Improved method for fluorescence labeling of sugar chains with sialic acid residues.
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Carbohydrate Metabolism↗
Biomedical subjects
T Ikenaka
Publications and source records attributed to T Ikenaka.
Identification of a disaccharide (Xyl-Glc) and a trisaccharide (Xyl2-Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor-like domain of human factors VII and IX and protein Z and bovine protein Z.
We have recently described a unique trisaccharide linked to a serine residue in the first epidermal growth factor-like domains of bovine blood coagulation factors VII (Ser-52) and IX (Ser-53) (Hase, S., Kawabata, S., Nishimura, H., Takeya, H., Sueyoshi, T., Miyata, T., Iwanaga, S., Takao, T., Shimonishi, Y., and Ikenaka, T. (1988) J. Biochem. (Tokyo) 104, 867-868). The sugar chain identified in these clotting factors consists of 1 mol of hexose (glucose (Glc] and 2 mol of pentose (xylose (Xyl]. We report here that human factors VII and IX and protein Z and bovine protein Z also contain such carbohydrate moieties linked to a serine residue at the same position found in bovine factors VII and IX. A glycopeptide derived from each of these proteins was subjected to amino acid sequence and component sugar analyses and fast atom bombardment mass spectrometric analysis. The results indicate that the glycopeptide derived from human factor IX contains 1 mol each of Glc and Xyl. The reducing end of this disaccharide was identified as Glc by analyzing the disaccharide generated by hydrazinolysis. In contrast, human factor VII and protein Z yielded two different glycopeptides which contained Glc and Xyl at molar ratios of 1:1 and 1:2, respectively, suggesting microheterogeneity of these O-linked sugar chains. Bovine protein Z glycopeptide contained 1 mol of Glc and 2 mol of Xyl. These sugar compositions were confirmed by analyses of the intact proteins. In relation to the trisaccharide sugar chain previously discovered in bovine factors VII and IX, these findings indicate the existence of a Xyl2-Glc-Ser and a Xyl-Glc-Ser structure in the first epidermal growth factor-like domains of human factors VII and IX and protein Z in addition to that of bovine protein Z. Whether these carbohydrate moieties contribute to the biological activities of these proteins is unknown.
Amino Acid Sequence↗
Amino acid sequences and disulfide bridges of serine proteinase inhibitors from bitter gourd (Momordica charantia LINN.) seeds.
Three serine proteinase inhibitors, MCTI-I, MCTI-II, and MCEI-I, were isolated from bitter gourd (Momordica charantia LINN.) seeds. MCTI-I and MCTI-II were inhibitors for trypsin and MCEI-I was an elastase inhibitor. Their amino acid sequences and the positions of disulfide bridges of MCTI-II were determined to be as follows. (sequence; see text)
Amino Acid Sequence↗
Purification and characterization of chitinase produced by Streptomyces erythraeus.
A chitinase was purified from the culture filtrate of Streptomyces erythraeus (SE). The enzyme (SE chitinase) has a molecular weight of 30,000 and pI 3.7, and shows optimal activity at pH 5.0 with an optimal ionic strength of less than 0.2 M NaCl. SE chitinase could hydrolyze chitin and its derivatives, but could not hydrolyze cell walls of Micrococcus lysodeikticus. The substrate specificity of SE chitinase was compared with those of hen egg white (HEW) and SE lysozymes. The binding mode of the chitinase to substrates was investigated using chitooligosaccharides and their derivatives. The results showed that the binding mode of SE chitinase to the substrate is similar to that of HEW and SE lysozymes.
Amino Acids↗
Structures of the sugar chains of interferon-gamma produced by human myelomonocyte cell line HBL-38.
Interferon-gamma produced by the human myelomonocyte cell line HBL-38 contained galactose, mannose, fucose, N-acetylglucosamine, and N-acetylneuraminic acid as sugar components. Sugar chains were liberated from interferon-gamma by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine under new reaction conditions in which no sialic acid residue was hydrolyzed. The pyridylamino (PA-) derivatives of the sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Seven major PA-sugar chains were isolated and the structure of each purified PA-sugar chain was identified by stepwise exoglycosidase digestion and 500-mHz 1H-NMR spectroscopy. The results indicated that the structures of the major PA-sugar chains were of the biantennary type, to which 0 to 2 mol of fucose and 1 to 2 mol of N-acetylneuraminic acid were linked as shown below. (formula; see text)
Asialoglycoproteins↗
Studies on the sugar chains of interferon-gamma from human peripheral-blood lymphocytes.
Sugar chains of interferon-gamma (IFN-gamma) from human peripheral-blood lymphocytes (PBL) were liberated by hydrazinolysis. After N-acetylation, the reducing end residues of the sugar chains were tagged with 2-aminopyridine and the pyridylamino (PA-) derivatives were purified by gel filtration and reversed-phase HPLC. Five major PA-sugar chains were obtained. The structure of each PA-sugar chain was estimated by comparing its elution times on anion exchange, reversed-phase, and size-fractionation HPLC with those of PA-sugar chains of IFN-gamma from the human myelomonocyte cell line HBL-38 (Yamamoto, S. et al. (1989) J. Biochem. 105, 547-555) as standards, and also by comparison of their elution times after partial desialylation. The results showed that IFN-gamma (PBL) contained mono- and disialo-biantennary structures with 0 or 1 mol of fucose residue, as found for IFN-gamma (HBL-38), but the N-acetylneuraminyl alpha 2-6 linkage was dominant in IFN-gamma (PBL), unlike IFN-gamma (HBL-38), which contains both N-acetylneuraminyl alpha 2-3 and alpha 2-6 linkages.
Carbohydrates↗
Amino acid sequence of chitinase from Streptomyces erythraeus.
The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.
Amino Acid Sequence↗
Actions of three human alpha-amylases expressed in yeast on modified substrates and the amino acid residues causing their different actions.
The mode of action of an alpha-amylase (yHXA) which was the gene product of a newly found human alpha-amylase gene expressed in yeast on synthetic substrates was compared with those of the gene products (yHSA and yHPA) of human salivary and pancreatic alpha-amylase gene in yeast. The substrates used were phenyl alpha-maltopentaoside (G5 phi) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2NH2 (AG5 phi), COOH (CG5 phi), or CH2I (IG5 phi). The digests were subjected to HPLC to determine the amounts of products. The HPLC analysis revealed that yHXA and yHSA bound G5 phi to their active sites in similar manners to give the same products, while yHPA hydrolyzed it in a different way. Modifications of the non-reducing-end glucose of G5 phi caused change of the binding mode to the active sites of the enzymes. AG5 phi and CG5 phi were hydrolyzed by the enzymes to give more phenyl alpha-glucoside (G phi) and less phenyl alpha-maltoside (G2 phi), while IG5 phi gave more G2 phi and less G phi, compared with G5 phi. The substrate binding mode of yHXA changed more extensively than that of yHSA. The results suggested that there exists an amino acid replacement between yHXA and yHSA. The amino acid residues replaced are neither acidic nor basic, are located in subsite S3, and interact with the CH2OH residue of the non-reducing-end glucose residue of G5 phi.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
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.
Animals↗
Use of a post-column enzymatic reaction for detection of macroamylasemia by high-performance liquid chromatography.
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Amylases↗
Alpha-amylase assay with use of a benzyl derivative of p-nitrophenyl alpha-maltopentaoside, BG5P.
p-Nitrophenyl O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)-alpha-D-glucopyranoside (BG5P) is hydrolyzed by both human salivary alpha-amylase (HSA) and human pancreatic alpha-amylase (HPA) to O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-alpha-D-glucopyranose (BG3) and p-nitrophenyl alpha-maltoside (G2P). Glucoamylase and alpha-glucosidase cannot hydrolyze BG5P because of the modification of the OH group of the 6-position of the non-reducing-end glucose residue with the benzyl group. Taking advantage of these characteristics of the substrate, BG5P, we developed a method to assay the total alpha-amylase activity in human fluids using glucoamylase and alpha-glucosidase as the coupled enzymes. This method is simple and can be used as the standard method for routine clinical assays of alpha-amylase activity.
Chromatography, High Pressure Liquid↗
Synthesis of p-nitrophenyl 6(5)-O-benzyl-alpha-maltopentaoside, a substrate for alpha amylases.
p-Nitrophenyl alpha-maltopentaoside, having a benzyl group on O-6 of the terminal (nonreducing) D-glucosyl group was prepared by use of a reductive ring-opening reaction. Highly regioselective reduction of p-nitrophenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl)-(1----4)- tris[O-(2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl)-(1----4)]-2,3,6-tri- O- benzoyl-alpha-D-glucopyranoside by dimethylamine-borane and p-toluenesulfonic acid, followed by debenzoylation, gave p-nitrophenyl O-(6-O-benzyl-alpha-D-glucopyranosyl)-(1----4)-tris[O-alpha-D-glucopyran osyl- (1----4)]-alpha-D-glucopyranoside. An experiment was done on the mode of action of human pancreatic and salivary alpha amylases on this derivative. The compound is suitable as a substrate for the assay of alpha amylase when used with glucoamylase and alpha-D-glucosidase as coupling enzymes.
Carbohydrate Conformation↗
Separation of human salivary alpha-amylase isozymes by high-performance liquid chromatography with a continuous monitor system of the activity.
Human salivary alpha-amylase isozymes were rapidly separated from each other by high-performance liquid chromatography with a postcolumn assay. The eluate from the HPLC column was mixed continuously with an intramolecularly quenched fluorescent substrate, p-nitro-phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside delivered by a pump. The mixture was incubated in a reaction coil, and the fluorescence intensity was continuously measured by a fluorescence detector. The assay was based on the marked increase in fluorescence with the enzymatic cleavage of the glycosidic bond of the substrate that links the fluorogenic and quenching moieties.
