© 2007 The Japanese Biochemical Society.
Free Oligosaccharides with Lewis x Structure Expressed in the Segmentation Period of Zebrafish Embryo
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
*To whom correspondence should be addressed. Tel: 81-6-6850-5380, Fax: 81-6-6850-5383, E-mail: suhase{at}chem.sci.osaka-u.ac.jp
Received March 8, 2007; Accepted May 22, 2007
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Abstract |
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We previously reported that zebrafish
1-3fucosyltrasferase 1 (zFT1) was expressed in embryos at the segmentation period, and was capable of synthesizing the Lewis x epitope [Galß1-4(Fuc1-3)GlcNAc] [Kageyama et.al, J. Biochem., 125, 838–845 (1999)]. In the current study, we attempted to detect the enzyme products of zFT1 in zebrafish embryos. Oligosaccharides were prepared from the zebrafish embryos at 12, 18 and 48 h after fertilization and labelled with a fluorophore, 2-aminopyridine, for highly sensitive detections. Pyridylamino (PA)-oligosaccharides that were 1-3/4fucosidase sensitive and time-dependently expressed at 18 h after fertilization were identified as candidates for the in vivo products synthesized by zFT1. Structures of these oligosaccharides were determined by a combination of exoglycosidase digestions and two-dimensional HPLC sugar mapping to be the biantennary complex-type structures with two Lewis x epitopes: (Galß1-4)0,1,2-{Galß1-4(Fuc1-3)GlcNAcß1-2Man1-6[Galß1-4(Fuc1-3)GlcNAcß1-2Man1-3]}Manß1-4GlcNAc, and (Galß1-4)0,1-{Galß1-4(Fuc1-3)GlcNAcß1-2Man1-6[Galß1-4(Fuc1-3)GlcNAcß1-2Man1-3]} Manß1-4GlcNAcß1-4GlcNAc. The presence of Lewis x structure of these oligosaccharides together with their expression time suggests that they are products of zFT1. Remarkably, most of these oligosaccharides were free form. Furthermore, we detected an endo-ß-N-acetylglucosaminidase activity in the 18 h embryo. These results suggest that the oligosaccharides synthesized by zFT1 are present in the embryo at the segmentation period in free form, owing to the liberation from glycoproteins with endo-ß-N-acetylglucosaminidase(s) and/or glycoamidase(s).
Key Words: Lewis x, zebrafish, embryogenesis, oligosaccharide, pyridylamination
Abbreviations:
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]; DEAE, diethylaminoethyl; EDTA, ethylene diaminetetra acetic acid; Fuc, L-fucose; Gal, D-galactose; GalNAc, N-acetyl-D-galactosamine; Glc, D-glucose; GlcNAc, N-acetyl-D-glucosamine; GN1 type oligosaccharide, N-linked oligosaccharide with a single GlcNAc at the reducing-end; GN2 type oligosaccharide, N-linked oligosaccharide with GlcNAcß1-4GlcNAc at the reducing-end; HPLC, high-performance liquid chromatography; LTA, Lotus tetagonolobus agglutinin; Man, D-mannose; MALDI-TOF-MS, matrix assisted laser desorption ionization time of flight mass spectrometry; PA, pyridylamino; zFT1, zebrafish -1,3-fucosyltransferase 1; zFT2, zebrafish -1,3-fucosyltransferase 2
Present address: Department of Functional Glycomics, Life Science Research Center, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan