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.     

Structure determination for octasaccharides derived from the carbohydrate-protein linkage region of chondroitin sulphate chains in the proteoglycan aggrecan from bovine articular cartilage

Five octasaccharides derived from the protein carbohydrate linkage region of chondroitin sulphate (CS) have been isolated from the large aggregating proteoglycan (aggrecan) extracted from the bovine articular cartilage of 6-year-old to 8-year-old animals. Following the purification of aggrecan the attached CS chains were digested with CS ABC endolyase and subsequently released from the protein core by beta-elimination. The individual oligosaccharides were purified by strong anion-exchange chromatography and their structures determined by very high-field one-dimensional and two-dimensional 1H-NMR spectroscopy. They were found to be octasaccharides, comprised of tetrasaccharide repeat-region extensions to the core tetrasaccharide linkage region structure. They have the following structures: deltaUA(beta1-3)GalNAc(beta1-4)GlcA(beta1-3)GalNAc(beta1-4)+ ++GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc(beta1-4)GlcA(beta1-3)GalNAc6S(b eta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc(b eta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNA c6S(beta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol and deltaUA(beta1-3)GalNAc4S(beta1-4)GlcA(beta1-3)GalNA c6S(beta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol. They differ only in the nature of the sulphation of the GalNAc residues of the tetrasaccharide-repeat-region extension, which forms the first two disaccharides of the repeat region. No sulphation of any of the uronic acid residues has been identified and in one oligosaccharide neither of the GalNAc residues were sulphated. The majority of the linkage regions contained GalNAc residues which were fully 6-sulphated. However, in a significant amount, only one of the residues was 6-sulphated while the other was either unsulphated or 4-sulphated. There was no evidence either for sulphation of the linkage region galactose residues or for phosphorylation of the xylose residue, through which the chain is attached to the core protein.     

Delayed formation of proteoglycan aggregate structures in human articular cartilage disease states

Proteoglycans are major components of many extracellular matrices. In cartilage, they provide reversible resistance to compression and exist as molecules with molecular weights (MWs) of 1-3 x 10(6). There is a central protein core of MWs approximately 2 x 10(5) (refs 1, 2) with specialized subregions, one containing mainly the chondroitin sulphate chains, another most of the keratan sulphate chains, and a third is a largely globular structure interacting specifically with both hyaluronic acid and a link protein to form stable aggregate structures such as those identified in human articular cartilage. In embryonic and tissue culture systems, proteoglycans are isolated as aggregate structures in as little as 5--10 min after synthesis (sulphation) with no nonaggregating precursor detected. However, Heineg?rd and Hascall have characterized the small proportion of nonaggregating proteoglycan present in bovine nasal septum cartilage and found that it contained more peptide than the aggregating proteoglycan. Work by Upholt et al. has suggested that the MW of unprocessed protein core, synthesized by a wheat-germ translating system from chick sternal cartilage mRNA, is approximately 340,000, leaving open the possibility of intermediates. I report here the presence, in some human cartilages, of a proteoglycan population that initially will not aggregate with the hyaluronic acid but subsequently can be chased into aggregate.     

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.     

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.     

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.     

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 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.     

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.     

Enzymatic synthesis of site-specifically (alpha 1-3)-fucosylated polylactosamines containing either a sialyl Lewis (x), a VIM-2, or a sialylated and internally difucosylated sequence

By using two different reaction pathways, we generated enzymatically three sialylated and site-specifically alpha 1-3-fucosylated polylactosamines. Two of these are isomeric hexasaccharides Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc and Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4) GlcNAc, containing epitopes that correspond to VIM-2 and sialyl Lewis (x), respectively. The third one, nonasaccharide Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, is a sialylated and internally difucosylated derivative of a trimeric N-acetyllactosamine. All three oligosaccharides have one fucose-free N-acetyllactosaminyl unit and can be used as acceptors for recombinant alpha 1-3-fucosyltransferases in determining the biosynthesis pathways leading to polyfucosylated selectin ligands.     

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.     

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.     

Characterization of an N-acetylglucosamine-6-O-sulfotransferase from human respiratory mucosa active on mucin carbohydrate chains

A microsomal GlcNAc-6-O-sulfotransferase activity from human bronchial mucosa, able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate onto methyl-N-acetylglucosaminides or terminal N-acetylglucosamine residues of carbohydrate chains from human respiratory mucins, has been characterized. The reaction products containing a terminal HO3S-6GlcNAc were identified by high performance anion-exchange chromatography. Using methyl-beta-N-acetylglucosaminide as a substrate, the optimal activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, 5 mM AMP in a 30 mM MOPS (3-(N-morpholino) propanesulfonic acid) buffer at pH 6.7. The apparent Km values for adenosine 3'-phosphate 5'-phosphosulfate and methyl-beta-N-acetylglucosaminide were observed at 9.1 x 10(-6) M and 0.54 x 10(-3) M, respectively. The enzyme had more affinity for carbohydrate chains with a terminal GlcNAc residue than for methyl-beta-N-acetylglucosaminide; it was unable to catalyze the transfer of sulfate to position 6 of the GlcNAc residue contained in a terminal Galbeta1-4GlcNAc sequence. However, oligosaccharides with a nonreducing terminal HO3S-6GlcNAc were substrates for a beta1-4 galactosyltransferase from human bronchial mucosa. These data point out that GlcNAc-6-O-sulfotransferase must act before beta1-4 galactosylation in mucin-type oligosaccharide biosynthesis.     

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.     

Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc

The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in Gal beta 1, 4GlcNAc beta 1,6(Gal beta 1,3) GalNAc alpha-O-Bn, the enzyme had a higher affinity ( > 3-fold) for the Gal linked to GlcNAc. (q) With respect to Gal beta 1,- 3GlcNAc beta-O-Bn (3.0 mM), fetuin triantennary asialo glycopeptide (2.4 mM), bovine IgG diantennary glycopeptide (2.8 mM), asialo Cowper's gland mucin (0.06 mM), and the acrylamide copolymers (0.125 mM each) containing Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, Gal beta 1,3GalNAc alpha-, Gal beta 1,3Gal beta-, or Gal alpha 1,3Gal beta- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by alpha 1,2-L-FT of the galactosyl residue which occurs on the antennary structure of the bovine IgG glycopeptide was adversely affected by the presence of an alpha 1,6-L-fucosyl residue located on the distant glucosaminyl residue that is directly attached to the asparagine of the protein backbone. This became evident from the 4-fold activity of alpha 1,2-L-FT toward bovine IgG glycopeptide after approximately 5% removal of alpha 1,6-linked Fuo.     

Enzymatic properties of beta-D-galactoside alpha2 leads to 6 sialytransferase from bovine colostrum

The substrate specificity and kinetic properties of a pure sialyltransferase from bovine colostrum have been examined. The transferase appears to incorporate sialic acid into the sequence, NeuAcalpha2 leads to 6Galbeta1 leads to 4GlcNAc, which is commonly found in glycoproteins. It has a strict substrate specificity for CMP-NeuAc and forms only the alpha2 leads to 6 sialyl linkage with beta-D-galactosides. N-Acetyllactosamine (Galbeta1 leads to 4GlcNAc) and asialo-glycoproteins containing the N-acetyllactosaminyl linkage at the nonreducing ends of the oligosaccharides prosthetic groups are the best acceptor substrates. Isomers of N-acetyllactosamine with beta1 leads to 3 or beta1 leads to 6 glycosidic linkages are less than 1% as effective as acceptor substates as the beta1 leads to 4-linked isomer. Lactose (Galbeta1 leads to 4Glc) is also a poor acceptor, indicating the importance of the 2-acetamido group in the N-acetylglucosaminyl residues. The unnatural substrate beta-methyl-L-arabinopyrano-side, a five-carbon analog of beta-methyl-D-galactoside which contains no 6-hydroxyl, also acts as a poor acceptor of the transferase and the sialylated product has been partially characterized. Kinetic properties of the enzyme in the presence and absence of inhibitors suggest that the transferase has an equilibrium random order mechanism.     

Immunological characterisation of the acrosomal filament in the marine shrimp Sicyonia ingentis

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi-, 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.     

Processing glycosidases of Saccharomyces cerevisiae

The properties of the N-glycan processing glycosidases located in the endoplasmic reticulum of Saccharomyces cerevisiae are described. alpha-Glucosidase I encoded by CWH41 cleaves the terminal alpha1, 2-linked glucose and alpha-glucosidase II encoded by ROT2 removes the two alpha1,3-linked glucose residues from the Glc3Man9GlcNAc2 oligosaccharide precursor while the alpha1,2-mannosidase encoded by MNS1 removes one specific mannose to form a single isomer of Man8GlcNAc2. Although trimming by these glycosidases is not essential for the formation of N-glycan outer chains, recent studies on mutants lacking these enzymes indicate that alpha-glucosidases I and II play an indirect role in cell wall beta1,6-glucan formation and that the alpha1,2-mannosidase is involved in endoplasmic reticulum quality control. Detailed structure-function studies of recombinant yeast alpha1,2-mannosidase are described that serve as a model for other members of this enzyme family that has been conserved through eukaryotic evolution.     

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.     

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were analysed by sequential exoglycosidase digestion and gel filtration chromatography, following reductive tritiation. In addition, selected tryptic glycopeptides obtained from frog retinal rod outer segment membranes were examined by electrospray mass spectrometry (ES-MS), fast atom bombardment mass spectrometry (FAB-MS), amino acid sequence and composition analysis, and carbohydrate composition analysis. The amino acid sequence data demonstrated that the glycopeptides were derived from rhodopsin and confirmed the presence of two N-glycosylation sites, at residues Asn2 and Asn15. The predominant glycan (approximately 60% of total) had the structure GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6) Man beta 1-4GlcNAc beta 1-4GlcNAc-(Asn), with the remaining structures containing 1-3 additional hexose residues, as reported previously for bovine rhodopsin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with the sialylated oligosaccharides being present exclusively at the Asn2 site. FAB-MS analysis of oligosaccharides released from the Asn2 site gave, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in the context of rod outer segment membrane renewal.