Sprouting of the fructan- and starch-storing geophyte Lachenalia minima: Effects on carbohydrate and water content within the bulbs

N-linked glycans of wall-bound exo- β -glucanases from mung bean and barley seedlings, namely Mung-ExoI and Barley-ExoII, were characterized. The N-linked glycans of Mung-ExoI and Barley-ExoII were liberated by gas-phase hydrazinolysis followed by re-N-acetylation. Their structures were determined by two-dimensional sugar-mapping analysis and MALDI-TOF mass spectrometry. N-glycans from both glucanases were of paucimannosidic-type (small complex-type) structures, Man α 1-6(±Man α 1-3)(Xyl β 1-2)Man β 1-4GlcNAc β 1-4(±Fuc α 1-3) GlcNAc, which are known as typical vacuole-type N-glycans. The results suggest that N-glycans of cell-wall glucanase were produced by partial trimming of complex-type N-glycans by exoglycosidases during its transport from Golgi apparatus to cell walls or in the cell walls.     

Accumulation of free complex-type N-glycans in MKN7 and MKN45 stomach cancer cells

During the N-glycosylation reaction, it has been shown that 'free' N-glycans are generated either from lipid-linked oligosaccharides or from misfolded glycoproteins. In both cases, occurrence of high mannose-type free glycans is well-documented, and the molecular mechanism for their catabolism in the cytosol has been studied. On the other hand, little, if anything, is known with regard to the accumulation of more processed, complex-type free oligosaccharides in the cytosol of mammalian cells. During the course of comprehensive analysis of N-glycans in cancer cell membrane fractions [Naka et al. (2006) J. Proteome Res. 5, 88-97], we found that a significant amount of unusual, complex-type free N-glycans were accumulated in the stomach cancer-derived cell lines, MKN7 and MKN45. The most abundant and characteristic glycan found in these cells was determined to be NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1-3Manbeta1-4GlcNAc. Biochemical analyses indicated that those glycans found were cytosolic glycans derived from lysosomes due to low integrity of the lysosomal membrane. Since the accumulation of these free N-glycans was specific to only two cell lines among the various cancer cell lines examined, these cytosolic N-glycans may serve as a specific biomarker for diagnosis of specific tumours. A cytosolic sialidase, Neu2, was shown to be involved in the degradation of these sialoglycans, indicating that the cytosol of mammalian cells might be equipped for metabolism of complex-type glycans.     

Structural analysis of N-linked glycans in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Man(alpha)1-6(Man(alpha)1-3)Man(beta)1-4GlcNAc(beta)1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.     

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

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

Characterization of wheat germ agglutinin ligand on soluble glycoproteins in Caenorhabditis elegans

Some mutants of Caenorhabditis elegans show altered patterns of ectopic binding with wheat germ agglutinin (WGA). Some of these mutants also have defects of morphogenesis and movement during development. To clarify the structures of WGA-ligands in C. elegans that may be involved in developmental events, we have analyzed glycan structures capable of binding WGA. We isolated glycoproteins from wild-type C. elegans by WGA-affinity chromatography, and analyzed their glycan structures by a combination of hydrazine degradation and fluorescent labeling. The glycoproteins had oligomannose-type and complex-type N-glycans that included agalacto-biantenna and agalacto-tetraantenna glycans. Although the complex-type glycans carried beta-GlcNAc residues at their non-reducing ends, they did not bind to the WGA-agarose-resin. Thus, it was suggested that these N-glycans were not responsible for WGA-binding of the isolated glycoproteins. Hydrazinolysis of the glycoproteins also released a considerable amount of GalNAc monosaccharide. It was surmised that N-acetylgalactosamine was derived from mucin-type O-glycans with the Tn-antigen structure (GalNAcalpha1-O-Ser/Thr). WGA-blotting assay of neoglycoproteins revealed that a cluster of Tn-antigens was a good ligand for WGA. These results suggested that the WGA-ligand in C. elegans is a cluster of alpha-GalNAc monosaccharides linked to mucin-like glycoprotein(s). The observations reported in this paper emphasize the possible significance of mucin-type O-glycans in the development of a multicellular organism.     

N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits

Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the site-specific location of the various glycans.     

