Copper Tolerance of a New Strain of Penicillium ochro-chloron

Pyridylamino derivatization is suitable for the microanalysis of sugars, but there is a problem in that by-products of the labeling reaction and fluorescent substances from samples occasionally interfere with the detection of pyridylamino sugars. Especially, interference by them is serious in monosaccharide analysis. I have developed a convenient purification method for pyridylamino monosaccharides by the use of a spin column with boronate-conjugated resin.     

Extraction method for preparing pyridylamino sugar derivatives and application to porcine gastric mucus glycoprotein analysis

For the sensitive detection of free sugars and oligosaccharides, the use of their pyridylamino derivatives has now found general acceptance. To remove excess 2-aminopyridine from this derivative in a reaction mixture, gel filtration and ion-exchange chromatography were conducted. It was found in the present study that contaminated 2-aminopyridine could be selectively removed from the reaction mixture by adjusting the pH with saturated sodium bicarbonate at above 8.5 followed by extraction with benzene. By using this method, fewer purification steps and less time are required, with minimum loss of pyridylamino sugar derivatives.     

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.     

Water Soluble Pigments Containing Xylose and Glucose in Gametangia of the Green Alga, Bryopsis maxima

Several water-soluble pigments were purified from gametangiaof Bryopsis maxima by liquid chromatography and characterizedby pyridylamination and high-performance anion-exchange chromatography.The structure of the main red pigment is proposed based on thedata of infrared spectrum, Mass spectrum, ¹H and ¹³C NMR spectraand pyridylamino analysis. As a consequence, this pigment containeda tetrapyrrole with phytol and a sugar chain comprised of xyloseand glucose. The sequence of the sugars in the chain was determinedbased on its Mass spectrum. The pigment was similar to chlorophyll-originpigments observed in other plants. No aldehyde group, however,was present at C5 in the open tetrapyrrole chain. (Received August 3, 1994; Accepted November 10, 1994)     

Determination of lectin-sugar binding constants by microequilibrium dialysis coupled with high performance liquid chromatography

We used high performance liquid chromatography to determine the concentrations of free ligands after equilibrium dialysis to examine lectin-sugar interactions. The binding of p-nitrophenyl 1-thio-alpha-mannoside, p-nitrophenyl N-acetyl-1-thio-beta-glucosaminide, and the pyridylamino derivative of Man6GlcNAc2 to concanavalin A and Triticum vulgaris lectin was examined. The binding constant, Ka, and the concentration of total binding sites, [L]t, were calculated from the trace amounts of sugars and lectins using the equation, [S]/[LS] = 1/Ka[L]t + [S]/[L]t, where [S] is the concentration of free ligand and [LS] the concentration of bound ligand.     

Application of 2-aminopyridine fluorescence labeling to glycosaminoglycans

A pyridylamination method was applied to glycosaminoglycans and the characteristics of the resulting pyridylamino glycosaminoglycans were examined. First, glycosaminoglycan chains, which uniformly possess a xylose residue at their reducing termini, were liberated from proteoglycan by successive digestion with protease and endo-beta-xylosidase. Then the glycosaminoglycan chains were coupled with 2-aminopyridine by reductive amination with sodium cyanoborohydride for 15 h according to the method of Hase, S. et al. [J. Biochem. 95, 197-203 (1984)]. The pyridylamination reaction caused neither depolymerization, de-N-acetylation, nor de-N- or de-O-sulfation. The pyridylamino glycosaminoglycan chains had an intact linkage region (GlcA-Gal-Gal-Xyl) between the carbohydrate chain and the peptide core of the proteoglycan. These pyridylamino glycosaminoglycans should be useful as substrates for endo-type glycosidases that act on glycosaminoglycan chains and as markers for studies of glycosaminoglycan metabolism.     

