Complete structure of glycopeptides isolated from IgG immunoglobulins of cow colostrum

Bovine immunoglobulins (IgG1 type) have been isolated from colostral whey. Hydrolysis by pronase, trypsin and (or) chymotrypsin yield several glycopeptides structural studies of which lead to the following results. 1. IgG1 colostral immunoglobulins possess two glycan moieties which are linked to the peptidic chain by an N-(beta-aspartyl)-N-acetylglucosaminylamine bound. 2. The peptidic sequence around the linkage region has been determined by classical methods and is as follows: Thr-Lys-Pro-Arg-Glu-Glu-Gln-Phe-Asn(Glycan)-Ser-Thr-Tyr-Arg. 3. The following procedures: partial acidic hydrolysis, periodic oxidation, hydrazinolysis-nitrous deamination, methylation and use of specific glycosidases allowed us to determine the structure of the glycan moieties which fit with the general following scheme: (see article) Thus they could be related to the general glycan structure so-called of "N-acetyllactosamine type" because they possess the pentasaccharidic core common to numerous glycoproteins Man alpha 1 leads to [Man alpha 1 leads to 6] Man beta 1 leads to 4 GlcNAc beta 1 leads to 4 GlcNAc beta 1 leads to Asn on which are conjugated 2 N-acetyllactosamine residues. Besides they present a microheterogeneity which is due to the varying number of additional N-acetylneuraminic acid and fucose residues. 4. These structures are compared to various immunoglobulin structures proposed by others: bovine serum IgG and human serum IgG, IgE and IgA.     

Enzymatic synthesis of N-linked oligosaccharides terminating in multiple sialyl-Lewis(x) and GalNAc-Lewis(x) determinants: clustered glycosides for studying selectin interactions

Galactosyltransferase, sialyltransferase, and fucosyltransferase were used to create a panel of complex oligosaccharides that possess multiple terminal sialyl-Le(x) (NeuAc alpha 2-3Gal[Fuc alpha 1-3] beta 1-4GlcNAc) and GalNAc-Le(x) (GalNAc[Fuc alpha 1-3]beta 1-4GlcNAc). The enzymatic synthesis of tyrosinamide biantennary, triantennary, and tetraantennary N-linked oligosaccharides bearing multiple terminal sialyl-Le(x) was accomplished on the 0.5 mumol scale and the purified products were characterized by electrospray MS and 1H NMR. Likewise, biantennary and triantennary tyrosinamide oligosaccharides bearing multiple terminal GalNAc-Le(x) determinants were synthesized and similarly characterized. The transfer kinetics of human milk alpha 3/4-fucosyltransferase were compared for biantennary oligosaccharide acceptor substrates possessing Gal beta 1-4GlcNAc, GalNAc beta 1-4GlcNAc, and NeuAc alpha 2-3Gal beta 1-4GlcNAc which established NeuAc alpha 2-3Gal beta 1-4GlcNAc as the most efficient acceptor substrate. The resulting complex oligosaccharides were chemically tethered through the tyrosinamide aglycone to the surface of liposomes containing phosphatidylthioethanol, resulting in the generation of glycoliposomes probe which will be useful to study relationships between binding affinity and the micro- and macro-clustering of selectin ligand.     

Structural studies of the sugar chains of hen ovomucoid. Evidence indicating that they are formed mainly by the alternate biosynthetic pathway of asparagine-linked sugar chains

The carbohydrate moieties of hen ovomucoid were released as oligosaccharides by hydrazinolysis. The neutral oligosaccharide fraction which comprised about 85% of the total sugar was fractionated into eight oligosaccharide fractions by Bio-Gel P-4 column chromatography. Occurrence of novel penta-antennary oligosaccharides in the larger three fractions was reported in the preceding paper (Yamashita, K., Kamerling, J.P., and Kobata, A. (1982) J. Biol. Chem. 257, 12809-12814). Structural studies of the remaining smaller oligosaccharides indicated that they all have Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc as their common core. The alpha-mannosyl residues occur either free or as one of the following five groups: GlcNAc beta 1 leads to 2Man, GlcNAc beta 1 leads to 4Man, GlcNAc beta 1 leads to 4(GlcNAc beta 1 leads to 2)Man, GlcNAc beta 1 leads to 6(GlcNAc beta 1 leads to 2)Man, and GlcNAc beta 1 leads to 6(GlcNAc beta 1 leads to 4)(GlcNAc beta 1 leads to 2) Man. In most oligosaccharides, a beta-N-acetylglucosamine residue is linked at the C-4 position of the beta-mannosyl residue of the core. The structural characteristic of the sugar chains of hen ovomucoid indicated that they are not formed by the ordinary processing pathway of the asparagine-linked sugar chains.     

