Novikoff ascites tumor cells contain N-acetyllactosaminide beta 1 leads to 3 and beta 1 leads to 6 N-acetylglucosaminyltransferase activity

Novikoff ascites tumor cell homogenate was found to catalyze the transfer of [14C]N-acetylglucosamine from UDP-[14C]GlcNAc to asialo-alpha 1-acid glycoprotein. Mucins appeared to be poor acceptors. Methylation and hydrolysis of the product formed in an incubation with UDP-GlcNAc and asialo-alpha 1-acid [3H]glycoprotein yielded 2,4,6-trimethyl [3H]galactose and 2,3,4-trimethyl [3H]galactose, indicating that N-acetylglucosaminyl residues were introduced to position C-3 and C-6 of the terminal galactoses on the glycoprotein. It is concluded that Novikoff cells contain two N-acetylglucosaminyltransferases which might be involved in the synthesis of linear and branched forms of cell surface polylactosaminoglycans and blood group I/i antigenic structures.     

Separation into two major forms of beta(1-3)N-acetylglucosaminyltransferase from human serum

The major beta(1-3)N-acetylglucosaminyltransferase [beta(1-3)GlcNAc-transferase] activity in human serum was isolated by DEAE- and CM-Sepharose column chromatography. This enzyme fraction consisted of two forms of the enzyme, which were separated from each other on a DEAE-Sepharose column and designated as GNAc-TI and GNAc-TII, respectively. They have the same molecular weights (about 90,000), optimum pH values (between pH 7.5 and 8.5) and apparent Km values for N-acetyllactosamine and lactose (7.1-8.8 mM and 10.9-11.5 mM, respectively). Glycosidase and methylation analyses of the reaction products demonstrated that both the enzyme catalyze exclusively the transfer of one N-acetylglucosamine to position C-3 of the terminal galactose of lactose in the beta linkage.     

Cell-free biosynthesis of erythroglycan in a microsomal fraction from K-562 cells.

Particulate membrane preparations from K-562 [human CML (chronic-myelogenous-leukaemia)-derived] cells catalyse the transfer of [3H]galactose from UDP-[3H]-galactose and [3H]N-acetylglucosamine from UDP-[3H]N-acetylglucosamine into an endogenous product that on digestion with Pronase yields long-chain glycopeptides (mol.wt. 7000--10 000) called 'erythroglycan'. Incorporation of either labelled sugar increased up to 60 min of incubation time. The labelled erythroglycan was isolated by chromatography on Sephadex G-50 and characterized by digestion with endo-beta-galactosidase from Escherichia freundii, followed by analysis on Bio-Gel P-2 and paper chromatography. This digestion gave the following four products: (1) a disaccharide with the sequence beta GlcNAc-beta Gal; (2) a trisaccharide with the sequence betaGal-betaGlcNAc-beta Gal; (3) a larger oligosaccharide containing galactose and N-acetylglucosamine; and (4) a putative protein-linkage region.     

Branch specificity of bovine colostrum CMP-sialic acid: N-acetyllactosaminide alpha 2----6-sialyltransferase. Interaction with biantennary oligosaccharides and glycopeptides of N-glycosylproteins

By use of 500-MHz 1H NMR spectroscopy, the branch specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase towards a biantennary glycopeptide and oligosaccharides of the N-acetyllactosamine type, differing in completeness and structure of their core portion, was investigated. In agreement with earlier reports (Van den Eijnden, D. H., Joziasse, D. H., Dorland, L., Van Halbeek H., Vliegenthart, J. F. G., and Schmid, K. (1980) Biochem. Biophys. Res. Commun. 92, 839-845), it appears that the enzyme strongly prefers the galactosyl residue at the Man alpha 1----3Man branch of the biantennary glycopeptide for attachment of the first sialic acid residue. This branch specificity is fully preserved with the structure (formula; see text) Reduction of the reducing N-acetylglucosaminyl residue in this structure, however, leads to a decreased branch specificity, whereas removal of this residue results in a random attachment of sialic acid to the galactoses at both branches. The decrease in branch specificity is accompanied by a reduction in the rate of sialic acid transfer to the galactose at the alpha 1----3 branch. Our results indicate that the presence of the aforementioned N-acetylglucosaminyl residue is a minimal structural requirement for branch specificity of the sialyltransferase. We propose that in the interaction of the sialyltransferase with its substrates, this N-acetylglucosaminyl residue functions as a recognition site mediating the correct positioning of the substrate on the enzyme.     

The distribution of repeating [Gal beta 1,4GlcNAc beta 1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1

The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species.     

The presence of N-acetyllactosamine and lactose: beta (1-3)N-acetylglucosaminyltransferase activity in human urine

Normal human urine was found to contain beta (1-3)N-acetylglucosaminyltransferase catalyzing the transfer of N-acetylglucosamine from UDP-GlcNAc to N-acetyllactosamine and lactose. Lacto-N-tetraose which carries the terminal Gal beta (1-3)GlcNAc structure was a poor acceptor. The product of the transferase reaction with N-acetyllactosamine as acceptor was identified by methylation analysis as GlcNAc beta (1-3)Gal beta (1-4)GlcNAc. The beta-linkage of the GlcNAc in the synthesized trisaccharide was confirmed by the action of the specific beta-N-acetylhexosaminidase. The enzyme requires Mn2+ ions for its activity, shows a broad pH optimum from 7 to 9, and appears to have a molecular weight of about 200,000 as estimated by Sephadex gel filtration.     

Galactose transport in Streptococcus thermophilus

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Galactose transport in Streptococcus thermophilus.

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Biosynthesis in vitro of Ii core glycosphingolipids from neolactotetraosylceramide by beta 1-3- and beta 1-6-N-acetylglucosaminyltransferases from mouse T-lymphoma

The N-acetylglucosaminyltransferases probably involved in the biosynthesis in vitro of Ii core glycosphingolipids have been solubilized from a membrane preparation of mouse lymphoma P-1798 and partially characterized. The detergent-extracted membrane supernatant contains both beta 1-3- and beta 1-6-N-acetylglucosaminyltransferase activities that transfer [3H]GlcNAc from UDP-[3H]GlcNAc to the terminal galactose of neolactotetraosylceramide (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; nLcOse4ceramide), to form the Ii core structures. The linkage of [3H]N-acetylglucosamine incorporated into the terminal galactose of nLcOse4Cer was determined from identification of 2,4,6-tri-O-methyl[3H]galactose and 2,3,4-tri-O-methyl[3H]galactose after hydrolysis of the permethylated enzymatic products, GlcNAc beta-[3H]Gal-GlcNAc-Gal-Glc-ceramide. In addition to the presence of beta-N-acetylglucosaminyltransferases, we have detected a galactosyltransferase activity in this soluble supernatant fraction that catalyzes the transfer of [14C]galactose from UDP-[14C]galactose to lactotriaosylceramide (GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; LcOse3ceramide) to form nLcOse4ceramide, the acceptor in the N-acetylglucosaminyltransferase-catalyzed reaction.     

Effects of various inhibitors on β-galactosidase purified from the thermoacidophilic <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis> subsp. <Emphasis Type="Italic">Rittmannii</Emphasis> isolated from Antarctica

β-Galactosidase purified from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica is a member of the GH42 family. The enzyme was not effected by various concentrations of its reaction product glucose, but was greatly inhibited by the other reaction product galactose using both substrates, ONPG and lactose. Linewever-Burk plot analysis derived from both ONPG and lactose hydrolysis results showed that galactose is a mixed-type inhibitor of the purified β-galactosidase. The enzyme was slightly activated by Mg²⁺ (13% at 20 mM), while inhibited at higher concentrations of Ca⁺² (33% at 10 mM), Zn⁺² (86% at 8 mM) and Cu⁺² (87% at 4 mM). The enzyme activity was not significantly altered by the metal ion chelators EDTA and 1,10-phenanthroline up to 20 mM, indicating that this enzyme is not a metalloenzyme. 2-Mercaptoethanol and DTT were found to enhance β-galactosidase activity, while p-chloromercuribenzoic acid (PCMB) completely inhibited enzymatic activity (97% at 1 mM; 99.7% at 2 mM), indicating at least one essential Cys residue modified by the reagents in the active site of β-galactosidase. Iodoacetamide and Nethylmaleimide had little effect on the β-galactosidase. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme strongly (19.8% at 1 mM; 71.9% at 10 mM), also showing the participation of serine for enzyme activity.     

Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18.

The glycosylation of human cytokeratin (CK) 8 and 18 was studied after metabolic labeling of HT29 colonic cells with [3H]glucosamine. Labeling of CK8/18 was not inhibited by tunicamycin, suggesting that glycosylation was not N-linked. Acid hydrolysis of CK8 and CK18, purified from [3H]glucosamine-labeled cells, generated free glucosamine. In the presence of UDP-[3H]galactose, galactosyltransferase catalyzed the labeling of cytokeratin 8 and 18. beta-Elimination of the [3H]galactose- labeled CK8/18 generated the disaccharide N-acetyllactosaminitol, indicating that cytokeratin 8 and 18 contain single O-linked N-acetylglucosamine residues. Using chemical analysis, the stoichiometry of glycosylation was found to be 1.5 and 2 molecules of N-acetylglucosamine/protein molecule of CK8 and CK18, respectively. Peptide maps of [3H]glucosamine-labeled CK8/18 showed that multiple peptides were labeled with the amino sugar. The biosynthetic and degradation rates of the carbohydrate moiety were faster than the protein core as determined by metabolic radiolabeling or pulse-chase experiments, respectively. Our results show that CK8 and 18 are glycosylated at multiple sites with a single O-linked N-acetylglucosamine. Furthermore, CK8/18 glycosylation is a dynamic process which is likely to have functional relevance.     

Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser and GlcAβ1-3Gal(4-O-sulfate)β1-3Galβ1-4Xylβ1-O-Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with α-N-acetylgalactosaminidase and β-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was α-linked, as in the product previously synthesized using serum enzymes, and not β-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAβ1-4GlcNAcα1-4]2-GlcAβ1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides. This revised version was published online in November 2006 with corrections to the Cover Date.     

Lactose Uptake Driven by Galactose Efflux in Streptococcus thermophilus: Evidence for a Galactose-Lactose Antiporter

Galactose-nonfermenting (Gal^-) Streptococcus thermophilus TS2 releases galactose into the extracellular medium when grown in medium containing excess lactose. Starved and de-energized Gal^- cells, however, could be loaded with galactose to levels approximately equal to the extracellular concentration (0 to 50 mM). When loaded cells were separated from the medium and resuspended in fresh broth containing 5 mM lactose, galactose efflux occurred. De-energized, galactose-loaded cells, resuspended in buffer or medium, accumulated [¹⁴C]lactose at a greater rate and to significantly higher intracellular concentrations than unloaded cells. Uptake of lactose by loaded cells was inhibited more than that by unloaded cells in the presence of extracellular galactose, indicating that a galactose gradient was involved in the exchange system. When de-energized, galactose-loaded cells were resuspended in carbohydrate-free medium at pH 6.7, a proton motive force (Δp) of 86 to 90 mV was formed, whereas de-energized, nonloaded cells maintained a Δp of about 56 mV. However, uptake of lactose by loaded cells occurred when the proton motive force was abolished by the addition of an uncoupler or in the presence of a proton-translocating ATPase inhibitor. These results support the hypothesis that galactose efflux in Gal^-S. thermophilus is electrogenic and that the exchange reaction (lactose uptake and galactose efflux) probably occurs via an antiporter system.     

Radiometric assays of N-acetylglucosaminylphosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase with substrates labeled in the glucosamine moiety

The assay of fibroblast and leukocyte-N-acetylglucosaminylphosphotransferase with alpha-methylmannoside acceptor and commercially available UDP-[3H or 14C]N-acetylglucosamine donor was modified to yield low background and consequently high sensitivity and reliability comparable to those obtained with the synthetically made [beta-32P]UDP-N-acetylglucosamine donor. This was achieved by an additional elution step that removed free [3H or 14C]N-acetylglucosamine which appeared to be the breakdown product responsible for the high background. In addition, the [3H or 14C]N-acetylglucosamine-1-phospho-6-alpha-methylmannoside product of the transfer reaction was then isolated and, following desalting, could serve as a substrate for the assay of alpha-N-acetylglucosaminyl phosphodiesterase. Cell preparations of patients with I-cell disease and pseudo-Hurler polydystrophy demonstrated severe to moderate deficiency of transferase activity and normal phosphodiesterase activity toward the respective substrates labeled with 3H or 14C in the glucosamine moiety.     

Modulation of two distinct galactosyltransferase activities in populations of mouse peritoneal macrophages

We have examined two galactosyltransferase activities in membrane preparations obtained from resident macrophages, from resident macrophages maintained in culture for 24 hr, and from thioglycollate (TG)-elicited macrophages. Transfer of galactose from uridine diphosphate (UDP)-galactose to N-acetylglucosamine is 2.6 times higher in membranes prepared from TG macrophages (107 +/- 5.5 nmol/hr/mg) than in membranes prepared from resident macrophages (41 +/- 2.0 nmol/hr/mg). Membranes obtained from resident macrophages cultured for 24 hr exhibit a 2.5 times higher activity (102 +/- 4.4 nmol/hr/mg) than membranes from resident cells plated for 4 hr. Transferase activity in membranes derived from TG macrophages is not significantly affected by overnight culture. The transferase reaction product, isolated on Bio-Gel P-4 and analyzed by galactosidase treatments, was identified as galactosyl-beta 1, 4-N-acetylglucosamine. The enzyme, therefore, is UDP-galactose:2-acetamido-2-deoxy-D-glucose 4 beta-galactosyltransferase. This is supported by the fact that this galactosyltransferase activity is specifically inhibited by high concentrations of N-acetylglucosamine (200 mM). We have also examined the transfer of galactose to N-acetyllactosamine. Membranes from TG-elicited macrophages contain a UDP-galactose:galactosyl-beta 1, 4-N-acetylglucosamine 3 alpha-galactosyltransferase which synthesizes the trisaccharide, galactosyl-alpha 1, 3-galactosyl-beta 1,4-N-acetylglucosamine. This product was identified by gel filtration chromatography, high performance liquid chromatography, and galactosidase digestions. This alpha-galactosyltransferase activity was not detected in membranes prepared from resident macrophages. These results indicate that glycosyltransferase activities are modulated in populations of mouse macrophages, and that these changes correlate with changes in cell surface lactosaminoglycans reported previously.     

Characterization of a high molecular weight glycoprotein secreted by the peri-implantation bovine conceptus

Cow conceptuses were flushed from uteri on Day 17 of pregnancy and cultured with [3H]glucosamine and [14C]leucine. A high molecular weight glycoprotein (HMWG) having an Mr = 765,000 was isolated by a combination of anion-exchange and gel-filtration chromatography. Selective chemical and enzymatic degradations were performed. The HMWG was resistant to Pronase and peptide: N-glycanase F. Only endo-beta-galactosidase and harsh alkaline reducing conditions were successful in dissociating carbohydrate from the protein core, suggesting that carbohydrate chains are N-linked to Asn and contain beta-galactosidic linkages. The intact molecule could bind to an affinity column of Datura stramoniom lectin, suggesting the presence of beta(1-4)-linked oligomers of N-acetylglucosamine. The susceptibility of HMWG to endo-beta-galactosidase suggests that at least some of these oligomers are substituted with galactose to form N-acetyllactosamine. Binding of HMWG to lectin could be inhibited partially with N-acetyllactosamine or completely with a mixture of N, N'-diacetylchitobiose and N, N', N"-triacetylchitotriose. In summary, properties of the HMWG suggest it contains lactosaminoglycan components and is almost identical to an HMWG secreted by the Day 16 ovine conceptus. Thus, embryos of these two ruminant species secrete similar molecules during early pregnancy.     

