Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Structural characterization of the oligosaccharide chains of native and crystallized boar seminal plasma spermadhesin PSP-I and PSP-II glycoforms.

The PSP-I/PSP-II heterodimer is the major protein of boar seminal plasma. Both subunits are glycoproteins of the spermadhesin family and each contains a single N-glycosylation site. After enzymatic release of the oligosaccharides from isolated PSP-I and PSP-II, mainly neutral and monosialylated oligosaccharides, and small amounts of disialylated oligosaccharides, were recovered from both proteins. Twenty-two neutral oligosaccharides, 11 monosialylated glycans and three disialylated carbohydrate chains were characterized using mass spectrometric and NMR techniques. PSP-I and PSP-II share the same glycans but differ in their relative molar ratios. Most glycan structures are proximally alpha1-6-fucosylated, diantennary complex-type bearing nonsialylated or alpha2-6-sialylated N-acetyllactosamine or di-N-acetyllactosamine antennae. The majority of nonsialylated N-acetyllactosamine antennae bear terminal alpha1-3-linked Gal residues. In addition, the N-acetylglucosamine residue of nonsialylated N-acetyl and di-N-acetyllactosamine antennae can be modified by an alpha1-3-linked fucose residue. Structures of higher antennarity, as well as structures 3,6-branched at galactose residues, were found in smaller amounts. In one oligosaccharide, N-acetylneuraminic acid is substituted by N-glycolylneuraminic acid. Mass spectrometric analysis of PSP-I and PSP-II glycoforms isolated from crystallized PSP-I/PSP-II heterodimer showed the coexistence of major PSP-I and PSP-II glycoforms in the hexagonal crystals. Oligosaccharides with the NeuNAcalpha2-6GalNAcbeta1-4GlcNAc-R motif block adhesive and activation-related events mediated by CD22, suggesting a possible immunoregulatory activity for PSP-I/PSP-II.     

The beta 1----2-D-xylose and alpha 1----3-L-fucose substituted N-linked oligosaccharides from Erythrina cristagalli lectin. Isolation, characterisation and comparison with other legume lectins

The carbohydrate moieties of Erythrina cristagalli lectin were released as oligosaccharides by hydrazinolysis, followed by N-acetylation and reduction with NaB3H4. Fractionation of the tritium-labelled oligosaccharide mixture by Bio-Gel P-4 column chromatography and high-voltage borate electrophoresis revealed that it is composed of five neutral oligosaccharides. Structural studies by sequential exoglycosidase digestion in combination with methylation analysis and two-dimensional 1H-NMR showed that the major component was the fucose-containing heptasaccharide Man alpha 3(Man alpha 6)(Xyl beta 2)Man beta 4GlcNAc beta 4(Fuc alpha 3)GlcNAcol. This is the first report of such a structure in plant lectins. Small amounts of the corresponding afucosyl hexasaccharide were also identified, as well as three other minor components. The structure of the heptasaccharide shows the twin characteristics of a newly established family of N-linked glycans, found to date only in plants. The characteristics are substitution of the common pentasaccharide core [Man alpha 3(Man alpha 6)Man beta 4GlcNAc beta 4GlcNAc] by a D-xylose residue linked beta 1----2 to the beta-mannosyl residue and an L-fucose residue linked alpha 1----3 to the reducing terminal N-acetylglucosamine residue. The oligosaccharide heterogeneity pattern for Erythrina cristagalli lectin was also found for the lectins from four other Erythrina species and the lectins of two other legumes, Sophora japonica and Lonchocarpus capassa.     

Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human alpha1-acid glycoprotein mediates growth of Streptococcus oralis

