were also identified in a major 230 000-Da cell-surface glycoprotein (the nerve growth factor inducible large external, or NILE, glycoprotein) of PC12 cells and appear to account for much or all of the difference in size between this glycoprotein as compared to the immunochemically cross-reactive 205 000-Da species present in postnatal brain. Glycoproteins containing poly(N-acetyllactosaminyl) oligosaccharides were selectively labeled by treatment of PC12 cells with endo-beta-galactosidase to expose N-acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP-[14C]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed the presence of a number of distinct PC12 cell glycoproteins that contain these oligosaccharides and have apparent molecular weights in the range of 25 000-250 000. Treatment of PC12 cells with nerve growth factor (NGF) altered the relative labeling of several of the glycoprotein bands, with a time course similar to the effects of NGF on neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of glycoproteins in human placenta: Processing of oligosaccharides

The processing of the high-mannose asparagine-linked oligosaccharides synthesized by first-trimester human placenta has been investigated. Tissue was pulsed for 1 h with [2-³H]mannose and chased for zero, 45, 90, and 180 min in media containing unlabeled mannose. Glycopeptides, prepared by Pronase digestion of the delipidated membrane pellets at each time point, were treated with endo-β-N-acetylglucosaminidase-H to release the high-mannose asparagine-linked oligosaccharides. The largest major processing intermediate isolated was Glc₁Man₉GlcNAc, which was converted into Man₉GlcNAc, and then into Man₈GlcNAc, Man₇GlcNAc, Man₆GlcNAc, and Man₅GlcNAc. There was also a minor pathway in which mannosyl residues were removed prior to the glucose. By carrying out the detailed structural characterization of the individual processing intermediates, it was possible to demonstrate that processing of the Man₉GlcNAc to Man₅GlcNAc proceeded by the nonrandom removal of the α1,2-linked mannosyl residues. Specifically, of 12 possible sequences of removal of the four α1,2-linked mannosyl residues present in Man₉GlcNAc, first-trimester human placenta utilized only two of these in the processing of asparagine-linked oligosaccharides. It is suggested that the limited number of processing pathways reflects a high degree of specificity of these reactions in human placenta.     

Resistance to Thrips palmi (Thysanoptera: Thripidae) in beans

Resistance in beans, Phaseolus vulgaris L., to the melon thrips Thrips palmi Karny was studied under field conditions at two sites in Colombia. Genotypes were rated for resistance on the basis of visual damage scores, bean production ratings (a visual estimate of pod and seed set), and grain yields. Of 1,138 genotypes tested, only 60 (5.3%) were rated as resistant. Repeated testing allowed us to identify potential sources of resistance in five germplasm accessions (G 02402, G 02852, G 03177, G 03569, and G 04055), one commercial variety ('Brunca'), six elite breeding lines (A 216, DOR 714, EMP 486, FEB 115, FEB 161, and FEB 162), 41 recombinant inbred lines derived from the BAT 881 x G 21212 cross, and seven recombinant inbred lines derived from a cross between DOR 364 and BAT 477. Resistance was not associated with maturity, growth habit, pubescence, and seed color or seed size. In general, correlations between visual damage scores and bean production ratings and between damage scores and yield were high and significant meaning that selection on damage rating is useful to identify genotypes that may have tolerance as a mechanism of resistance. The continuous distribution of damage scores of 139 recombinant inbred lines suggested that the inheritance of resistance to the melon thrips might be quantitative. Overall, resistance levels in beans can be considered as moderate, because none of the genotypes tested received damage scores of <3 on a 1-9 scale and none was ever rated as highly resistant in terms of bean production ratings.     

Tests for resistance to Aphis fabae in field beans (Viciafaba)

Two lines of Viciafaba were as resistant to Aphis fabae as Rastatt, one of their progenitors, when compared with susceptible control varieties in glasshouse experiments. In short experiments, fewer adult aphids settled on plants of resistant varieties, which subsequently bore smaller total numbers of aphids. In a longer experiment, the aphids multiplied more slowly on the resistant varieties, which survived after susceptible beans had been killed. The bean varieties did not vary in numbers of aphids on the stem or growing point but resistant varieties had markedly smaller proportions of the total on the abaxial surface of the leaves; in particular the smaller veins supported fewer aphids. V. narbonensis was very resistant to A. fabae but was susceptible to Myzus persicae, indicating that its resistance mechanism is distinct from that within V. faba. In infested field trials most of the variation in yield of bean varieties was attributable to the size of A. fabae colonies, and resistant varieties outyielded susceptible varieties. Yields of resistant varieties in the presence of large numbers of aphids were, however, less than those from trials without aphids. In uninfested trials susceptible varieties usually gave the largest yields. The significance of these observations is discussed in relation to policies and methods for breeding for resistance to A. fabae in V. faba.     

Synthesis of natural beta-D-(1-->3)-glucopyranosyl oligosaccharides

beta-D-(1-->3)-Glucan core structure derivatives corresponding to schizophyllan, epiglucan and lentinan were synthesized efficiently. 4,6-O-Benzylidenated glucopyranosyl acceptors were found to be helpful in the attempted beta-D-(1-->3) bond formation. The epiglucan pentasaccharide showed a weak anti-tumor activity in preliminary mice tests.     