Pentose cycle and reducing equivalents in rat mammary-gland slices

1. Slices of mammary gland of lactating rats were incubated with glucose labelled uniformly with (14)C and in positions 1, 2, 3 and 6, and with (3)H in all six positions. Glucose carbon atoms are incorporated into CO(2), fatty acids, lipid glycerol, the glucose and galactose moieties of lactose, lactate, soluble amino acids and proteins. C-3 of glucose appears in fatty acids. The incorporation of (3)H into fatty acids is greatest from [3-(3)H]glucose. (3)H from [5-(3)H]glucose appears, apart from in lactose, nearly all in water. 2. The specific radioactivity of the galactose moiety of lactose from [1-(14)C]- and [6-(14)C]-glucose was less, and that from [2-(14)C]- and [3-(14)C]-glucose more, than that of the glucose moiety. There was no randomization of carbon atoms in the glucose moiety, but it was extensive in galactose. 3. The pentose cycle was calculated from (14)C yields in CO(2) and fatty acids, and from the degradation of galactose from [2-(14)C]glucose. A method for the quantitative determination of the contribution of the pentose cycle, from incorporation into fatty acids from [3-(14)C]glucose, is derived. The rate of the reaction catalysed by hexose 6-phosphate isomerase was calculated from the randomization pattern in galactose. 4. Of the utilized glucose, 10-20% is converted into lactose, 20-30% is metabolized via the pentose cycle and the rest is metabolized via the Embden-Meyerhof pathway. About 10-15% of the triose phosphates and pyruvate is derived via the pentose cycle. 5. The pentose cycle is sufficient to provide 80-100% of the NADPH requirement for fatty acid synthesis. 6. The formation of reducing equivalents in the cytoplasm exceeds that required for reductive biosynthesis. About half of the cytoplasmic reducing equivalents are probably transferred into mitochondria. 7. In the Appendix a concise derivation of the randomization of C-1, C-2 and C-3 as a function of the pentose cycle is described.     

Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal beta-linked N-acetylgalactosamine

Many studies have shown that the human blood fluke Schistosoma mansoni contains glycoproteins whose oligosaccharide side chains are antigenic in infected hosts. We report here that adult male schistosomes synthesize glycoproteins containing complex-type N-linked chains that have structural features not commonly found in mammalian glycoproteins. Adult male worms were incubated in media containing either [3H]mannose, [3H]glucosamine, or [3H]galactose, and the metabolically radiolabeled oligosaccharides on newly synthesized glycoproteins were analyzed. Schistosomes synthesize triantennary- and biantennary-like complex-type asparagine-linked chains that contain mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Interestingly, none of the complex-type chains contain sialic acid, and few of the chains contain galactose. Since N-acetylgalactosamine is not a common constituent of mammalian-derived N-linked chains, we investigated the position and linkage of this residue in the schistosome-derived glycopeptides. Virtually all of the N-acetylgalactosamine was beta-linked and in a terminal position. The unusual features of the S. mansoni glycoprotein oligosaccharides support the possibility that they may be involved in the host immune response to infection.     

Enzymes responsible for synthesis of corneal keratan sulfate glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta3GnT7/beta4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.     

Escherichia coli Heat-Labile Enterotoxin Binds to Glycosylated Proteins with Lactose by Amino Carbonyl Reaction

The binding of Escherichia coli heat-labile enterotoxin (LT) type I to glycosylated proteins with lactose (Galβ1-4Glc) by amino carbonyl reaction was studied by the Western blot assay and by the microtiter well binding assay. LT bound to a lactose-α-lactalbumin amino carbonyl product (Lac-LA), whereas cholera toxin did not. The binding ability of Lac-LA was abolished by β-galactosidase treatment, indicating that the terminal galactose is essential for the binding of LT. The binding of LT to Lac-LA was inhibited by galactose and lactose, and most effectively inhibited by lactulose (Galβ1-4Fru), which is a structural analog of the Amadori rearrangement product of the amino carbonyl reaction between lactose and an ε-amino group of a lysine residue (lactuloselysine). The results suggest that LT recognizes the portion of lactuloselysine in Lac-LA. LT also bound to a melibiose (Galα1-6Glc)-α-lactalbumin amino carbonyl product (Mel-LA), but the binding ability of Mel-LA was weaker than that of Lac-LA, suggesting that the β1-4 linked terminal galactose is dispensable but preferable for the binding. Furthermore, LT bound to the amino carbonyl products of lactose with β-lactoglobulin, caseins, bovine serum albumin, and ovalbumin. These results indicate that LT binds to the amino carbonyl products between proteins and sugars containing the terminal galactose, such as lactose.