Yuan C. Lee

Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection

The method of anion exchange chromatography followed by pulsed amperometric detection (AE-PAD; Johnson, D. C., and Polta, T. Z. (1986) Chromatogr. Forum 1, 37-44) has been applied to the compositional analysis of glycoconjugates. Using 22 mM NaOH as a column effluent, underivatized fucose, galactosamine, glucosamine, galactose, glucose, and mannose were readily separated in 15 min at a flow rate of 1 ml/min. The limit of quantification of the monosaccharides was better than 100 pmol (signal to noise ratio 184:1). AE-PAD was employed to quantify the monosaccharides of several glycoproteins, glycopeptides, and oligosaccharides after hydrolysis with 2 M trifluoroacetic acid. Both neutral and amino sugars could be rapidly estimated in a single chromatographic step using AE-PAD. Complete release of N-acetylglucosamine required more vigorous hydrolysis conditions (Lee, Y. C. (1972) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 28, pp. 63-73, Academic Press, New York). In both glycopeptides and oligosaccharides, approximately one less residue of Man than predicted was determined. Both AE-PAD and liquid chromatographic analysis of borate-monosaccharide complexes with fluorometric detection (Mikami, H., and Ishida, Y. (1983) Bunseki Kagaku 32, E207-E210) gave similar quantification of mannose and other sugars. The capability of rapid, sensitive quantification of underivitized monosaccharides should facilitate structural analysis of glycoconjugates.

Affinity enhancement by multivalent lectin-carbohydrate interaction.

The binding of simple carbohydrate ligands by proteins often requires affinity enhancement to attain biologically relevant strength. This is especially true for endocytotic receptors and the molecules that engage in the first-line of defense. For such purposes, nature often utilizes a mode of affinity enhancement that arises from multiple interactions between the binding proteins and the carbohydrate ligands, which we term glycoside cluster effect. In this review article we give a number of examples and describe important factors in the multi-valent interactions that govern the degree of affinity enhancement.

Synthesis of 3-(2-aminoethylthio)propyl glycosides

Abstract Anomeric pairs of 3-(2-aminoethylthio)propyl d -galactopyranoside ( 4, 4a ), d -glucopyranoside ( 5, 5a ), and 2-acetamido-2-deoxy- d -glucopyranoside ( 6, 6a ) were prepared by addition of 2-aminoethanethiol to the corresponding, anomeric, allyl glycosides. The allyl α-glycosides were prepared by refluxing the sugars with allyl alcohol in the presence of an acid catalyst; the allyl β-glycosides were prepared by the reaction of acetylated glycosyl bromides with allyl alcohol in the presence of mercuric cyanide, followed by O -deacetylation. The rate of thiol addition to the allylic group was found to be different for each glycoside.

Microheterogeneity of the carbohydrate group of Aspergillus oryzae α-amylase☆☆☆★

Abstract α-Amylase from Aspergillus oryzae was purified by gel filtration and ion-exchange chromatography. It appeared to be homogeneous by ultracentrifugation, electrophoresis, and ion-exchange chromatography, and contained glucosamine, mannose, galactose, arabinose, and xylose. Exhaustive proteolysis of the amylase yielded Ser · Asp(NH · Oligosaccharide) and Asp(NH · Oligosaccharide). By chromatography on Dowex 50-X2, the latter glycopeptide could be fractionated into several peaks, each having a different sugar composition. This implies a heterogeneity of the carbohydrate group of the α-amylase.

High-performance anion-exchange chromatography of oligosaccharides using pellicular resins and pulsed amperometric detection

High-performance liquid chromatography using pellicular quaternary amine-bonded resins was used to separate a variety of neutral, sialylated, and phosphorylated oligosaccharides. At pH 4.6, sialylated compounds were separated according to number of negative charges, sialic acid linkage [alpha(2,3) compared to alpha(2,6)], and position of sialic acid linkage along a linear saccharide chain. At pH 13, the neutral sugar portion of the sialylated chain had a significant effect on the separation, due to oxyanion formation. Specifically, sialylated tetrasaccharides containing the Gal beta(1,3)GlcNAc sequence were retained much more than their Gal beta(1,4)GlcNAc- or Gal-beta(1,4)GalNAc-sialylated counterparts. Linear phosphorylated oligosaccharides could be completely separated according to number of charges and net carbohydrate content. Partial separation of linear-chain positional isomers, differing in either location of Man-6-PO4 in the chain or linkage position of Man or Man-6-PO4, was accomplished. Branched-chain phosphorylated compounds could be completely separated according to which antennae contained the Man-6-PO4. The electrochemical current generated by oxidation of sialylated, phosphorylated, and neutral oligosaccharides was compared to that of a glucose. The relative molar response factors for neutral, sialylated, and phosphorylated oligosaccharides ranged from 0.2 to 3.2. Neutral oligosaccharides gave the following molar responses for each group of structurally related compounds: (1) mono- and disaccharide, 1-1.3; (2) linear tri- and tetrasaccharides, 1.5-2.0; and (3) branched pentasaccharide-nonasaccharides, 2.4-3.1. Response factors for the sialyated compounds were not as consistent and were affected by linkage position of sialic acid. For oligosaccharides of the same size, increasing phosphorylation resulted in a twofold decrease in response factor for each added phosphate group. Therefore, conversion of sialylated and phosphorylated oligosaccharides to their neutral counterparts, using alkaline phosphatase or neuraminidase, respectively, was required for quantitative analysis of oligosaccharide mixtures using electrochemical response. Using this approach, complete separation of the parent neutral structures was obtained, the relative proportions of the neutral species were quantified, and the amount of sialic acid released was easily determined in a neuraminidase digest.

Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines

In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N-acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N-acetylglucosaminidase that removes a terminal N-acetylglucosamine from the N-glycan. The innermost N-acetylglucosamine residue attached to asparagine residue is also modified with α(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP-N-acetylneuraminic acid, required for sialylation. Inhibition of N-acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans. Published in 2004.

The synthesis of 4-methylumbelliferyl α-ketoside of N-acetylneuraminic acid and its use in a fluorometric assay for neuraminidase

Abstract 4-Methylumbelliferyl α-ketoside of N-acetylneuraminic acid was synthesized by reacting the sodium salt of 4-methylumbelliferone with the 2-chloro-2-deoxy derivative of peracetylated methyl N-acetylneuraminate, followed by preparative silica gel chromatography, deblocking, and purification by gel filtration on Sephadex G-25. The final product was isolated as either the sodium or ammonium salt, and its suitability as a substrate for neuraminidase was evaluated. The optimal pH values for various neuraminidases were 5.6 in acetate buffer (Arthrobacter ureafaciens), 5.0–5.1 in acetate buffer (Clostridium perfringens), and 4.4 in phosphate-citrate buffer (human fibroblasts). Km values for these enzymes at the optimal pH were 6 × 10−4 m (Arthrobacter), 1 × 10−4 m (Clostridium), and 3 × 10−4 m (human fibroblasts).

Inhibition of adhesion of type 1 fimbriated Escherichia coli to highly mannosylated ligands.

The inhibitory potencies of a number of mannosides, di‐ and trivalent mannosides, a set of mannose‐terminating dendrimers, and five types of mannose‐bearing neoglycoproteins were determined by using a binding assay that measures the binding of 125I‐labeled, highly mannosylated neoglycoprotein to a type 1 fimbriated Escherichia coli (K12) strain in suspension. The IC50 values (the concentration of inhibitor that causes 50 % reduction in the bound 125I‐ligand to E. coli) obtained by this method were much lower than the equivalent values obtained by hemagglutination or in assays that involve microplate immobilization. Two important factors that strongly influence the affinity to E. coli adhesin are: 1) the presence of an α‐oriented aglycon that has a long aliphatic chain or an aromatic group immediately next to the glycosyl oxygen, and 2) the presence of multiple mannosyl residues that can span a distance of 20 nm or longer on a relatively inflexible structure. The two best inhibitors, which are a highly mannosylated neoglycoprotein with the longest linking arm between a mannose and protein amino group and the largest mannosylated dendrimer (fourth generation), exhibited sub‐nM IC50 values.

Rapid and sensitive determination of sphingosine bases and sphingolipids with fluorescamine.

Abstract A rapid and sensitive method is described for determining sphingosine and sphingolipids in the 1–100 nanomole range. Sphingosine is released from the sphingolipids by hydrolysis with hydrochloric acid in aqueous methanol, and then reacted with fluorescamine at pH 8.0. The same fluorescence intensities were obtained with equimolar concentrations of sphingosine, psychosine, cerebroside, and sphingomyelin. A hexosamine-containing sphingolipid, ganglioside, gave about twice the expected fluorescence. This result is explained by the fact that hexosamines and other primary amines react with fluorescamine. However, the method was easily modified to determine sphingosine in gangliosides by extracting the hydrophobic base from the hydrolysis mixture with ether. The procedure should have broad application in the field of sphingolipid chemistry and biochemistry.

Preparation of cluster glycosides ofN-acetylgalactosamine that have subnanomolar binding constants towards the mammalian hepatic Gal/GalNAc-specific receptor

Preparation of di-and tri-valent cluster glycosides containingN-acetyl-d-galactosamine (GalNAc) is described. Oligopeptides that contain a protected amino group and two or three free carboxyl groups are activated by methyl chloroformate and then coupled to 6-aminohexyl 2-acetamido-2-deoxy-β-d-galactopyranoside. The concentrations of the divalent GalNAc glycosides needed to produce 50% inhibition of the binding of asialoorosomucoid to the isolated, purified rat liver receptor specific for galactose and GalNAc and to the receptor on the hepatocyte surface were of the order of 10−8 M and 10−9 M, respectively. The binding affinity of the trivalent glycoside was 10-to 20-fold stronger than the divalent glycosides towards both the soluble receptor and intact hepatocyte.