Automatic and effortful processes in auditory memory reflected by event-related potentials. Age-related findings

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man alpha1 --> 6 (Man alpha1 --> 3) Man beta1 --> 4GlcNAc beta1 --> 4GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal beta1 --> 3Gal beta1 --> 4GlcNAc beta1 --> 2Man alpha1 --> 6 (Gal beta1 --> 3Gal beta1 --> 4GlcNAc beta1 --> 2Man alpha1 --> 3) Man beta1 --> 4GlcNAc beta1 --> 4 (Fuc alpha1 --> 6)GlcNAc-PA; 6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively.     

Expression and characterization of a lactosaminoglycan-carrying glycoprotein of Zajdela hepatoma cell surface--structural analysis of the carbohydrate moiety

In poorly differentiated hepatoma cells, a glycoprotein carrying lactosaminoglycans is identified, and the structure of its glycan moiety is proposed. After membrane solubilization, protein fractionation by gel filtration, and electroelution, this glycoprotein (GPIII) was identified by its affinity for Datura stramonium lectin and its content in large glycopeptides. As shown by PAGE, GPIII has an apparent molecular mass of 100 kDa and is highly glycosylated (36%). It appears as an integral membrane glycoprotein. It is absent from normal hepatocytes, in that no heavy glycopeptides could be detected that bound to Datura lectin or to specific antiserum. The glycan moiety of GPIII has been analyzed according to carbohydrate composition, glycosidase treatment, affinity chromatography on immobilized pokeweed, Datura and Griffonia lectins, and by NMR and methylation analyses. The glycan is a N-linked tetraantennary lactosaminoglycan of 6.6 kDa, containing Gal, GlcNAc, Man, and NeuNAc in a 16:14:3:4 molar ratio, with an average of three repeating units/branch. Its beta-Gal residues are in the penultimate position and are linked in beta1-4 at least in four structural elements (three peripheral and one internal). It contains a very branched structure with Gal alpha1-3Gal beta1-4GlcNAc side chains linked in the C6 position to an inner Gal residue in a main branch. Alpha-Gal and NeuNAc residues [mainly NeuNAc alpha(2-3) linkage] are expressed as the nonreducing terminal groups. A possible structural model is proposed for this heterogeneous lactosaminoglycan, although no definitive structure can be established. That this lactosaminoglycan-carrying glycoprotein GPIII is not expressed in hepatocytes suggests its expression to be linked to the undifferentiated and/or malignant state of this hepatoma.     

Branching beta 1-6N-acetylglucosaminetransferases and polylactosamine expression in mouse F9 teratocarcinoma cells and differentiated counterparts

beta-All-trans-retinoic acid (RA)-induced endodermal differentiation of mouse F9 teratocarcinoma cells is accompanied by changes in glycoprotein glycosylation, including expression of i antigen (i.e. polylactosamine) and leukophytohemagglutinin-reactive oligosaccharides (i.e. -GlcNAc beta 1-6Man alpha 1-6-branched N-linked). We have used the F9 teratocarcinoma cells as a model to study developmental regulation of glycosyltransferase activities which are responsible for the biosynthesis of beta 1-6GlcNAc-branched N- and O-linked oligosaccharides and polylactosamine. Growth of F9 cells in the presence of 10(-6) M RA for 4 days increased core 2 GlcNAc transferase and GlcNAc transferase V activities by 13- and 6-fold, respectively, whereas the activities of GlcNAc transferase I, beta 1-3GlcNAc transferase (i), beta 1-4Gal transferase, and beta 1-3Gal transferase increased 2-4-fold. Induction of glycosyltransferase activities by RA was dose-dependent and showed a biphasic response with approximately half of the increase observed 3 days after RA treatment and the remainder occurred by day 4. PYS-2, a parietal endoderm cell line, showed levels of glycosyltransferase activities similar to those of RA-treated F9 cells. Glycosyltransferase activities in the RA-resistant F9 cell line (RA-3-10) were low and showed only a small induction by RA. These observations suggest that differentiation of F9 cells is closely associated with induction of multiple glycosyltransferase activities, with most pronounced increases in GlcNAc transferase V and 2',5'-tetradenylate (core 2) GlcNAc transferase. The increase in GlcNAc transferase V was also reflected by the 4-6-fold increase in the binding of 125I-leukophytohemagglutinin to several cellular glycoproteins, which occurred after 3 days of RA treatment. The endo-beta-galactosidase-sensitive polylactosamine content of membrane glycoproteins and, in particular, the LAMP-1 glycoprotein was markedly increased after RA treatment of F9 cells. Consistent with these observations, fucosylated polylactosamine (i.e. dimeric Lex) was also increased in RA-treated cells. Analysis of the aryl oligosaccharides produced by F9 cells cultured in the presence of aryl alpha-D-GalNAc showed that RA treatment enhanced the synthesis of disialyl core 2 O-linked oligosaccharides and increased the polylactosamine content of the aryl oligosaccharides by > 20-fold. The results suggest that differentiation of F9 cells into endoderm is closely associated with increased GlcNAc transferase V and core 2 GlcNAc transferase activities, enzymes which control the level of beta 1-6GlcNAc-branched N- and O-linked oligosaccharides, the preferred substrates for polylactosamine addition.     

Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells

The native structures of the Asn-linked oligosaccharides and the O-glycans at Ser126 of human erythropoietin expressed from recombinant BHK cells have been elucidated. Enzymatically released N-glycans were studied by methylation analyses, fast-atom-bombardment mass spectrometry as well as one- and two-dimensional 1H-NMR spectrometry at 600 MHz. Many (82.7%) were found to be tetraantennary N-acetyllactosamine-type (22.8% with one, 3.6% with two and 0.4% with three N-acetyllactosamine repeats) being tetrasialylated (41%), trisialylated (29.6%) and disialylated (12.2%). A few (9.7%; 4.1% 2,4-branched, 5.6%, 2,6-branched) of the chains were triantennary (5.4% trisialyl, 4.3% disialyl) and 4.6% were of the disialyl diantennary type. Almost all of the innermost GlcNAc residues were alpha 1-6 fucosylated and NeuAc was exclusively alpha 2-3 linked to Gal beta 1-4GlcNAc-R; 60% of the protein was found to be O-glycosylated at Ser126; structures were monosialylated (70%) or disialylated (30%) forms of the Gal beta 1-3GalNAc core type. Glycosylation patterns at individual Asn-Xaa-Thr/Ser sites were determined by analytical high-pH anion-exchange chromatography with pulsed amperometric detection. Only tetraantennary chains with 0-3 N-acetyllactosamine repeats were detected at Asn38 and Asn83, while almost all of the di- and triantennary oligosaccharides were attached to Asn24. Batch analysis of different preparations of recombinant erythropoietin revealed the high reproducibility of the production procedure. Structures containing terminal GalNAc-GlcNAc were detected in small amounts in a few batches.     

Structural changes in the oligosaccharide moiety of human IgG with aging

In order to elucidate the relationship between glycosylation of IgG and aging, oligosaccharide structures of human IgG purified from sera of men and women aged 18 to 73 years were investigated. Oligosaccharides were liberated quantitatively from IgG by hydrazinolysis followed by N-acetylation and were tagged with p-aminobenzoic acid ethyl ester. The oligosaccharide structures were then analyzed by HPLC in conjunction with sequential exoglycosidase digestion. All IgG samples were shown to contain a series of biantennary complex type oligosaccharides which consisted of +/-Galbeta1-4GlcNAcbeta1-2Manalpha1-6(+/-GlcNAcbeta 1-4)(+/-Galbeta1-4GlcNAcbeta1-2Man(alpha)1-3)Man(beta)1-+ ++4GlcNAcbeta1-4(+/- Fucalpha1-6)GlcNAc and their mono- and disialo glycoforms in different ratios. In female IgG samples only, the incidence of non-galactosylated oligosaccharides with non-reducing terminal GlcNAc residues increased with aging (r>0.8), whereas that of digalactosylated oligosaccharides decreased (r<-0.8). A weaker correlation was observed between aging and the incidence of neutral and monosialo oligosaccharides in female IgG (r=0.461 and r= -0.538, respectively) and between aging and the incidence of oligosaccharides with a bisecting GlcNAc in both male and female IgG samples (r=0.566 and r=0.440, respectively). In addition, a significant change with aging in the galactosylation of IgG oligosaccharides was observed in females in their thirties, fifties, and sixties (p<0.02, p<0.01, and p<0.04, respectively). These findings may contribute to our understanding of autoimmune diseases such as rheumatoid arthritis in which glycosylation is involved.     

Effect of glycosylation on the in vivo circulating half-life of ribonuclease

The circulating half-lives of the four isozymes of bovine pancreatic ribonuclease (RNases A, B, C, and D) have been determined in normal and in nephrectomized rats. The isozymes differ only in their glycosyl content. While A contains no sugars, B has a simple oligosaccharide (GlcNAc2 Man4-5),and C and D each have a complex oligosaccharide (GlcNAc4 Man 2-3 Gal2 Fuc NeuAc2, and GlcNAc4 Man3 Gal2 Fuc NeuAc4, respectively) attached to Asn-34 of the polypeptide chain. All four isozymes were cleared rapidly in normal rats (t 1/2 = 2 to 3 min), as expected on the basis of the established role of the kidneys in removing low molecular weight proteins from circulation. In nephrectomized rats, however, a much slower clearance was observed, thus permitting the evaluation of the role of the carbohydrate chains in the catabolism of the isozymes. The clearance curves can be analyzed in terms of two processes, a rapid initial one, shown to represent the equilibration of the injected enzyme into extravascular space, and a second one which is interpreted as the catabolic clearance of the enzyme. The haf-life of the RNase isozymes was calculated from this second process and found to be in the range 528 to 577 min for RNase A, 15 min for RNase B, 681 to 862 min for RNase C, and 839 to 941 min for RNase D. The rapidly cleared RNase B was treated with alpha-mannosidase to remove 3 of the 4 mannosyl residues, leaving only a trisaccharide (GlcNAc2-betaMan) attached to the protein. The half-life of this RNase B derivatives was found to be in the range 616 to 733 min. From these results it is concluded (a) that the addition of complex oligosaccharides to a protein does not have any significant direct effect on its circulating half-life (RNases C and D compared to RNase A), and (b) that in the rat there exists a mechanism for clearing glycoproteins based on specific recognition of exposed alpha-mannosyl residues (RNase B compared to the other isozymes and to alpha-mannosidase-treated RNase B).     

Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. I. Role of glucose in the initial glycosylation of invertase in the endoplasmic reticulum

Oligosaccharides on invertase restricted to the endoplasmic reticulum (ER) in alg3,sec18 yeast at 37 degrees C were found to be 20% wild type Man8GlcNAc and 80% Man1 alpha-->2Man1 alpha-->2Man1 alpha-->3(Man1 alpha-->6)Man1 beta-->4GlcNAc2 (Verostek, M.F., Atkinson, P.H., and Trimble, R. B. (1991) J. Biol. Chem. 266, 5547-5551). These results suggested that alg3 was slightly leaky, but did not address whether the oligosaccharide-lipid Man9GlcNAc2 and Man5GlcNAc2 precursors were glucosylated in alg3 yeast. Therefore, an alg3,sec18,gls1 strain was constructed to delete the GLS1-encoded glucosidase I responsible for trimming the terminal alpha 1,2-linked glucose from newly transferred Glc3ManxGlcNAc2 oligosaccharides. Invertase activity was overexpressed 5-10-fold on transforming this strain with a multicopy plasmid (pRB58) carrying the SUC2 gene, and preparative amounts of the ER form of external invertase, derepressed and accumulated at 37 degrees C, were purified. The N-linked glycans were released by sequential treatment with endo-beta-N-acetylglucosaminidase H (endo H) and peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase. Oligosaccharide pools were sized separately on Bio-Gel P-4, which showed that endo H released about 17% of the carbohydrate as Glc3Man8GlcNAc, while peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase released the remainder as Hex8GlcNAc2 and Man5GlcNAc2 in a 1:4 ratio. Glycan structures were assigned by 500-MHz two-dimensional DQF-COSY 1H NMR spectroscopy, which revealed that the endo H-resistant Hex8GlcNAc2 pool contained Glc3Man5GlcNAc2 and Man8GlcNAc2 in a 6:4 ratio, the latter a different isomer from that formed by the ER alpha 1,2-mannosidase (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Recovery of Glc3Man8GlcNAc and not the ER form of Man8GlcNAc provided an internal control indicating the absence of glucosidase I, which was confirmed by incubation of [3H]Glc3[14C]Man9GlcNAc with solubilized membranes from either alg3,sec18,gls1 or alg3,sec18,GLS1 strains. Chromatographic analysis of the products showed that [3H]Glc was removed only in the presence of the GLS1 gene product. Thus, the vast majority of the N-linked glycosylation in the ER of alg3 yeast (> 75%) occurs by transfer of Man5GlcNAc2 without prior addition of the 3 glucoses normally found on the lipid-linked precursor.     

Structure of the N-linked oligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae

The periplasmic invertase was purified from Saccharomyces cerevisiae och1::LEU2 disruptant cells (delta och1), which have a defect in elongation of the outer chain attached to the N-linked core oligosaccharides (Nakayama, K., Nagasu, T., Shimma, Y., Kuromatsu, J., and Jigami, Y. (1992) EMBO J. 11, 2511-2519). Structural analysis of the pyridylaminated (PA) neutral oligosaccharides released by hydrazinolysis and N-acetylation confirmed that the och1 mutation causes a complete loss of the alpha-1,6-polymannose outer chain, although the PA oligosaccharides (Man9GlcNAc2-PA and Man10GlcNAc2-PA), in which one or two alpha-1,3-linked mannose(s) attached to the endoplasmic reticulumn (ER)-form core oligosaccharide (Man8GlcNAc2) were also detected. Analysis of the delta och1 mnn1 strain oligosaccharides released from total cell mannoprotein revealed that the delta och1 mnn1 mutant eliminates the alpha-1,3-mannose attached to the core and accumulates predominantly a single ER-form oligosaccharide species (Man8GlcNAc2), suggesting a potential use of this strain as a host cell to produce glycoproteins containing mammalian high mannose type oligosaccharides. The delta och1 mnn1 alg3 mutants accumulated Man5GlcNAc2 and Man8GlcNAc2 in total cell mannoprotein, confirming the lack of outer chain addition to the incomplete corelike oligosaccharide and the leaky phenotype of the alg3 mutation. All the results suggest that the OCH1 gene encodes an alpha-1,6-mannosyltransferase that is functional in the initiation of alpha-1,6-polymannose outer chain addition to the N-linked core oligosaccharide (Man5GlcNAc2 and Man8GlcNAc2) in yeast.     

Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 2. Characterization of E-selectin binding fractions, and structural requirements for physiological binding to E-selectin

E-selectin binding gangliosides were isolated from myelogenous leukemia HL60 cells, and the E-selectin binding pattern was compared with that of human neutrophils as described in the preceding paper in this issue. The binding fractions were identified as monosialogangliosides having a series of unbranched polylactosamine cores. Structures of fractions 12-3, 13-1, 13-2, and 14, which showed clear binding to E-selectin under the conditions described in the preceding paper, were characterized by functional group analysis by application of monoclonal antibodies, 1H-NMR, FAB-MS, and electrospray mass spectrometry with collision-induced dissociation of permethylated fractions. Fractions 12-3, 13-1, and 13-2 were characterized by the presence of a major ganglioside with the following structure: NeuAc alpha 2-->3Gal beta 1-->4 GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer. Fractions 12-3 and 13-2 contained, in addition, small quantities (10-15%) of extended SLex with internally fucosylated structures: NeuAc alpha 2-->3 Gal beta 1-->4-(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4 (+/- Fuc alpha 1-->3)GlcNA c beta 1-->3 Gal beta beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->Glc Beta Cer. Fraction 13-1, showing stronger E-selectin binding activity than 12-3 and 13-2, contained only a trace quantity (< 1%) of SLex. Fraction 14, which also showed clear binding to E-selectin, was characterized by the presence of the following structures, in addition to two internally monofucosylated structures (XX and XXI, Table 2, text): NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer; andNeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4 (Fuc alpha 1--3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1--4Glc beta Cer. SLex determinant was completely absent. Thus, the E-selectin binding epitope in HL60 cells is carried by unbranched terminally alpha 2-->3 sialylated polylactosamine having at least 10 monosaccharide units (4 N-acetyllactosamine units) with internal multiple fucosylation at GlcNAc. These structures are hereby collectively called "myeloglycan". Monosialogangliosides from normal human neutrophils showed an essentially identical pattern of gangliosides with selectin binding property. Myeloglycan, rather than SLex, provides a major physiological epitope in E-selectin-dependent binding of leukocytes and HL60 cells.     

Conformational studies on the selectin and natural killer cell receptor ligands sulfo- and sialyl-lacto-N-fucopentaoses (SuLNFPII and SLNFPII) using NMR spectroscopy and molecular dynamics simulations. Comparisons with the nonacidic parent molecule LNFPII

This investigation is focused on the conformational behavior of the blood group Lewisa (Le(a)-active pentasaccharide lacto-N-fucopentaose II (LNFPII) and its sulfated and sialylated analogs, SuLNFPII and SLNFPII. The latter two are more potent oligosaccharide ligands for the animal lectins, E- and L-selectin, and the natural killer cell receptor, NKR-P1, than are the shorter chain analogs based on the trisaccharide Le(a) domain. We report here that the three oligosaccharides based on the fucopentasaccharide have very similar average solution conformations as determined from NMR spectroscopical parameters, in particular 13C chemical shift differences. From restrained simulated annealing and restrained molecular dynamics (MD) simulations performed in order to determine the most probable conformational distributions around the glycosidic linkages we derive models for these oligosaccharides that are in good agreement with experimental parameters, such as rotating-frame Overhauser effects (ROE's) and long-range 1H,13C coupling constants across the glycosidic linkages. In these model structures the Le(a) domain at the non-reducing end of the longer chain oligosaccharides approximates the same rigid structure as in the shorter analogs. The Gal beta 1-4Glc linkage at the reducing end is also rather rigid, showing only little more flexibility than the Le(a) domain. However, the NeuAc alpha 2-3Gal linkage in SLNFPII, and the GlcNAc beta 1-3Gal linkage in all three oligosaccharides are flexible, in each case fluctuating mainly between two minimum energy structures: (phi = -81 degrees, psi = 8 degrees) and (phi = -160 degrees, psi = -20 degrees) for the NeuAc alpha 2-3Gal linkage, as reported previously for the isomeric sequence 3'-sialyl Le(x), and (phi = -25 degrees, psi = -26 degrees) and (phi = 20 degrees, psi = 24 degrees) for the GlcNAc beta 1-3Gal linkage. The flexibility of the latter linkage may allow the lactosyl domain at the reducing end to fit with little strain into extended carbohydrate binding sites on the recognition proteins, and, for the purposes of drug designs, it will be important to establish which conformational distribution is assumed for the GlcNAc beta 1-3Gal linkage in these longer chain oligosaccharides in the bound state.     

Carbohydrate binding specificity of the B-cell maturation mitogen from Artocarpus integrifolia seeds

Artocarpin, a mannose-specific lectin, is a homotetrameric protein (M(r) 65,000) devoid of covalently attached carbohydrates and consists of four isolectins with pI in the range 5-6.5. Investigations of its carbohydrate binding specificity reveal that among monosaccharides, mannose is preferred over glucose. Among mannooligosaccharides, mannotriose (Man alpha 1-3[Man alpha 1-6]Man) and mannopentaose are the strongest ligands followed by Man alpha 1-3Man. Extension of these ligands by GlcNAc at the reducing ends of mannooligosaccharides tested remarkably improves their inhibitory potencies, while substitution of both the alpha 1-3 and alpha 1-6 mannosyl residues of mannotriose and the core pentasaccharide of N-linked glycans (Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc) by GlcNAc or N-acetyllactosamine in beta 1-2 linkage diminishes their inhibitory potencies. Sialylated oligosaccharides are non-inhibitory. Moreover, the substitution of either alpha 1-3 or alpha 1-6 linked mannosyl residues of M5Gn or both by mannose in alpha 1-2 linkage leads to a considerable reduction of their inhibitory power. Addition of a xylose residue in beta 1-2 linkage to the core pentasaccharide improves the inhibitory activity. Considering the fact that artocarpin has the strongest affinity for the xylose containing hepasaccharide from horseradish peroxidase, which differs significantly from all the mannose/glucose-specific lectins, it should prove a useful tool for the isolation and characterization of glycoproteins displaying such structure.     

Sialylated N-glycans in adult rat brain tissue--a widespread distribution of disialylated antennae in complex and hybrid structures

This paper extends our earlier work on the analysis of neutral N-glycans from adult rat brain to glycans carrying NeuAc residues as their sole charged groups. These structures comprised at least 40% of the total (acidic and neutral) N-glycan pool. Compounds were identified by a combination of endoglycosidase and exoglycosidase digestions, anion-exchange chromatography, normal and reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption/ionisation-mass spectrometry and combined gas chromatography/mass spectrometry. Mono-, di- and trisialylated components, together with components substituted with four (or more) NeuAc residues, showed abundances of approximately 12, 10, 7 and 7%, respectively, relative to the total N-glycan pool. In addition, neuraminidase digestion resulted in the neutralisation of a fraction of highly charged species, possibly indicating the presence of N-glycans substituted with short chains of polysialic acid. Sialylated bi-, tri- [mainly the (2,4)-branched isomer], tetraantennary complex, polylactosamine and hybrid structures were detected. Typically, for 'brain-type' N-glycosylation, these sialylated structures were variously modified by the presence of core alpha1-6-linked and outer-arm alpha1-3-linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewis(x) and NeuAc alpha2-3 substituted Galbeta1-4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal beta1-3-linked galactose residues was observed. The presence of beta1-3-linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAc alpha2-3Galbeta1-3(NeuAc alpha2-6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N-glycans with more than one beta1-3galactose residue and (2,4)-branched triantennary oligosaccharides containing three such residues. Disialylated hybrid glycans containing beta1-3-linked galactose and 'polylactosamine' N-glycans with one to three terminal beta1-3galactose residues were additional novel features. The N-glycans modified by polysialylation lacked outer-arm fucose and bisecting GlcNAc residues but all contained one or more terminal beta1-3-linked galactose residues. These may be representative, therefore, of the polysialylated N-glycans expressed mainly on neural cell-adhesion molecules and known to be present in adult rat brain. The diversity of presentation of terminal sialylated groups in rat brain implies potential specificity for possible charge or lectin-mediated interactions. The distinguishing sets of sialylated structures described here are indicative of differences in the natural glycosylation processing pathways in different cell types within the central nervous system, a specificity that may be further magnified on the individual glycoproteins.     

Structural and functional characterisation of the signal recognition particle-specific 54 kDa protein (SRP54) of tomato

The major oligosaccharide chains of bovine, frog and human rhodopsins are identical in structure. These molecules are unique in terms of their abridged size as compared to other asparagine-linked glycoproteins, a property which must be reflected in the activities and properties of the glycosyltransferases of the retina that are concerned with their biosynthesis. One of the unusual characteristics of the major oligosaccharide chain is its lack of branching. A key enzymatic step required for branching to occur would be the transfer of a residue of N-acetylglucosamine to the terminal unsubstituted mannose residue of the major rhodopsin oligosaccharide. We have investigated the kinetic properties of enzymes that would catalyse this process. The N-acetylglucosaminyl-transferases (GlcNAc-transferases) of Golgi-enriched fractions of bovine retina, human retina, rat liver Golgi and a partially purified GlcNAc-transferase II from rat liver were examined using as acceptors rhodopsin, opsin and the oligosaccharide isolated from rhodopsin. The identification of the substrates and products of the reactions was carried out by chromatographic means. From an evaluation of the Vmax/K(m) ratios it was observed that bovine and human retinas have very limited abilities compared to the rat liver enzymes to carry out the transfer of GlcNAc to these acceptors, showing from 100- to about 800-fold lower efficiency in this regard. A comparison of the activities obtained with intact rhodopsin or opsin and the rhodopsin oligosaccharide indicated that the activity of GlcNAc-transferase II was not appreciably influenced by the polypeptide portion of the molecule. It was also observed that prior galactosylation of rhodopsin blocked the addition of GlcNAc to rhodopsin. It is suggested that these properties contribute to the assembly of the abridged structures that have been observed in the oligosaccharides of rhodopsins of all species thus far examined.     

Overview of N- and O-linked oligosaccharide structures found in various yeast species

Yeast and most higher eukaryotes utilize an evolutionarily conserved N-linked oligosaccharide biosynthetic pathway that involves the formation of a Glc3Man9GlcNAc2-PP-dolichol lipid-linked precursor, the glycan portion of which is co-translationally transferred in the endoplasmic reticulum (ER) to suitable Asn residues on nascent polypeptides. Subsequently, ER processing glycohydrolases remove the three glucoses and, with the exception of Schizosaccharomyces pombe, a single, specific mannose residue. Processing sugar transferases in the Golgi lead to the formation of core-sized structures (Hex<15GlcNac2) as well as cores with an extended poly-alpha1,6-Man 'backbone' that is derivatized with various carbohydrate side chains in a species-specific manner (Hex50-200GlnNAc2). In some cases these are short alpha1,2-linked Man chains with (Saccharomyces cerevisiae) or without (Pichia pastoris) alpha1,3-Man caps, while in other yeast (S. pombe), the side chains are alpha1,2-linked Gal, some of which are capped with beta-1,3-linked pyruvylated Gal residues. Charged groups are also found in S. cerevisiae and P. pastoris N-glycans in the form of mannose phosphate diesters. Some pathogenic yeast (Candida albicans) add poly-beta1,2-Man extension through a phosphate diester to their N-glycans, which appears involved in virulence. O-Linked glycan synthesis in yeast, unlike in animal cells where it is initiated in the Golgi using nucleotide sugars, begins in the ER by addition of a single mannose from Man-P-dolichol to selected Ser/Thr residues in newly made proteins. Once transported to the Golgi, sugar transferases add one (C. albicans) or more (P. pastoris) alpha1,2-linked mannose that may be capped with one or two alpha1,3-linked mannoses (S. cerevisiae). S. pombe is somewhat unique in that it synthesizes a family of mixed O-glycans with additional alpha1,2-linked Man and alpha1,2- and 1, 3-linked Gal residues.     