Testing the new animal phylogeny: first use of combined large-subunit and small-subunit rRNA gene sequences to classify the protostomes

Although the small-subunit ribosomal RNA (SSU rRNA) gene is widely used in the molecular systematics, few large-subunit (LSU) rRNA gene sequences are known from protostome animals, and the value of the LSU gene for invertebrate systematics has not been explored. The goal of this study is to test whether combined LSU and SSU rRNA gene sequences support the division of protostomes into Ecdysozoa (molting forms) and Lophotrochozoa, as was proposed by Aguinaldo et al. (1997) (Nature 387:489) based on SSU rRNA sequences alone. Nearly complete LSU gene sequences were obtained, and combined LSU + SSU sequences were assembled, for 15 distantly related protostome taxa plus five deuterostome outgroups. When the aligned LSU + SSU sequences were analyzed by tree-building methods (minimum evolution analysis of LogDet-transformed distances, maximum likelihood, and maximum parsimony) and by spectral analysis of LogDet distances, both Ecdysozoa and Lophotrochozoa were indeed strongly supported (e.g., bootstrap values >90%), with higher support than from the SSU sequences alone. Furthermore, with the LogDet-based methods, the LSU + SSU sequences resolved some accepted subgroups within Ecdysozoa and Lophotrochozoa (e.g., the polychaete sequence grouped with the echiuran, and the annelid sequences grouped with the mollusc and lophophorates)-subgroups that SSU-based studies do not reveal. Also, the mollusc sequence grouped with the sequences from lophophorates (brachiopod and phoronid). Like SSU sequences, our LSU + SSU sequences contradict older hypotheses that grouped annelids with arthropods as Articulata, that said flatworms and nematodes were basal bilateralians, and considered lophophorates, nemerteans, and chaetognaths to be deuterostomes. The position of chaetognaths within protostomes remains uncertain: our chaetognath sequence associated with that of an onychophoran, but this was unstable and probably artifactual. Finally, the benefits of combining LSU with SSU sequences for phylogenetic analyses are discussed: LSU adds signal, it can be used at lower taxonomic levels, and its core region is easy to align across distant taxa-but its base frequencies tend to be nonstationary across such taxa. We conclude that molecular systematists should use combined LSU + SSU rRNA genes rather than SSU alone.     

Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

Background

Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.

Results

The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Le^x and H type 2 antennae in sialylated complex-type N-glycans.

Conclusion

The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.     

Novel poly-GalNAcbeta1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing beta1,4-N-acetylgalactosaminyltransferase and alpha1,3-fucosyltransferase

Glycans containing the GalNAcbeta1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Galbeta1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAcbeta1-4GlcNAcbeta1-)n. These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Galbeta1-4GlcNAcbeta1-)n. We also stably expressed human alpha1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with alpha1,3-fucosylated LDN (LDNF) GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-R as well as novel "poly-LDNF" structures (-3GalNAcbeta1-4(Fucalpha 1-3)GlcNAcbeta1-)n. The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common alpha-subunit in L8-GalNAcT cells. The alpha-subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO4-4GalNAcbeta1-4GlcNAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.     

Phylogeny of protostome moulting animals (Ecdysozoa) inferred from 18 and 28S rRNA gene sequences

Reliability of reconstruction of phylogenetic relationships within a group of protostome moulting animals was evaluated by means of comparison of 18 and 28S rRNA gene sequences sets both taken separately and combined. Reliability of reconstructions was evaluated by values of the bootstrap support of major phylogenetic tree nodes and by degree of congruence of phylogenetic trees inferred by various methods. By both criteria, phylogenetic trees reconstructed from the combined 18 and 28S rRNA gene sequences were better than those inferred from 18 and 28S sequences taken separately. Results obtained are consistent with phylogenetic hypothesis separating protostome animals into two major clades, moulting Ecdysozoa (Priapulida + Kinorhyncha, Nematoda + Nematomorpha, Onychophora + Tardigrada, Myriapoda + Chelicerata, Crustacea + Hexapoda) and unmoulting Lophotrochozoa (Plathelminthes, Nemertini, Annelida, Mollusca, Echiura, Sipuncula). Clade Cephalorhyncha does not include nematomorphs (Nematomorpha). Conclusion was taken that it is necessary to use combined 18 and 28S data in phylogenetic studies.     

