Free oligosaccharides with Lewis x structure expressed in the segmentation period of zebrafish embryo.
We previously reported that zebrafishalpha1-3fucosyltrasferase 1 (zFT1) was expressed in embryos at the segmentation period, and was capable of synthesizing the Lewis x epitope [Gal beta1-4(Fuc alpha1-3)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 alpha1-3/4fucosidase 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: (Gal beta1-4)(0,1,2)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]}Man beta1-4GlcNAc, and (Gal beta1-4)(0,1)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]} Man beta1-4GlcNAc beta1-4GlcNAc. 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-beta-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-beta-N-acetylglucosaminidase(s) and/or glycoamidase(s).
Moriguchi
Kazunobu
K
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Takemoto
Tatsuya
T
Aoki
Takafumi
T
Nakakita
Shin-ichi
S
Natsuka
Shunji
S
Hase
Sumihiro
S
D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005647:Fucosyltransferases / Q000378:metabolism; D007983:Lewis Blood Group Antigens; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology* / Q000378:metabolism
D007983:Lewis Blood Group Antigens; C494219:Lewis a oligosaccharide; D009844:Oligosaccharides; D005647:Fucosyltransferases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase