Moriguchi Kazunobu, Takemoto Tatsuya, Aoki Takafumi, Nakakita Shin-ichi, Natsuka Shunji, Hase Sumihiro
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
J Biochem. 2007 Aug;142(2):213-27. doi: 10.1093/jb/mvm128.
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).
我们之前报道过，斑马鱼α1-3岩藻糖基转移酶1（zFT1）在胚胎的体节期表达，并且能够合成Lewis x表位[Gal β1-4(Fuc α1-3)GlcNAc] [Kageyama等人，《生物化学杂志》，125，838 - 845（1999）]。在当前研究中，我们试图检测斑马鱼胚胎中zFT1的酶产物。从受精后12、18和48小时的斑马鱼胚胎中制备寡糖，并用荧光团2-氨基吡啶进行标记，以进行高灵敏度检测。在受精后18小时对α1-3/4岩藻糖苷酶敏感且呈时间依赖性表达的吡啶氨基（PA）-寡糖被鉴定为zFT1在体内合成产物的候选物。通过外切糖苷酶消化和二维高效液相色谱糖图谱相结合的方法确定这些寡糖的结构为具有两个Lewis x表位的双天线复合型结构：(Gal β1-4)(0,1,2)-{Gal β1-4(Fuc α1-3)GlcNAc β1-2Man α1-6[Gal β1-4(Fuc α1-3)GlcNAc β1-2Man α1-3]}Man β1-4GlcNAc，以及(Gal β1-4)(0,1)-{Gal β1-4(Fuc α1-3)GlcNAc β1-2Man α1-6[Gal β1-4(Fuc α1-3)GlcNAc β1-2Man α1-3]} Man β1-4GlcNAc β1-4GlcNAc。这些寡糖中Lewis x结构的存在及其表达时间表明它们是zFT1的产物。值得注意的是，这些寡糖中的大多数是游离形式。此外，我们在18小时的胚胎中检测到一种内切β-N-乙酰氨基葡萄糖苷酶活性。这些结果表明，由于内切β-N-乙酰氨基葡萄糖苷酶和/或糖酰胺酶从糖蛋白上释放，zFT1合成的寡糖在体节期以游离形式存在于胚胎中。