Streptococcus oralis is the agent of a large number of infections in immunocompromised patients, but little is known regarding the mechanisms by which this fermentative organism proliferates in vivo. Glycoproteins are widespread within the circulation and host tissues, and could provide a source of fermentable carbohydrate for the growth of those pathogenic organisms with the capacity to release monosaccharides from glycans via the production of specific glycosidases. The ability of acute phase serum alpha1-acid glycoprotein to support growth of S.oralis in vitro has been examined as a model for growth of this organism on N-linked glycoproteins. Growth was accompanied by the production of a range of glycosidases (sialidase, N-acetyl-beta-D-glucosaminidase, and beta-D-galactosidase) as measured using the 4-methylumbelliferone-linked substrates. The residual glycoprotein glycans remaining during growth of this organism were released by treatment with hydrazine and their analysis by HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with only terminal N-acetylglucosamine residues attached to asparagines of the protein backbone remaining when growth was complete. Monosaccharides were released sequentially from the glycans by S.oralis glycosidases in the order sialic acid, galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the actions of exo-glycosidic activities, including mannosidases which have not previously been reported for S.oralis. All released monosaccharides were metabolized during growth with the exception of fucose which remained free in culture supernatants. Direct release of oligosaccharides was not observed, indicating the absence of endo-glycosidases in S.oralis. We propose that this mechanism of deglycosylation of host glycoproteins and the subsequent utilization of released monosaccharides is important in the survival and persistence of this and other pathogenic bacteria in vivo.     

Core structures of polysialylated glycans present in neural cell adhesion molecule from newborn mouse brain.

Polysialylation of the neural cell adhesion molecule (N-CAM) is known to destabilize cell-cell adhesion and to promote plasticity in cell-cell interactions. To gain more insights into the molecular mechanisms regulating the selective expression of polysialic acid on distinct glycan chains, the underlying core structures of polysialylated N-CAM glycans from newborn mouse brain were examined. Starting from low picomolar amounts of oligosaccharides, a multistep approach was used that was based on various mass spectrometric techniques with minimized sample consumption. Evidence could be provided that polysialylated murine N-CAM glycans comprise diantennary, triantennary and tetraantennary core structures carrying, in part, type-1 N-acetyllactosamine antennae, sulfate groups linked to terminal galactose or subterminal N-acetylglucosamine residues and, as a characteristic feature, a sulfated glucuronic acid unit which was bound exclusively to C3 of terminal galactose in Manalpha3-linked type-2 antennae. Hence, our results reveal that part of the murine N-CAM carbohydrates are modified within a single oligosaccharide by polysialic acid plus a HSO3-GlcA-moiety, which is likely to represent a HNK1-epitope. As HNK1-carbohydrates are also known to modulate cell-cell interactions, the simultaneous presence of both carbohydrate epitopes may reflect a new mechanism involved in the fine-tuning of N-CAM functions.     

Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin.

The carbohydrate binding specificity of Mr = 30,000 lectin (CBP30) from baby hamster kidney (BHK) cells has been studied by inhibition of binding of the radiolabeled lectin to asialofetuin-Sepharose using model oligosaccharides and glycopeptides. CBP30 binds type I or II Gal beta(1----3(4))GlcNAc chains but not Gal(beta 1----3)GalNAc. The inhibitory potency of straight chain polylactosamine structures or complex-type branched glycans is increased in proportion to the number of Gal(beta 1----3(4)) units present. Fucosylation or sialylation of terminal galactose residues or further substitution by (alpha 1----3)-linked galactose or N-acetylgalactosamine does not affect binding whereas substitution of the penultimate N-acetylglucosamine residue drastically reduces binding. Thus, blood group A, H type I or H type II structures, shows high affinity whereas Lex, Lea, and Leb structures bind poorly. CBP30 binds to murine Engelbreth-Holm-Swarm (EHS) tumor laminin and human amniotic fluid fibronectin but not human plasma fibronectin. Binding involves polylactosamine glycans as well as tri- and tetraantennary complex-type glycans present in EHS laminin and amniotic fluid fibronectin but absent in plasma fibronectin. Proteolytic fragments of EHS laminin (E1X/Nd, P1, E8, and E3) bind CBP30, but only fragment E8 supports attachment and spreading of BHK cells. BHK cell adhesion to EHS laminin or fragment E8 was not disturbed by CBP30-specific antibodies, but at relatively high concentrations (45 micrograms/ml) CBP30 inhibited spreading and partially attachment of cells on laminin.     

Interactions of cartilage proteoglycans with hyaluronate. Inhibition of the interaction by modified oligomers of hyaluronate.

Oligomers of hyaluronic acid were prepared by digestion of hyaluronic acid from rooster combs with testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35), leech head hyaluronidase (hyaluronate 3-glycanohydrolase, EC 3.2.1.36), and with fungal hyaluronidase (hyaluronate lyase from Streptomyces hyalurolyticus). The oligomers were fractionated by gel permeation, using Sephadex G-50. Oligomers isolated after incubation of the hyaluronic acid with the testicular hyaluronidase were further modified. To prepare oligomers with N-acetylglucosamine at both ends, terminal nonreducing glucuronic acid residues were removed with beta-glucuronidase. Reducing terminal N-acetylglucosamine residues were removed by reaction under mildly alkaline conditions. The reducing terminal N-acetylglucosamine residues were also reduced with sodium borohydride to form N-acetylglucosaminitol. The potentials of the various oligosaccharides to bind to the proteoglycan from bovine nasal septum cartilage were estimated by determining their effectiveness as inhibitors of the proteoglycan-hyaluronate interaction. The present study shows that, to bind maximally to the proteoglycan, the hyaluronate oligosaccharide must be at least 10 sugar residues in length and be terminated at the nonreducing and reducing ends with a glucuronate residue and an N-acetylglucosamine residue, respectively. Sugar residues extended beyond this basic decasaccharide, do not interact with the hyaluronate binding site on the proteoglycan.     

Core fucosylation of N-linked glycans in leukocyte adhesion deficiency/congenital disorder of glycosylation IIc fibroblasts

Leukocyte adhesion deficiency/congenital disorder of glycosylation IIc (LAD II/CDG IIc) is a genetic disease characterized by a decreased expression of fucose in glycoconjugates, resulting in leukocyte adhesion deficiency and severe morphological and neurological abnormalities. The biochemical defect is a reduced transport of guanosine diphosphate-L-fucose (GDP-L-fucose) from cytosol into the Golgi compartment, which reduces its availability as substrate for fucosyltransferases. The aim of this study was to determine the effects of a limited supply of GDP-L-fucose inside the Golgi on core fucosylation (alpha1,6-fucose linked to core N-acetylglucosamine [GlcNAc]) of N-linked glycans in LAD II fibroblasts. The results showed that, although [3H]fucose incorporation was generally reduced in LAD II cells, core fucosylation was affected to a greater extent compared with other types of fucosylation of N-linked oligosaccharides. In particular, core fucosylation was found to be nearly absent in biantennary negatively charged oligosaccharides, whereas other types of structures, in particular triantennary neutral species, were less affected by the reduction. Expression and activity of alpha1,6-fucosyltransferase (FUT8) in control and LAD II fibroblasts were comparable, thus excluding the possibility of a decreased activity of the transferase. The data obtained confirm that the concentration of GDP-L-fucose inside the Golgi can differentially affect the various types of fucosylation in vivo and also indicate that core fucosylation is not dependent only on the availability of GDP-L-fucose, but it is significantly influenced by the type of oligosaccharide structure. The relevant reduction in core fucosylation observed in some species of oligosaccharides could also provide clues for the identification of glycans involved in the severe developmental abnormalities observed in LAD II.     

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.     

Structural determination of the oligosaccharide side chains from a glycoprotein isolated from the mucus of the coral Acropora formosa

An extracellular mucous glycoprotein has been isolated from the hard coral Acropora formosa. The glycoprotein contains sulfated oligosaccharide side chains attached through O-glycosidic linkages to serine and threonine, the principal amino acids (77%) in the polypeptide. The oligosaccharide side chains consist of D-arabinose, D-mannose, and N-acetyl-D-glucosamine with smaller amounts of D-galactose, L-fucose, and N-acetyl-D-galactosamine, but no sialic or uronic acids. Alkaline borohydride reductive cleavage resulted in a mixture of oligosaccharide alditols. Six oligosaccharides were purified by high performance liquid chromatography. The structures of these oligosaccharides, which do not resemble those of any other glycoprotein so far examined, were determined by a combination of gas chromatography/mass spectrometry analysis of methylation products and NMR spectroscopy. All oligosaccharides contain a reducing terminal mannitol residue with N-acetylglucosamine linked to carbon 2, 4, or 6 of the mannitol. There is no evidence for linkage of N-acetylglucosamine to any other glycoses in the glycoprotein. Galactose was detected in two oligosaccharides linked to the 4-position of mannitol. Arabinose (Ara) was found in only one oligosaccharide. This was probably due to hydrolysis of the labile arabino-furanoside linkages. Evidence is presented which indicates the arabinose occurs primarily at the terminal position of oligosaccharide side chains. The structures of the oligosaccharides isolated from the glycoprotein were: (Formula: see text).     

