3D STRUCTURE DETERMINATION OF RECOMBINANT BETA-1-GALACTOSYLTRANSFERASEFERASE

半乳糖基转移酶家族的酶具有多种功能， 即：它们参与糖转移、细胞间黏附和 在发展过程中形成的格局。糖的受体和供体 某些酶的特异性可被α-乳清蛋白调节 (La)，一种乳腺特异性钙结合蛋白，具有序列 与C型溶菌酶结构相似(1)。自.以来 半乳糖基转移酶家族的酶是多功能的，我们是 目前正在调查各区域的结构和功能 β-1，4半乳糖转移酶(β-1，4 GT)及其相互作用 用LA来说明不同的功能。目前，使用 遗传工程和晶体结构测定方法，目的是 我们实验室的主要任务是确定三维结构的 含跨膜全长蛋白催化结构域(TM) 结构域及其复合体β-1，4-GT及其底物修饰蛋白 洛杉矶。这些研究的目的是定义糖核苷酸和糖 β-1，4-GT催化域的受体结合特性 家庭，确定与LA互动的区域(S) 改变酶的糖受体专一性的糖。 此外，我们的目标是确定所涉区域的特征。 细胞黏附及其与层粘连蛋白和其他细胞基质的相互作用 蛋白质。这些结构信息将有益于 了解糖基转移酶在细胞表面的作用。 早些时候在我们的实验室中，TM结构域的功能分析是 在哺乳动物中表达和定位TM突变体 TM域的单元和一些结构要求是 与功能相关(2)。对于结构和功能 茎区域、糖供体和受体结合的分析 催化结构域的区域，以及糖依赖的研究 β-1，4-GT与重组LA的蛋白质-蛋白质相互作用 蛋白质已经在大肠杆菌中表达，蛋白质是从 包涵体，体外折叠，并分析底物结合 酶活性(3)。这些结果表明， 催化结构域位于β-1，4-GT的COOH末端部分， 可以进一步细分为N-末端和C-末端的一半。少校 糖受体的结合区位于糖链的N-末端。 催化结构域，而糖核苷酸供体的结合是 定位到C-末端的一半，并且为了催化 发生时，两半必须在催化表面重叠。O4-H 的GlcNAc必须与半乳糖的C1非常接近 (UDP-α-半乳糖)，以及转化 C_1处的构型必须发生才能产生β-糖苷键。 Cys 134和Cys 247之间的二硫键在 催化作用，但不适用于折叠或结合底物。重组人 β-1，4-GT和大鼠LAS的短、长两种形态，并在体内产生 对大鼠LAS进行了结晶，用于结构测定。该结构 对其中一些蛋白质的测定正在进行中。

The enzymes of galactosyltransferase family exhibit diverse functions, namely: they are involved in sugar transfer, cell-cell adhesion and pattern formation during development. The sugar acceptor and donor specificity of some of the enzymes can be modulated by alpha-lactalbumin (LA), a mammary gland specific calcium binding protein that has sequence and structural similarity to the c- type lysozymes (1). Since the enzymes of galactosyltransferase family are multifunctional, we are currently investigating the structure and function of various regions of the beta-1,4 galactosyltransferase(beta-1,4 GT) and its interactions with LA to account for the diverse functions. Currently, using the genetic engineering and crystal structure determination methods, the aim of our laboratory is to determine the three-dimensional structure of the catalytic domain, full length protein containing transmembrane (TM) domain and the complex beta-1,4 GT and its substrate modifier protein LA. The studies are aimed to define the sugar nucleotide and sugar acceptor binding specificities of the catalytic domain of beta-1,4 GT family, identify the region(s) that interact with LA in the presence of sugar which change the sugar acceptor specificity of the enzyme. Furthermore, our aim is to characterize the region that is involved in cell adhesion and interactions with laminin and other cell-matrix proteins. This structural information will be beneficial in understanding the role of glycosyltransferases at the cell surface. Earlier in our laboratory, the functional analysis of the TM domain was carried out by expressing and localizing the TM mutants in mammalian cells and some of the structural requirements of the TM domain were correlated with the function (2). For the structure and function analysis of the stem region, the sugar donor and acceptor binding regions of the catalytic domain, and for the studies on sugar-dependent protein-protein interactions between beta-1,4 GT and LA the recombinant proteins have been expressed in E.coli, proteins renatured from the inclusion bodies, folded in vitro, and analyzed for substrate bindings and enzymatic activities (3). These results have shown that the catalytic domain lies at the COOH-terminal portion of beta-1,4 GT and can be further subdivided into the N and C-terminal halves. The major binding region for the sugar acceptor lies in the N-terminal half of the catalytic domain, while the binding of the sugar nucleotide donor is localized to the C-terminal half, and in order that the catalysis to occur the two halves have to overlap at the catalytic surface. The O4-H of the GlcNAc has to be in the close proximity of the C1 of galactose (UDP-alpha-galactose) at the catalytic site, and the inversion of the configuration at C1 has to occur to generate a beta-glycosidic linkage. The disulfide bond between Cys 134 and Cys 247 is required during catalysis, but not for folding or binding substrates. The recombinant beta-1,4 GT and rat LAs, the short and long forms, and in vivo produced rat LAs have been crystalized for structure determination. The structure determination of some of these proteins are in progress.