Structure of the carbohydrate units of human amniotic fluid fibronectin

Human amniotic fluid fibronectin was found to contain three types of carbohydrates: complex-type N-glycosidic glycans, lactosaminoglycans, and O-glycosidic glycans. The structures of the complex-type glycans were established by carbohydrate and methylation analysis, Smith degradation, sequential exoglycosidase treatments, lectin chromatography, and DEAE-Sephadex chromatography. Lactosaminoglycans were analyzed by fast atom bombardment mass spectrometry, and the O-glycosidically-linked oligosaccharides by gas-liquid chromatography-mass spectrometry and high-pressure liquid chromatography. The results show that amniotic fluid fibronectin contains 2 mol of biantennary and 2-3 mol of triantennary, complex-type N-glycosidic glycans. Unlike the N-glycosidic glycans of human adult plasma fibronectin, which contain only traces of fucose and are completely sialylated, the glycans from amniotic fluid fibronectin are fucosylated and only partially sialylated. The complex-type N-glycosidic glycans present in amniotic fluid fibronectin also include a fractional amount (0.1 mol) of glycans with a polylactosaminyl structure. In addition, 4 mol of O-glycosidic oligosaccharides, which have not previously been described in fibronectins, were found in amniotic fluid fibronectin. The major oligosaccharides in this fraction have the structures Gal beta 1----3GalNAcol, NeuNAc alpha 2----3Gal beta 1----3GalNAcol and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcol. O-glycosidically linked oligosaccharides were also detected in human adult plasma fibronectin but in smaller amounts than in amniotic fluid fibronectin. These results show that amniotic fluid fibronectin differs from plasma fibronectin with regard to the number of glycans attached to the polypeptide and that the glycans present in these two fibronectins differ in structure.     

Evidence from Hox genes that bryozoans are lophotrochozoans

Bryozoans, or moss animals, are small colonial organisms that possess a suspension-feeding apparatus called a lophophore. Traditionally, this "phylum" has been grouped with brachiopods and phoronids because of the feeding structure. Available molecular and morphological data refute this notion of a monophyletic "Lophophorata." Alternative hypotheses place bryozoans either at the base of the Lophotrochozoa or basal to the Lophotrochozoa/Ecdysozoa split. Surprisingly, the only molecular data bearing on this issue are from the 18S nuclear ribosomal gene. Here we report the results of a Hox gene survey using degenerate polymerase chain reaction primers in a gymnolaemate bryozoan, Bugula turrita. Putative orthologs to both the Post2 and the Lox5 genes were found, suggesting that bryozoans are not a basal protostome group but closely allied to other lophotrochozoan taxa. We also found the first definitive evidence of two Deformed/Hox4 class genes in a nonvertebrate animal.     

A transient tobacco expression system coupled to MALDI-TOF-MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants

Tobacco-based transient expression was employed to elucidate the impact of differential targeting to subcellular compartments on activity and quality of gastric lipase as a model for the production of recombinant glycoproteins in plants. Overall N-linked glycan structures of recombinant lipase were analyzed and for the first time sugar structures of its four individual N-glycosylation sites were determined in situ by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) on a trypsin digest without isolation or deglycosylation of the peptides. Three glycosylation sites contain both complex-type N-glycans and high-mannose-type structures, the fourth is exclusively linked to high-mannose glycans. Although the overall pattern of glycan structures is influenced by the targeting, our results show that the type of glycans found linked to a given Asn residue is largely influenced by the physico-chemical environment of the site. The transient tobacco system combined with MALDI-TOF-MS appears to be a useful tool for the evaluation of glycoprotein production in plants.     

Characterization of the N-linked glycans of Giardia intestinalis

This article reports the first rigorous evidence for the existence of N-glycans in Giardia intestinalis, a parasite that is a widespread human pathogen, being a major cause of enteric disease in the world. Excreted/secreted molecules of G. intestinalis are known to stimulate the immune system. Structural strategies based on MALDI and electrospray mass spectrometry were employed to examine the excreted/secreted molecules for their N-glycan content. These revealed that the major oligosaccharides released by peptide N-glycosidase F are complex-type structures and correspond to bi-, and triantennary structures without core (alpha1,6) fucosylation. The major nonreducing epitopes in these complex-type glycans are: Galbeta1-4GlcNAc (LacNAc) and NeuAc alpha2-6Galbeta1-4GlcNAc (sialylated LacNAc).     