Structure, biosynthesis, and function of asparagine-linked glycans on plant glycoproteins

In plants, glycoproteins with asparagine-linked glycans (oligosaccharides) are found in vacuoles, in the extracellular space or matrix, and associated with the endo-membrane system (endoplasmic reticulum, Golgi apparatus, plasma membrane, tonoplast). These glycans are of the high-mannose type, with a structure identical to that found in other organisms (mammals, yeast), or of the complex type with a β1–2 linked xylosyl residue not found in mammalian complex glycans. Asparagine-linked glycans play multiple roles by modifying the physicochemical properties of the polypeptides to which they are attached.     

Identification of a novel UDP-Glc:GlcNAc beta1-->4-glucosyltransferase in Lymnaea stagnalis that may be involved in the synthesis of complex-type oligosaccharide chains

Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L. stagnalis were incubated in media containing either [(3)H]-mannose, [(3)H]-glucosamine, or [(3)H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L. stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L. stagnalis contains a beta1-->4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is beta6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is beta2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel beta1-->4-gluco-syltransferase present in the prostate gland may be involved in the biosynthesis of Glcbeta1-->4GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans.     

Sulfated O-linked glycans of the vitelline coat as ligands in gamete interaction in the ascidian, Halocynthia roretzi

In the ascidian Halocynthia roretzi, sperm-egg binding is probably mediated through the interaction between alpha-L-fucosidase present on the sperm surface and anionic saccharide chains of the egg vitelline coat. To characterize biologically active glycans, total glycans were chemically released from the glycopeptide fraction of the vitelline coat. The fraction of uncharged glycans and two fractions of negatively charged glycans were separated by diethylaminoethyl-anion exchange chromatography. In a competitive inhibition assay of fertilization, both anionic fractions showed inhibitory activity, with more anionic glycans being most potent, while uncharged glycans were biologically inactive. Chemical desulfation combined with a competitive inhibition assay of fertilization and ion analysis determined that sulfate groups were responsible for anionic character and crucial for biological activity. Monosaccharide analysis of anionic fractions showed a high content of N-acetylgalactosamine, galactose, xylose and the presence of arabinose, mannose, N-acetylglucosamine, glucose and rhamnose. Glycans were O-linked and galactose and xylose residues were detected at reducing termini. Linkage analysis suggested that 1,4-linked xylose, 1,3-linked galactose and N-acetylgalactosamine residues, substituted to different degrees by sulfate groups on the C-3 and C-4 carbons, respectively, constituted the core structures of anionic glycans.     

Structures of the carbohydrate moiety attached to one site in the first domain of turkey ovomucoid: elucidation by 1H-n.m.r. spectroscopy

1H-N.m.r. spectroscopy was used to elucidate the primary structures of the carbohydrate moiety attached to asparagine at residue 53 in the first domain of turkey ovomucoid, a serine proteinase inhibitor. The carbohydrate moiety is a heterogeneous mixture of three structurally closely related complex-type oligosaccharides. Of the total carbohydrate moiety, 61% is tetra-antennary with terminal galactose and with an intersecting N-acetylglucosamine, and containing an additional N-acetylglucosamine (10') attached to mannose (4'). Another 23% is tri-antennary with terminal galactose and with an intersecting N-acetylglucosamine. The remaining 16% is tri-antennary with terminal galactose (6 and 8 only), with an intersecting N-acetylglucosamine.     

