Structure and function of oligosaccharide of glycoprotein : Approach by recombinant technology and organic synthesis.

低聚糖改变蛋白质的结构、动力学和物理性质，通过氮到天冬氨酸或氧到丝氨酸或苏氨酸与蛋白质共价结合。随着基因克隆技术的突破，蛋白质的一级结构从编码该蛋白的基因序列中推断出来。根据得到的序列，可以预测蛋白质的某些性质，包括N-连接的葡聚糖的潜在糖硅化位点。然而，寡糖的结构和功能不能从核苷酸序列中推断出来。本研究采用分子克隆、定点突变和低聚糖有机合成等技术，对白血病抑制因子(LIF)、胎盘催乳素(PL)和绒毛膜促性腺激素(CG)等糖蛋白的生物活性进行了研究。我们已经在大鼠体内克隆了编码LIF的基因。LIF是细胞因子超家族的一员，在多种组织和细胞中具有不同的功能。在7个可能的N-糖硅化位点中，有6个位于9，34，63，73，96和116位。在DA-1a细胞增殖系统中评价其活性时，这些寡糖参与了生物学功能。PL-lm是胎盘催乳素家族的成员，有两个可能的N-糖化位点(79和128)。用谷氨酰胺取代ASN 79和ASN 128的突变体对NB2细胞的增殖有明显的抑制作用。马CG是促性腺激素的一个独特成员，因为它同时具有促黄体生成素和促卵泡激素的活性。缺失α-亚基N-糖链的心电突变体(Asn56)表现出类黄体生成素活性的完全丧失，但表现出较强的促卵泡刺激素样活性。在心电b亚基的C端部也有O-糖链。缺乏O-连接低聚糖的突变体也获得了很强的FSH样活性。因此，我们论证了糖蛋白中寡糖的生物学意义。

Oligosaccharides modify the structure, dynamics and physical properties of the proteins to which they are covalently bound through nitrogen to aspargine or oxygen to serine or threonine. After the breakthrough of cDNA cloning, the primary structure is deduced from the sequence of nucleotides in the cDNA encoding the protein. From the derived sequence, certain properties of the protein can be predicted including potential glycosilation sites for N-linked glycans. Oligosaccharide structure and function, however, can not be deduced from the nucleotide sequence. In this research project, we have studied on the biological activity of several glycoproteins including Leukemia lnhibitory Factor (LIF), Placental Lactogen (PL) and Chorionic Gonadotropin (CG) using techniques including molecular cloning, site-directed mutagenesis and organic synthesis of oligosaccharides. We have cloned the cDNA which encodes LIF in the rat. LIF is a member of a cytokine superfamily and has diverse functions in the various tissues and cells. Of seven possible N-glycosilation sites, six sites are glycosilated at positions of 9,34,63,73,96 and 116. These oligosaccharides are involved in the biological function when the activity was evaluated in the DA-1a cell proliferation system. PL-lm is a member of the placental prolactin family and has two possible N-glycosilation sites (79 and 128). The mutants of PLs of which substituting the Asn 79 and Asn 128 with Gln showed significant decrease of Nb2 cell proliferation. Equine CG (eCG) is a unique member of gonadotropin, because it possesses both LH and FSH activities. The eCG mutant which lack N-glycan of a-subunit (Asn56) showed complete loss of LH-like activity but showed potent FSH-like activity. eCG has also O-glycans at the C-teminal portion of b-subunit. The mutant which lack O-linked oligosaccharides was also shoed potent FSH like activity. Thus, we demonstrated the biological importance of oligosaccharides in the glycoproteins.