Kiyoshi Okihara

350-kDa Royal Jelly Glycoprotein (Apisin), Which Stimulates Proliferation of Human Monocytes, Bears the β1-3Galactosylated N-Glycan: Analysis of the N-Glycosylation Site

While doing a structural analysis of minor component N-glycans linked to 350-kDa royal jelly glycoprotein (RJGP), which stimulates the proliferation of human monocytes, we found that a Galβ1-3GlcNAcβ1-4Man unit occurs on the insect glycoprotein. The structure of the fluorescence-labeled N-glycan was analyzed by sugar component analysis, IS-MS, and 1H-NMR. The structural analysis showed that the 350-kDa RJGP bears Galβ1-3GlcNAcβ1-4(GlcNAcβ1-2)Manα1-3 (Manα1-3Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc, suggesting this insect glycoprotein is one of the substrates for both β1-3 galactosyl and β1-4 N-acetylglucosamininyl transferases. To our knowledge, this is the first report that succeeded in identifing an insect glycoprotein bearing the β1-3 galactosylated N-glycan.

Flavonoid glycosides and limonoids from Citrus molasses

Molasses of tangerine orange (Citrus unshiu Markovich) is obtained as a waste product in the course of tangerine orange juice production. This molasses is expected to be a useful source of organic compounds such as flavonoids and limonoids. To elucidate a use for this molasses waste, we isolated and identified its organic constituents. Two new flavanonol glycosides were isolated from tangerine orange molasses, along with several flavonoids such as hesperidine, narirutin, eriodictyol, 3′,4′,5,6,7,8-hexamethoxy-3-O-β-d-glucopyranosyloxyflavone, and 3′,4′,5,6,7,8-hexamethoxy- 3-β-d-[4-O-(3-hydroxy-3-methylglutaloyl)]-glucopyranosyloxyflavone, and limonoids such as limonin, nomilin, and cyclic peptide, citrusin III. The structures of the new flavanonol glycosides were determined as (2R,3R)-7-O-(6-O-α-l-rahmnopyranosyl-β-d-glucopyranosyl)-aromadendrin and 7-O-(6-O-α- l-rahmnopyranosyl-β-d-glucopyranosyl)-3,3′,5,7-tetrahydroxy-4′-methoxyflavanone by means of spectral analyses using 1H-NMR, 13C-NMR, and 2D-NMR. Of these compounds, flavanone glycoside, hesperidin and narirutin were isolated as the main constituents. Thus, molasses is a promising source of flavonoid glycosides.

Inhibitory effects of propolis granular A. P. C on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in A/J mice

We examined the effect of propolis granular A. P. C on lung tumorigenesis in female A/J mice. Lung tumors were induced by the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) administered in drinking water for 7 weeks in mice maintained on an AIN-76A semi-synthetic diet. Propolis granular A. P. C (100 mg/kg body wt.) was administered orally daily for 6 days/week from 1 week before NNK administration and throughout the experiment. Sixteen weeks after the NNK treatment, the mice were killed and the number of surface lung tumors was measured. The number of lung tumors in mice treated with NNK alone for 7 weeks (9.4 mg/mouse) was significantly more than in that observed in control mice. Propolis granular A. P. C significantly decreased the number of lung tumors induced by NNK. These results indicate that propolis granular A. P. C is effective in suppressing NNK-induced lung tumorigenesis in mice.

Tumor antigen occurs in N-glycan of royal jelly glycoproteins : Honeybee cells synthesize T-antigen unit in N-glycan moiety

In our previous paper (Kimura, Y., et al., Biosci. Biotechnol. Biochem., 67, 1852–1856, 2003), we found that a complex type N-glycans containing β1-3 galactose residue occurs on royal jelly glycoproteins. During structural analysis of minor components of royal jelly N-glycans, we found complex type N-glycans bearing both galactose and N-acetylgalactosamine residues. Detailed structural analysis of pyridylaminated oligosaccharide revealed that the newly found N-glycan had a complex type structure harboring a tumor marker (T-antigen) unit: Galβ1-3GalNAcβ1-4GlcNAcβ1-2Manα1-6 (Galβ1-3GalNAcβ1-4GlcNAcβ1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc. To our knowledge, this may be the first report of the presence of the T-antigen unit in the N-glycan moiety of eucaryotic glycoproteins.

Occurrence of GalNAcβ1-4GlcNAc unit in N-glycan of royal jelly glycoprotein

Elsewhere, we characterized the structure of twelve N-glycans purified from royal jelly glycoproteins (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000)). Structural analysis showed that the typical high-mannose type structure (Man9-4GlcNAc2) accounts for about 72% of total N-glycans, a biantennary-type structure (GlcNAc2Man3GlcNAc2) about 8%, and a hybrid-type structure (GlcNAc1Man4GlcNAc2) about 3%. During structural analysis of minor N-glycans of royal jelly glycoproteins, we found that one had an N-acetyl-galactosaminyl residue at the non reducing end; most of such residues have been found in N-glycans of mammalian glycoproteins. By exoglycosidase digestion, methylation analysis, ion-spray (IS)-MS analysis, and 1H NMR spectroscopy, we identified the structure of the N-glycan containing GalNAc as; GlcNAcβ1-2Manα1-6(GalNAcβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1- 4GlcNAc. This result suggested that a β1-4 GalNAc transferase is present in hypopharyngeal and mandibular glands of honeybees.

Evidence for New β1-3 Galactosyltransferase Activity Involved in Biosynthesis of Unusual N-Glycan Harboring T-Antigen in Apis mellifera

In a previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 70, 2583–2587, 2006), we found that new complex type N-glycans harboring Thomsen-Friedenreich antigen (Galβ1-3GalNAc) unit occur on royal jelly glycoproteins, suggesting the involvement of a new β1-3galactosyltransferase in the synthesis of the unusual complex type N-glycans. So far, such β1-3galactosyltransferase activity, which can transfer galactosyl residues with the β1-3 linkage to β1-4 GalNAc residues in N-glycan, has not been found among any eucaryotic cells. But using GalNAc2GlcNAc2Man3GlcNAc2-PA as acceptor N-glycan, we detected the β1-3 galactosyltransferase activity in membrane fraction prepared from honeybee cephalic portions. This result indicates that honeybee expresses a unique β1-3 galactosyltransferase involved in biosynthesis of the unusual N-glycan containing a tumor related antigen in the hypopharyngeal gland.

First Evidence for Occurrence of Galβ1-3GlcNAcβ1-4Man Unit in N-Glycans of Insect Glycoprotein: β1-3Gal and β1-4GlcNAc Transferases Are Involved in N…

On a way of structural analysis of total N-glycans linked to glycoproteins in royal jelly (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000), Kimura, M. et al., Biosci. Biotechnol. Biochem., 66, 1985-1989 (2002)), we found that some complex type N-glycans containing a β1-3galactose residue occur on the insect glycoproteins. Up to date, it has been considered that naturally occurring insect glycoproteins do not bear the galactose-containing N-glycans, therefore, in this report we describe the structural analysis of the complex type N-glycans of royal jelly glycoproteins. By a combination of endo- and exo-glycosidase digestions, IS-MS analysis, and 1H-NMR spectroscopy, the structures of the β1-3 galactose-containing N-glycan were identified as the following; GlcNAcβ1-2Manα1-6[GlcNAcβ1-2(Galβ1-3GlcNAcβ1-4)Manα1-3]Manβ1-4GlcNAcβ1-4GlcNAc, Manα1-3Manα1-6[GlcNAcβ1-2(Galβ1-3GlcNAcβ1-4)Manα1-3]Manβ1-4GlcNAcβ1-4GlcNAc, and Manα1-6(Manα1-3)Manα1-6[GlcNAcβ1-2(Galβ1-3GlcNAcβ1-4)Manα1-3]Manβ1-4GlcNAcβ1-4GlcNAc. To our knowledge, this is the first report showing that the Galβ1-3GlcNAcβ1-4Man unit occurs in N-glycans of insect glycoproteins, indicating a β1-3 galactosyl transferase and β1-4GlcNAc transferase (GNT-IV) are expressed in the honeybee cells.