Wen D X, Livingston B D, Medzihradszky K F, Kelm S, Burlingame A L, Paulson J C
Cytel Corporation, San Diego, California.
J Biol Chem. 1992 Oct 15;267(29):21011-9.
The Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase forms the NeuAc alpha 2,3Gal beta 1,3(4)GlcNAc sequences found in terminal carbohydrate groups of glycoproteins and glycolipids. High energy collision-induced dissociation analysis of tryptic peptides from only 300 pmol of the purified Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase provided 25% of the total amino acid sequence and led to the successful cloning of this enzyme. The peptide sequence information was used to design short degenerate primers for use in the polymerase chain reaction. A long specific cDNA fragment was amplified which was used to isolate a clone from a rat liver cDNA library. The cloned cDNA encodes a 374-amino acid protein containing an amino-terminal signal-anchor sequence characteristic of all cloned glycosyltransferases and produced sialyltransferase activity when transiently expressed in COS-1 cells. When compared with two other cloned sialyltransferases, the primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase revealed a homologous region in all three enzymes consisting of a stretch of 55 amino acids located in their catalytic domains. This feature together with lack of homology in the remaining 85% of the sequence of the three sialyltransferases defines a pattern of sequence homology not found in cloned cDNAs of other glycosyltransferase families.
Galβ1,3(4)GlcNAcα2,3-唾液酸转移酶形成了在糖蛋白和糖脂末端碳水化合物基团中发现的NeuAcα2,3Galβ1,3(4)GlcNAc序列。对仅300皮摩尔纯化的Galβ1,3(4)GlcNAcα2,3-唾液酸转移酶的胰蛋白酶肽段进行高能碰撞诱导解离分析，提供了25%的总氨基酸序列，并成功克隆了该酶。肽段序列信息被用于设计用于聚合酶链反应的短简并引物。扩增出一个长的特异性cDNA片段，用于从大鼠肝脏cDNA文库中分离一个克隆。克隆的cDNA编码一个374个氨基酸的蛋白质，该蛋白质含有所有克隆的糖基转移酶特有的氨基末端信号锚定序列，并且在COS-1细胞中瞬时表达时产生唾液酸转移酶活性。与另外两种克隆的唾液酸转移酶相比，Galβ1,3(4)GlcNAcα2,3-唾液酸转移酶的一级结构显示，这三种酶在催化结构域中有一个由55个氨基酸组成的同源区域。这一特征以及这三种唾液酸转移酶其余85%序列中缺乏同源性，定义了在其他糖基转移酶家族的克隆cDNA中未发现的序列同源模式。
