Tomohiro Mega

Analysis of neutral sugars as glycamines using an amino acid analyzer.

Abstract A new method of the analysis of neutral sugars was developed based on the separation of their corresponding glycamines. Sugars and ammonia were combined by means of the reduction with sodlum cyanoborohydride. The glycamines thus obtained were quantitatively analyzed by an automatic amino acid analyzer. Satisfactery results were obtained in the analyses of the constituent sugars of several polysaccharides and glycoconjugates.

Methanolysis products of asparagine-linked N-acetylglucosamine and a new method for determination of N- and O-glycosidic N-acetylglucosamine in glycoproteins that contain asparagine-linked carbohydrates.

Abstract Amino acid analyzer and gas chromatographic analyses of methanolysates of model compounds of N-glycosidic and O-glycosidic N-acetylglucosamine shows that the main product of N-glycosidic N-acetylglucosamine is glucosamine and that of O-glycosidic N-acetylglucosamine is methyl α-glucosaminide. The amounts of N-acetylglucosamine in glycoproteins determined by methanolysis followed by gas-liquid chromatography represent the amount of O-glycosidic N-acetylglucosamine. A new method was developed to determine O-glycosidic and N-glycosidic N-acetylglucosamine in glycoproteins. Glucosamine and methyl α-glucosaminide in the methanolysates of glycoproteins are determined in an amino acid analyzer using the buffer system that is usually used for analysis of basic amino acids in the two-column system. The contents of O- and N-glycosidic N-acetylglucosamine of Taka-amylase A, ovalbumin, and egg white and yolk flavoproteins determined by the method agree reasonably well with the values calculated from the carbohydrate structures or compositions of the compounds. Therefore, this method can be used to determine the number of N-glycosidically linked carbohydrate chains together with the amount of O-glycosidic N-acetylglucosamine in glycoproteins.

Glucose trimming of N-glycan in endoplasmic reticulum is indispensable for the growth of Raphanus sativus seedling (kaiware radish).

Recently I found that glycosidase inhibitors such as castanospermine, deoxynojirimycin, swainsonine, 2-acetamindo 2,3-dideoxynojirimycin, and deoxymannojirimycin change the N-glycan structure of root glycoproteins, and that the glucosidase inhibitors castanospermine and deoxynojirimycin suppress the growth of Raphanus sativus seedlings (Mega, T., J. Biochem., 2004). The present study undertook to see whether the growth suppression is due to the inhibition of glucose trimming in endoplasmic reticulum (ER). The study, using three glucosidase inhibitors, castanospermine, N-methyl deoxynojirimycin, and deoxynojirimycin, upon the growth of R. sativus foliage leaf, made clear that glucose trimming is indispensable for plant growth, because the inhibition of glucose trimming correlated with leaf growth. On the other hand, processing inhibition in the Golgi apparatus by other glycosidase inhibitors had little effect on plant growth, although N-glycan processing was disrupted depending on inhibitor specificity. These results suggest that N-glycan processing after glucosidase processing is dispensable for plant growth and cell differentiation.

Plant-Type N-Glycans Containing Fucose and Xylose in Bryophyta (Mosses) and Tracheophyta (Ferns)

The presence of typical plant-type N-glycans (eg, M3FX, Gn2M3FX, and Lea2M3FX) in mosses, ferns, and other organisms was examined to determine which plant initially acquired glycosyltransferases to produce plant-type N-glycans during organic evolution. No M3FX-type N-glycan was detected in lichens (Cladonia humilis) or in any one of the three preland plants Enteromorpha prolifera, Ulva pertusa Kjellman, and Chara braunii Gmelin. In Bryophyta, M3FX-type N-glycan was detected at trace amounts in Anthocerotopsida (hornworts) and at certain amounts in Bryopsida (mosses), but not in Hepaticopsida (liverworts). Lea2M3FX was detected in some Bryopsida of relatively high M3FX content. Most Tracheophyta (ferns and higher plants) contained the three typical M3FX-type glycans as the main N-glycans in different ratios. These results suggest that organisms acquired xylosyltransferase and fucosyltransferase during the development of mosses from liverworts, and that later all plants retained both enzymes. Bryopsida have also obtained galactosyltransferase and fucosyltransferase to synthesize the Lea antigen.

Conversion of egg-white lysozyme to a lectin-like protein with agglutinating activity analogous to wheat germ agglutinin

Oligomers made of Asp-52-esterified lysozyme or native one showed agglutination for human, mouse, rat and chicken erythrocytes, and also for Hep G2 cells (liver carcinoma-derived cell line) [1] not weaker than wheat germ agglutinin (WGA). Oligomers of Asp-52-esterified lysozyme have about 4-fold stronger agglutinating activity than those of the native one. These results indicate that some enzymes can be converted to lectin-like proteins (neolectin) with binding characteristics similar to their substrate specificity.

Isolation of Lectins by Affinity Chromatography with Porcine Plasma Proteins Immobilized on Agarose

To develop a convenient method to isolate lectins, we prepared an affinity gel by coupling plasma proteins with agarose beads under conditions where the pH did not exceed 7.5. The validity of the use of this affinity gel in combination with elution using a hapten saccharide was confirmed by isolation of concanavalin A from Jack bean meal. Successful application of the method was demonstrated by isolation of two novel vegetable lectins from udo (Aralia cordate) and wasabi (Wasabia japonica). The method would be useful to isolate new lectins from various sources including plant and animal tissues.

On the structure of galactosylhydroxylysine and glycopeptides derived from bovine tracheal cartilage

Abstract Galactosylhydroxylysine isolated from the alkaline hydrolysate of bovine tracheal cartilage was shown to be O-β-D-galactopyranosylhydroxylysine. The structures of several hydroxylysine-containing glycopeptides suggested the existence of a unique sequence around the glycosylated site with one exception in that an amino acid other than arginine was present in the third position from the glycosylated residue. Isolation of glycopeptides with an identical hexapeptide sequence yet with a different carbohydrate side chain suggested that the attachment of glucose to a polypeptide-bound galactose is not regulated by a short amino acid sequence in the glycopeptide region.

Determination and characterization of succinyl tri-alanine p-nitroanilide hydrolyzing metalloendopeptidase in serum.

Serum succinyl (Ala)3-p-nitroanilide hydrolyzing elastase-like activity which elevates in patients with obstructive jaundice, is due to the joint action of two enzymes: first, succinyl (Ala)3-p-nitroanilide is cleaved to succinyl (Ala)2 and Ala-p-nitroanilide by metalloendopeptidase, and then Ala-p-nitroanilide is cleaved to Ala and p-nitroaniline by aminopeptidase. We adopt a new assay method for serum endopeptidase activity using HPLC.