Presence of an endo-beta-galactosidase degrading the linkage region between the chondroitin sulfate chain and core peptide of proteoglycan

Pyridylamino chondroitin sulfate, of which the reducing terminal xylose was coupled with a fluorescent 2-aminopyridine, was incubated at pH 4.0 with an extract from the mid-gut gland of Patnopecten. The high- and low-molecular-weight products were separated by ethanol precipitation, and identified by high-performance liquid chromatography analysis. The enzyme was found to expose a galactose residue at the reducing terminus of chondroitin sulfate, and also released the pyridylamino disaccharide, galactosylxylose, from the reducing terminal site of pyridylamino chondroitin sulfate. These results suggest that endo-beta-galactosidase activity, which hydrolyzes the galactosylgalactose linkage of peptidochondroitin sulfate, is present in the mid-gut gland of Patnopecten.     

Galactose recognition by a tetrameric C-type lectin, CEL-IV, containing the EPN carbohydrate recognition motif

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.     

Structural analyses of asparagine-linked oligosaccharides of porcine pancreatic kallikrein

The structures of asparagine-linked oligosaccharides of porcine pancreatic beta-kallikrein are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The mixture of pyridylamino oligosaccharides was separated by reverse-phase and amide-adsorption high-performance liquid chromatography. The pyridylamino oligosaccharides were separated into more than 50 kinds of oligosaccharides. The structures of 5 kinds of triantennary and 12 kinds of tetraantennary oligosaccharides were determined by the use of high-resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Furthermore, the structures of five kinds of oligomannose-type oligosaccharides were elucidated by a combination of exoglycosidase digestion and high-performance liquid chromatography. 1H NMR data for 14 out of the 17 kinds of N-acetyllactosamine-type oligosaccharides reported here have not previously been described in the literature. (1) It has been shown that fucose containing tri- and tetraantennary oligosaccharides is predominant in porcine pancreatic beta-kallikrein B. (2) It has also been shown that the heterogeneity of the structure in these types of oligosaccharides is derived from the variety of the positions of galactose residues linked to outer N-acetylglucosamine residues. (3) The distribution of oligosaccharides into two glycosylation sites, asparagine-95 and asparagine-239, of beta-kallikrein B was determined. It has been found that oligomannose-type oligosaccharides are exclusively present at asparagine-239, although N-acetyllactosamine-type oligosaccharides occur at both glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of T-antigen, a carcinoma marker from native human cells, by endo-alpha-N-acetylgalactosaminidase of Alcaligenes sp

The endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. could release T-antigen (Ga1 beta 1----3GalNAc), a carcinoma-associated marker from asialo glycophorin and human cells. The released T-antigen from human asialo erythrocytes was determined by thin layer chromatography and gas liquid chromatography. The released T-antigen from human gastric carcinoma cell Kato III was identified by high performance liquid chromatographies with a reverse-phase column and a size fractionation column. Released T-antigen could be analyzed quantitatively by a sensitive method including pyridylamino derivatization and following high performance liquid chromatography. This suggests that the enzyme is useful for detection and determination of T-antigen from cells.     

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.     

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.     

Improved Method for Drawing of a Glycan Map,and the First Page of Glycan Atlas,Which Is a Compilation of Glycan Maps for a Whole Organism

Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC) is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA-) glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.     

Purification and characterization of neutral alpha-mannosidase that is activated by Co2+ from Japanese quail oviduct

An alpha-mannosidase was purified from the magnum section of Japanese quail oviduct by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, mannan-Sepharose 4B chromatography, and hydroxyapatite chromatography. The purified alpha-mannosidase (referred to as neutral alpha-mannosidase) showed a single band on polyacrylamide gel with or without sodium dodecyl sulfate. Its molecular weight was found to be 330,000 by gel chromatography. Neutral alpha-mannosidase hydrolyzed p-nitrophenyl alpha-D-mannopyranoside and the pyridylamino derivative of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (Km value was 3 mM). Mannosyl alpha 1-2 linkages in the pyridylamino derivative of Man alpha 1-2 Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc were hardly hydrolyzed. Its optimum pH was found to be 7.0. The activity of the enzyme was activated by CO2+, and was potently inhibited by Cu2+, Hg2+, swainsonine, and 1-deoxymannojirimycin.     

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.     

A structural study of the asparagine-linked oligosaccharide moiety of duck ovomucoid

Asparagine-linked oligosaccharides of duck ovomucoid were released quantitatively from the protein by digestion with glycoamidase A (from almond), the reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated using two different types of high performance liquid chromatography (HPLC) on a reversed phase column and an amide adsorption column. More than sixteen different oligosaccharides were separated and the structures were characterized by a combination of the 2-dimensional sugar mapping technique using HPLC, exoglycosidase digestion, and proton nuclear magnetic resonance measurements (1- and 2-dimensional). Furthermore, the HPLC profile of duck ovomucoid oligosaccharides was compared with previously reported profiles obtained from quail and chicken ovomucoids.Abbreviations COSY chemical shift-correlated spectroscopy - DQF-COSY double quantum filtered COSY - DSS sodium, 4,4-dimethyl-4-silapentane 1-sulfonate - Gal D-galactose - GlcNAc or GN N-acetyl-D-glucosamine - HOHAHA homonuclear Hartmann-Hahn spectroscopy - Man or M D-mannose - NOE nuclear Overhauser enhancement - ODS octadecylsilyl - PA pyridylamino - ROESY rotating frame nuclear Overhauser effect spectroscopy - SDS/PAGE sodium dodecyl sulfate/polyacrylamide gel electrophoresis     

Analysis of asparagine-linked oligosaccharides from plasma membranes of rat normal liver and ascites hepatoma cells

Isolated plasma membranes from rat liver and ascites hepatoma cells were shown by SDS-polyacrylamide gel electrophoresis and concanavalin A reactivity to contain a variety of glycoproteins having asparagine-linked oligosaccharides. Membrane oligosaccharides were released by almond glycopeptidase digestion, and the pyridylamino derivatives were analyzed by high-performance liquid chromatography. Forty-four percent of the total carbohydrates in the original membranes were released and suggested to be of the complex type. Hepatoma membranes showed different oligosaccharide patterns from normal.     

Structure of a sugar chain of a protease inhibitor isolated from barbados pride (Caesalpinia pulcherrima Sw.) seeds

An asparagine-linked sugar chain of a protease inhibitor from barbados pride (Caesalpinia pulcherrima Sw.) was liberated by hydrazinolysis. After N-acetylation, the reducing end residue of this carbohydrate unit was coupled with 2-aminopyridine and the pyridylamino (PA-) derivative was purified by gel-filtration and reversed-phase HPLC. The structure of the resulting PA-sugar chain was determined mainly by stepwise exoglycosidase digestions and 500 MHz 1H-NMR spectroscopy and proved to be as follows: (formula; see text).     

A Novel Method for Detection of Endo-Xyloglucan Transferase

A new approach has been developed for quantification of theactivity of endo-xyloglucan transferase, a novel enzyme thatmediates the transfer of a segment of one xyloglucan moleculeto another xyloglucan molecule. Purified xyloglucans with definedmolecular-weight distributions and their fluorescent derivatives(pyridylamino xyloglucans) were used as substrates for the enzymaticreaction. The transferase activity was quantified by monitoringchanges in molecular-weight distributions of substrates by analkali compatible gel permeation chromatographic system, equippedwith a pulsed amperometric detector and a fluorescence detector.This new method was applied to the rapid detection and characterizationof a novel transferase derived from plant tissues. (Received February 28, 1992; Accepted September 28, 1992)     

Transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae has transglycosylation activity. Treatment of (Man)6(GlcNAc)2Asn with the enzyme in the presence of free N-acetylglucosamine gave a mixture of (Man)6GlcNAc and (Man)6GlcNAc beta 1----4GlcNAc mixture. N-Acetylglucosamine at the reducing end of the latter sugar chain was found by HPLC of the carbohydrate composition and of an exoglycosidase digest of the pyridylamino derivative of the reducing-end residue, and by 400 MHz 1H-NMR spectroscopy.