Isolation of Hox and Parahox genes in the hemichordate Ptychodera flava and the evolution of deuterostome Hox genes

Because of their importance for proper development of the bilaterian embryo, Hox genes have taken center stage for investigations into the evolution of bilaterian metazoans. Taxonomic surveys of major protostome taxa have shown that Hox genes are also excellent phylogenetic markers, as specific Hox genes are restricted to one of the two great protostome clades, the Lophotrochozoa or the Ecdysozoa, and thus support the phylogenetic relationships as originally deduced by 18S rDNA studies. Deuterostomes are the third major group of bilaterians and consist of three major phyla, the echinoderms, the hemichordates, and the chordates. Most morphological studies have supported Hemichordata+Chordata, whereas molecular studies support Echinodermata+Hemichordata, a clade known as Ambulacraria. To test these competing hypotheses, complete or near complete cDNAs of eight Hox genes and four Parahox genes were isolated from the enteropneust hemichordate Ptychodera flava. Only one copy of each Hox gene was isolated suggesting that the Hox genes of P. flava are arranged in a single cluster. Of particular importance is the isolation of three posterior or Abd-B Hox genes; these genes are only shared with echinoderms, and thus support the monophyly of Ambulacraria.     

Hybrid- and complex-type N-glycans are not essential for Newcastle disease virus infection and fusion of host cells

N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.     

Structural analysis of N-glycans of the planarian Dugesia japonica

To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N-glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N-glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2-aminopyridine at their reducing end. The labeled N-glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N-glycans, it was shown that the N-glycans of planarian include high mannose-type and pauci-mannose-type glycans. However, many of the major N-glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3-O-methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non-reducing-end mannose, as the glycans were resistant to α-mannosidase digestion. These results indicate that methylated high-mannose-type glycans are the most abundant structure in planarians.     

Specificity of DC-SIGN for mannose- and fucose-containing glycans

The dendritic cell specific C-type lectin dendritic cell specific ICAM-3 grabbing non-integrin (DC-SIGN) binds to "self" glycan ligands found on human cells and to "foreign" glycans of bacterial or parasitic pathogens. Here, we investigated the binding properties of DC-SIGN to a large array of potential ligands in a glycan array format. Our data indicate that DC-SIGN binds with K(d)<2muM to a neoglycoconjugate in which Galbeta1-4(Fucalpha1-3)GlcNAc (Le(x)) trisaccharides are expressed multivalently. A lower selective binding was observed to oligomannose-type N-glycans, diantennary N-glycans expressing Le(x) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LacdiNAc-fucose), whereas no binding was observed to N-glycans expressing core-fucose linked either alpha1-6 or alpha1-3 to the Asn-linked GlcNAc of N-glycans. These results demonstrate that DC-SIGN is selective in its recognition of specific types of fucosylated glycans and subsets of oligomannose- and complex-type N-glycans.     

Characterization of the N-linked oligosaccharides of megalin (gp330) from rat kidney

Megalin (gp 330) is a large cell surface receptor expressed on the apical surfaces of epithelial tissues, that mediates the binding and internalization of a number of structurally and functionally distinct ligands. In this paper we report the first detailed structural characterization of megalin-derived oligosaccharides. Using strategies based on mass spectrometric analysis, we have defined the structures of the N-glycans of megalin. The results reveal that megalin glycoprotein is heterogeneously glycosylated. The major N-glycans identified belong to the following two classes: high mannose structures and complex type structures, with complex structures being more abundant than high mannose structures. The major nonreducing epitopes in the complex-type glycans are: GlcNAc, Galbeta1-4GlcNAc (LacNAc), NeuAcalpha2-6Galbeta1-4GlcNAc (sialylated LacNAc), GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4GlcNAc (Sd(a)) and Galalpha1-3Galbeta1-4GlcNAc. Most complex structures are characterized by the presence of (alpha1,6)-core fucosylation and the presence of a bisecting GlcNAc residue.