Chromatography, High Pressure Liquid↗
Phosphorylation of the heavy chain of skeletal muscle myosin by casein kinase II: localization of the phosphorylation site to the amino terminus.
While the heavy chain of rabbit skeletal muscle myosin is not phosphorylatable by casein kinase II, it turned out to be phosphorylatable after removal of all of the light chains. The phosphorylation site for the kinase was determined to be Ser-1 and/or Ser-2 at the amino terminus.
Amino Acid Sequence↗
Pyridylamino sugar chain as an acceptor for galactosyltransferase.
Pyridylamino asialo-agalacto-biantennary sugar chain (PA-acceptor), prepared from human alpha 1-acid glycoprotein, was incubated with bovine milk galactosyltransferase. Transfer of galactose residues to PA-acceptor was detected by HPLC analysis, and thus PA-acceptor was shown to be useful for galactosyltransferase assay. Moreover, three species of products, i.e. PA-acceptor monogalactosylated on the Man alpha 1-3 branch of the trimannosyl core, PA-acceptor monogalactosylated on the Man alpha 1-6 branch, and digalactosylated PA-acceptor, were separated and identified by reversed-phase HPLC, so we could simultaneously determine the branch specificity (the ratio of galactosylation on Man alpha 1-3 branch to that on Man alpha 1-6 branch) of the galactosyltransferase. We fractionated the bovine milk galactosyltransferase on a DEAE-5PW column and confirmed that there was a heterogeneity in this enzyme preparation. Each fraction was assayed for acceptor specificity (the ratio of the activity towards N-acetylglucosamine to that towards PA-acceptor) and branch specificity using the PA-acceptor. However, we could not detect differences in the specificities among the fractions. In addition, we found that alpha-lactalbumin stimulated the galactosyltransferase activity towards PA-acceptor.
Aminopyridines↗
Structures of the sugar chains of recombinant human granulocyte-colony-stimulating factor produced by Chinese hamster ovary cells.
Recombinant human granulocyte-colony-stimulating factor (G-CSF) was purified from Chinese hamster ovary cells transfected with human G-CSF cDNA. The recombinant human G-CSF was treated with alkaline borohydride and the oligosaccharide-alditols liberated were fractioned by gel filtration on a Bio-Gel P-4 column, followed by high-performance liquid chromatography by use of a strong anion exchanger. Two oligosaccharide-alditols were obtained and their structures were identified by component analysis and 500-MHz 1H-NMR spectroscopy. The structures of the sugar chains were NeuAc alpha 2-3Gal beta 1-3GalNAcol and NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAcol.
Animals↗
Structures of sugar chains of ricin D.
The toxic lectin, ricin D, contains mannose, fucose, xylose, and N-acetylglucosamine as sugar components. Sugar chains are linked to Asn-10 of the A-chain, and to Asn-95 and Asn-135 of the B-chain (Funatsu, G. et al. (1978) Agric. Biol. Chem. 42, 501-503; Araki, T. & Funatsu, G. (1985) FEBS Lett. 191, 121-124). Asparagine-linked sugar chains of each glycopeptide from ricin D were liberated by hydrazinolysis followed by N-acetylation. The reducing end residues of the sugar chains were coupled with 2-aminopyridine and the pyridylamino (PA-) derivatives obtained were purified by gel-filtration and reversed-phase HPLC. Eight main PA-sugar chains were obtained from three glycopeptides and the structures of these sugar chains were determined by component analysis, stepwise exoglycosidase digestions, partial acetolysis, and 500 MHz 1H-NMR spectroscopy. The results show that oligomannose type sugar chains (Man6-7GlcNAc2) are linked to Asn-95; Man5-7 GlcNAc2 and M4X (structure, see below) to Asn-135 of the B-chain, and M3FX and M3X to Asn-10 of the A-chain. (Formula: see text).
Acetylation↗
Structures of sugar chains of a p-nitrophenyl acetate-hydrolyzing esterase from the microsomes of rat liver.
The structures of sugar chains of a p-nitrophenyl acetate-hydrolyzing esterase from the microsomes of rat liver were established. The enzyme contained mannose and glucosamine as sugar components. Asparagine-linked sugar chains of the esterase were liberated by hydrazinolysis. After N-acetylation of the hydrazinolysate, the reducing ends of the sugar chains were coupled with 2-aminopyridine. Fluorescent pyridylamino (PA-) derivatives of sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Eleven PA-sugar chains were obtained. The structures of the PA-sugar chains were first identified by HPLC using two series of separation systems by which 11 PA-oligomannose-type sugar chains with known structures could be separated. Further elucidation of the structures of each PA-sugar chain was performed by exoglycosidase digestions and partial acetolysis. The structures of two of the PA-sugar chains were further confirmed by 500 mHz 1H-NMR spectroscopy.
Animals↗