Transfer of glycerol by Endo-beta-N-acetylglucosaminidase F to oligosaccharides during chitobiose core cleavage

N-Linked oligosaccharides, when hydrolyzed by glycerol-containing preparations of endo-beta-N-acetylglucosaminidase (Endo) F from Flavobacterium meningosepticum were found to have glycerol attached to their reducing ends. The absence of a reducing end was confirmed by high-field 1H NMR spectroscopy, and the incorporated glycerol was verified through mass spectrometry and collisionally activated decomposition fast atom bombardment/mass spectrometry/mass spectrometry techniques. Periodate oxidation of [1(3)-14C]glycerol-labeled oligosaccharides indicated glycerol was glycosidically linked via its 1(3) carbon to the C1 of the reducing end N-acetylglucosamine. In a second, less favored reaction, the glycerol glycoside was hydrolyzed by Endo F using water as the terminal nucleophile, thus regenerating the N-acetylglucosamine reducing end. Glycerol could be removed from Endo F preparations without affecting enzyme stability, and chitobiosyl core hydrolysis in its absence provided intact oligosaccharides with normal N-acetylglucosamine reducing ends. The incorporation of labeled glycerol may provide a useful method for monitoring of Endo F release of oligosaccharides.     

Arraying glycomics: a novel bi-functional spacer for one-step microscale derivatization of free reducing glycans

Glycan array development is limited by the complexity of efficiently generating derivatives of free reducing glycans with primary amines or other functional groups. A novel bi-functional spacer with selective reactivity toward the free glycan and a second functionality, a primary amine, was synthesized. We demonstrated an efficient one-step derivatization of various glycans including naturally isolated N-glycans, O-glycans, milk oligosaccharides, and bacterial polysaccharides in microgram scale. No protecting group manipulations or activation of the anomeric center was required. To demonstrate its utility for glycan microarray fabrication, we compared glycans with different amine-spacers for incorporation onto an amine-reactive glass surface. Our study results revealed that glycans conjugated with this bi-functional linker were effectively printed and detected with various lectins and antibodies.     

A human lysosomal alpha-mannosidase specific for the core of complex glycans.

A novel lysosomal alpha-mannosidase, with unique substrate specificity, has been partially purified from human spleen by chromatography through concanavalin A-Sepharose, DEAE-Sephadex, and Sephacryl S-300. This enzyme can catalyze the hydrolysis of only 1 mannose residue, that which is alpha(1----6)-linked to the beta-linked mannose in the core of N-linked glycans, as found in the oligosaccharides Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc and Man alpha(1----6)Man beta(1----4) GlcNAc. The newly described alpha-mannosidase does not catalyze the hydrolysis of mannose residues outside of the core, even if they are alpha(1----6)-linked, and is not active on the other alpha-linked mannose in the core, which is (1----3)-linked. The narrow specificity of the novel mannosidase contrasts sharply with that of the major lysosomal alpha-mannosidase, which is able to catalyze the degradation of oligosaccharides containing diverse linkage and branching patterns of the mannose residues. Importantly, although the major mannosidase readily catalyzes the hydrolysis of the core alpha(1----3)-linked mannose, it is poorly active towards the alpha(1----6)-linked mannose, i.e. the very same mannose residue for which the newly characterized mannosidase is specific. The novel enzyme is further differentiated from the major lysosomal alpha-mannosidase by its inability to catalyze the efficient hydrolysis of the synthetic substrate p-nitrophenyl alpha-mannoside, and by the strong stimulation of its activity by Co2+ and Zn2+. Similarly to the major mannosidase, it is strongly inhibited by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, but not by deoxymannojirimycin. The presence of this novel alpha-mannosidase activity in human tissues provides the best explanation, to date, for the structures of the oligosaccharides stored in human alpha-mannosidosis. In this condition the major lysosomal alpha-mannosidase activity is severely deficient, but apparently the alpha(1----6)-mannosidase is unaffected, so that the oligosaccharide structures reflect the unique specificity of this enzyme.     

Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans

It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.     

Transient expression of sialylated glycans during glycoprotein processing by embryonal carcinomas

Embryonal carcinoma and early embryonic cells express unusually large and complex carbohydrates on their surfaces that are lost during differentiation. These carbohydrates are composed of alternating galactose and N-acetylglucosamine residues and have either linear or branched architectures. Compared to the glycans expressed by many differentiated cells these glycans are poorly sialylated. However, metabolic studies reveal that there is a transient expression of sialylated glycans during the processing of glycoproteins by embryonal carcinomas. After a short pulse with mannose the major complex-type glycan is a biantennary glycan with two sialic acids. During subsequent chase periods this glycan species is replaced by unsialylated glycans that have elongated branches composed of alternating galactose and N-acetylglucosamine residues.     

Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein

The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.