Sumihiro Hase

Structure analyses of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound.

Summary A potential aldehyde group of an oligosaccharide is combined with 2-aminopyridine by means of the reductive amination with sodium cyanoborohydride giving a fluorescent 2-aminopyridine derivative. The sugar derivative can be used for the purpose of the determination of 1, a degree of polymerization; 2, sequence of the sugar units; 3, the linkage points of the sugar units, and the preparation of finger prints.

Microquantitative analysis of neutral and amino sugars as fluorescent pyridylamino derivatives by high-performance liquid chromatography

A method for the quantitation of picomole amounts of neutral and amino sugars in glycoconjugates was developed. Glycoconjugates were hydrolyzed with a mixture of equal amounts of 4 M trifluoroacetic acid and 4 M hydrochloric acid, and the free amino groups were acetylated. Sugars were coupled with 2-aminopyridine. After the excess reagents were removed by gel-permeation high-performance liquid chromatography, the fluorescent pyridylamino derivatives of sugars were separated and quantified by high-performance liquid chromatography on a reversed-phase column. This method allowed the determination of 0.01-10 nmol of sugars. About 100 pmol of several glycoconjugates were analyzed by the present method, with satisfactory results.

Precolumn derivatization for chromatographic and electrophoretic analyses of carbohydrates

Sensitive detection and high chromatographic resolution are required for analysing the structures of oligosaccharides available in only limited amounts. Precolumn derivatization is one of the most suitable methods for this purpose as it can compensate for the shortcomings of the intrinsic nature of oligosaccharides. Recently published methods of precolumn derivatization are reviewed and advantages and problems are discussed.

Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins

A mixture of the pyridylamino (PA) derivatives of 12 oligomannose-type sugar chains was fractionated into five fractions (mannose5N-acetylglucosamine2-PA approximately mannose9N-acetylglucosamine2-PA) by size-fractionation HPLC with a MicroPak AX-5 column. Each fraction thus obtained was then analyzed by reversed-phase HPLC with a Cosmosil 5C18-P column. In this way, the 12 PA-oligomannose-type sugar chains were completely separated from each other. The method was used to identify the structures of oligomannose-type sugar chains of human C3, the third component of human complement.

Estimation of elution times on reverse-phase high-performance liquid chromatography of pyridylamino derivatives of sugar chains from glycoproteins.

Addition of a sugar residue to a pyridylamino (PA) sugar chain affects its elution time on reverse-phase HPLC and the contribution of the sugar residue is not influenced by the other sugar residues [S. Hase, S. Natsuka, H. Oku, and T. Ikenaka (1987) Anal. Biochem. 167, 321-326]. The partial relative elution times (Ei) of 22 sugar residues were calculated from the relative elution times (the elution time relative to Man5Glc-NAc2-PA) of three kinds of PA-sugar chain: the oligomannose and N-acetyllactosamine types and sugar chains with a xylose residue. The relative elution time of a PA-sugar chain can be calculated by summing the Ei values of all of the constituent sugar residues. The calculated relative elution times of 44 PA-sugar chains agreed well with the observed values. This method can be used to estimate the relative elution times of PA-sugar chains that are not yet available, and to estimate the structures of sugar chains in limited amounts of glycoproteins by a combination of sugar composition analysis and exoglycosidase digestion.

Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-α-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates

Polygalacturonate 4-α-galacturonosyltransferase (pectin synthase) was solubilized from pollen tubes of Petunia axillaris and characterized. To accomplish this, an assay method using fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as acceptor substrates was developed. When the pollen tube enzyme was solubilized with 0.5% (v/v) Triton X-100 and was incubated with PA-OGA and UDP-galacturonic acid (UDP-GalUA), successive transfer activity of more than 10 GalUAs from UDP-GalUA to the nonreducing end of PA-OGA was observed by diethylaminoethyl high-performance liquid chromatography. This activity was time- and enzyme concentration-dependent. The optimum enzyme activity was observed at pH 7.0 and 30°C. Among the PA-OGAs investigated, those with a degree of polymerization of more than 10 were preferred as substrates. The crude pollen tube enzyme had an apparentK m value of 13 μm for the PA-OGA with a degree of polymerization 11 and 170 μm for UDP-GalUA. The characteristics of the P. axillarispollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed.

Fluorescence method for the structural analysis of oligomannose-type sugar chains by partial acetolysis☆

Fluorescence labeling was used in the analysis of partial acetolysis products of oligomannose-type sugar chains with five to nine mannose residues. The principle of the method was the pyridylamination of fragments obtained by the partial acetolysis of pyridylamino sugar chains and the identification of the fragments with an HPLC apparatus equipped with a fluorescence spectrophotometer. The method was tested by analysis of eight oligomannose-type sugar chains with known chemical structures and was found to be effective for analysis of branching structures with samples of 0.5 nmol.

Separation of oligomannose-type sugar chains having one to five mannose residues by high-performance liquid chromatography as their pyridylamino derivatives

Ten oligomannose-type sugar chains (ManGlcNAc2-Man5GlcNAc2) were prepared from various glycoproteins and fluorescence labeled with 2-aminopyridine. The fluorescent pyridylamino (PA)-sugar chains were first separated into five fractions according to their molecular sizes by HPLC on a TSK gel Amide-80 column. Each fraction was then separated into the component PA-sugar chains by reversed-phase HPLC on a Capcell Pak C18 column according to their chemical structures. The method is useful for studying the substrate specificities of alpha-mannosidases with Man5GlcNAc2-PA as a substrate.

Analysis of tissue glycoprotein sugar chains by two-dimensional high-performance liquid chromatographic mapping.

Excellent separation of 45 pyridylamino derivatives of oligosaccharides were achieved by the two-dimensional combination of reversed-phase and size-fractionation high-performance liquid chromatography. The sugar chains of brain glycoproteins were derivatized into a mixture of pyridylamino-oligosaccharides from lyophilized brain tissue without any purification steps, and they were well separated by the system used. The pattern obtained was reproducible, and inter-individual variation was negligible. This finding demonstrated the possibility that this method could be applied to the detection of differences in the structure of glycoprotein sugar chains in crude preparations.

Structural analysis of N-glycans of the planarian Dugesia japonica

To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N‐glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N‐glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2‐aminopyridine at their reducing end. The labeled N‐glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N‐glycans, it was shown that the N‐glycans of planarian include high mannose‐type and pauci‐mannose‐type glycans. However, many of the major N‐glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3‐O‐methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non‐reducing‐end mannose, as the glycans were resistant to α‐mannosidase digestion. These results indicate that methylated high‐mannose‐type glycans are the most abundant structure in planarians.