Purification and characterization of UDP-N-acetylglucosamine: alpha1,3-D-mannoside beta1,4-N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase-IV) from bovine small intestine

A new beta1,4-N-acetylglucosaminyltransferase (GnT) which involves in branch formation of Asn-linked complex-type sugar chains has been purified 224,000-fold from bovine small intestine. This enzyme requires divalent cations, such as Mn2+, and catalyzes the transfer of GlcNAc from UDP-GlcNAc to biantennary oligosaccharide and produces triantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-3 arm. The purified enzyme shows a single band of Mr 58,000 and behaves as a monomer. The substrate specificity demonstrated that the beta1-2-linked GlcNAc residue on the Manalpha1-3 arm (GnT-I product) is essential for the enzyme activity. beta1-4-Galactosylaion to this essential beta1-2-linked GlcNAc residue or N-acetylglucosaminylation to the beta-linked Man residue (bisecting GlcNAc, GnT-III product) blocks the enzyme action, while beta1-6-N-acetylglucosaminylation to the Manalpha1-6 arm (GnT-V product) increases the transfer. Based on these findings, we conclude that the purified enzyme is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV (GnT-IV), that has been a missing link on biosynthesis of complex-type sugar chains.     

Phosphorylated oligosaccharides in lysosomal enzymes: identification of alpha-N-acetylglucosamine(1)phospho(6)mannose diester groups

In human fibroblasts, the recognition of lysosomal enzymes by cell surface receptors is mediated by mannose 6-phosphate residues located on oligosaccharides that can be cleaved by endo-beta-N-acetylglucosaminidase H. About half of these oligosaccharides, as isolated from beta-hexosaminidase and cathepsin D secreted by human skin fibroblasts, are anionic. Most of these are resistant to alkaline phosphatase. The resistance is due to alpha-N-acetylglucosamine residues linked to mannose 6-phosphate by a phosphodiester bond. The major phosphorylated oligosaccharides contain one and two and possibly three phosphate groups blocked by N-acetylglucosamine. Besides the blocked phosphate groups these oligosaccharides contain a common inner core consisting of Man alpha 1,6-(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta GlcNAc and either one or two alpha 1,2-linked mannose residues.     

Garlic (Allium sativum) lectins bind to high mannose oligosaccharide chains

Two mannose-binding lectins, Allium sativum agglutinin (ASA) I (25 kDa) and ASAIII (48 kDa), from garlic bulbs have been purified by affinity chromatography followed by gel filtration. The subunit structures of these lectins are different, but they display similar sugar specificities. Both ASAI and ASAIII are made up of 12.5- and 11.5-kDa subunits. In addition, a complex (136 kDa) comprising a polypeptide chain of 54 +/- 4 kDa and the subunits of ASAI and ASAIII elutes earlier than these lectins on gel filtration. The 54-kDa subunit is proven to be alliinase, which is known to form a complex with garlic lectins. Constituent subunits of ASAI and ASAIII exhibit the same sequence at their amino termini. ASAI and ASAIII recognize monosaccharides in mannosyl configuration. The potencies of the ligands for ASAs increase in the following order: mannobiose (Manalpha1-3Man) < mannotriose (Manalpha1-6Manalpha1-3Man) approximately mannopentaose < Man9-oligosaccharide. The addition of two GlcNAc residues at the reducing end of mannotriose or mannopentaose enhances their potencies significantly, whereas substitution of both alpha1-3- and alpha1-6-mannosyl residues of mannotriose with GlcNAc at the nonreducing end increases their activity only marginally. The best manno-oligosaccharide ligand is Man9GlcNAc2Asn, which bears several alpha1-2-linked mannose residues. Interaction with glycoproteins suggests that these lectins recognize internal mannose as well as bind to the core pentasaccharide of N-linked glycans even when it is sialylated. The strongest inhibitors are the high mannose-containing glycoproteins, which carry larger glycan chains. Indeed, invertase, which contains 85% of its mannose residues in species larger than Man20GlcNAc, exhibited the highest binding affinity. No other mannose- or mannose/glucose-binding lectin has been shown to display such a specificity.