Saccharopolyspora: an underexplored source for bioactive natural products

Actinomycetes are a rich source for secondary metabolites with a diverse array of biological activities. Among the various genera of actinomycetes, the genus Saccharopolyspora has long been recognized as a potential source for antibiotics and other therapeutic leads that belong to diverse classes of natural products. Members of the genus Saccharopolyspora have been widely reported from several natural sources including both terrestrial and marine environments. A plethora of this genus has been chemically investigated for the production of novel natural products with interesting pharmacological effects. Therefore, Saccharopolyspora is considered one of the pharmaceutical important genera that could provide further chemical diversity with potential lead compounds. In this review, the literature from 1976 until December 2018 was covered, providing a comprehensive survey of all natural products derived from this genus and their semi-synthetic derivatives along with their biological activities, whenever applicable. Moreover, the biological diversity of Saccharopolyspora species and their habitats were also discussed.     

Immunochemistry of highly branched N-glycans. Terminal N-acetyl-D-glucosamine (GlcNAc) cluster antigens contain epitopes composed of terminal GlcNAc residues linked to mannose

We have previously demonstrated by the immunoperoxidase method the presence of a chicken heterophile antigenic determinant (CHAD-1) in medullary lymphocytes of the bursa of Fabricius and thymus as well as in some nonlymphoid cells. It has been found that the anti-CHAD-1 antibody could be neutralized by absorption with several glycoproteins or glycopeptides containing highly branched, asparagine-linked oligosaccharides terminating in N-acetylglucosamine residues. In the present study, fetuin, desialo-fetuin, and a series of 27 highly purified oligosaccharides with well-defined structures were used to investigate the chemical composition and fine structure of the CHAD-1 epitope. It was shown that anti-CHAD-1 antibody binds to oligosaccharides with at least three terminal N-acetyl glucosamine residues at the nonreducing end. These residues may be linked beta 1-2, beta 1-4, or beta 1-6 to one, two, or three different mannose residues. The antibody combining site accommodates at least four carbohydrate residues. Oligosaccharides containing five or six terminal N-acetylglucosamine residues at the nonreducing end demonstrated the highest immunoreactivity with the anti-CHAD-1 antibody. Substitution of terminal N-acetylglucosamine residues with galactose, or with galactose and sialic acid, masks CHAD-1. On the basis of this work, epitopes that react with the anti-CHAD-1 antibody will be renamed terminal N-acetylglucosamine cluster antigens (TGCA). Anti-TGCA antibody has potential use in the monitoring of biosynthetic processing of asparagine-linked oligosaccharides and in studies of their cellular distribution and functions.     

Hypersensitive response of beans to Apion godmani (Coleoptera: Curculionidae)

High levels of resistance to Apion godinani Wagner have been reported in bean, Phaseolus vulgaris L., landraces from Mexico. We report on the role of hypersensitivity to A. godmani in five resistant and three susceptible bean genotypes. In susceptible genotypes (cultivars 'Canario 107','Jamapa', and 'Zacatecas 45'), the eggs and first instars of A. godmani were embedded in the pod mesocarp and usually were surrounded by healthy tissue. In contrast, in resistant landraces ('Amarillo 154', 'Amarillo 155', 'J-117', 'Puebla 36', and 'Pinto 168'), necrotic tissues developed concentrically around the oviposition site, encapsulating eggs and dead larvae. An inverse relationship between percentage egg and larval encapsulation at the early immature pod stages and percentage of damaged seeds at harvest was found. Results indicate that hypersensitivity in developing pods plays an important role in antibiosis to A. godmani in beans. This information will facilitate future genetic and biochemical research and provide much needed information concerning the phenotypic basis of resistance to A. godmani in bean.     

The goat mammary glandular epithelial (GMGE) cell line promotes polyfucosylation and N,N'-diacetyllactosediaminylation of N-glycans linked to recombinant human erythropoietin

We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Application of isotope dilution analysis to the determination of sulfate in glycoproteins and oligosaccharides

Isotope dilution analysis, using a probe of ³⁵S-labeled BaSO₄, is proposed for the determination of sulfate in hydrolysates of glycoproteins and other glycoconjugates. A scaled-down version of the method of Klockow is presented. A modified radioassay in strongly acidic conditions, effective in the range of 0.5 to 10 nmol sulfate per sample, has been developed.     

Breeding for partial resistance in dry beans (Phaseolus vulgaris) to bean rust (Uromyces appendiculatus)

Significant differences in susceptibility of 23 bean cultivars were found after natural infection by a single-pustule isolate of bean rust in field trials over three seasons. These differences were not related to differences in the number of adaxial or abaxial acicular leaf hairs, latent period in primary leaves or a difference in pustule size. Forty-five large-seeded bean lines with partial resistance to a single-pustule isolate were developed by recurrent selection for commercial evaluation.     

Nutritional and bioactive properties of three edible species of green algae,genus Caulerpa (Caulerpaceae)

The green algae genus Caulerpa is coenocytic, and the thallus consists of only one cell with many nuclei. It is widely distributed in the tropical seas. In the Southeast Asian waters, there are at least ten known species. Three species, particularly Caulerpa racemosa var. clavifera f. macrophysa (Kützing) Weber-van Bosse, C. racemosa var. laetevirens (Montagne) Weber-van Bosse, and Caulerpa lentillifera J. Agardh are widely consumed. The proximate analysis and secondary metabolite composition of these three species were determined to describe their lipid and nutritional values. Glycolipids and phospholipids were the major lipid classes, with significant levels of triacylglycerol. Polyunsaturated fatty acids (PUFA) were the major fatty acids of all the three species. Typical n-3 and n-6 PUFA such as 18:3n-3, 18:4n-3, 20:5n-3, 18;2n-6, and 20:4n-6 were found in significant amount in all these three species. All three species contained a red-pigmented secondary metabolite determined as caulerpin. All three extracts exhibited potent antimicrobial activity against human food pathogenic bacteria and anti-inflammatory activity against the murine macrophage cell line, RAW 264.7.