STRUCTURE-FUNCTION RELATIONSHIP OF BETA 1-4 GALACTOSYLTRANSFERASE

我们已经证明，牛β 1- 4半乳糖基转移酶，在Okayama-Berg表达载体中工程化， 在转染编码功能活性β 1的COS-7细胞后， 4半乳糖基转移酶蛋白。 402个残基长的蛋白质， 在COS-7细胞中表达的具有氨基端膜锚定结构域 和UDP-半乳糖、N-乙酰葡糖胺、葡萄糖和 α-乳白蛋白都是完整有效的 的活性 表达的蛋白质由α-乳白蛋白调节，以改变蛋白质的表达。 对葡萄糖的受体特异性，合成乳糖。 本 研究制备了缺失构建体，其将编码 缺乏氨基端膜锚定结构域的蛋白质， 前70个残基的蛋白质相当于分泌形式的 酶活性蛋白质。 缺失构建体，在 Okayama-Berg载体转染COS-7细胞后， 酶活性蛋白质。 另一方面，当在E. 大肠杆菌中，蛋白质的缩写形式，具有前70个氨基末端 缺失的残基具有酶活性。 的结果 氨基末端缺失构建体表明， 不缺失膜锚定结构域的β 1 - 4半乳糖基转移酶 当在哺乳动物细胞内表达时，其不稳定或失活。 膜锚定结构域在哺乳动物细胞中的作用可能是 将蛋白质定位到合适的位置， 失活或增强其稳定性。 α-乳清蛋白钙结合环序列的比较 与C型Ca 2+结合和非结合中的相应序列 溶菌酶显示，某些保守的残基， 与Ca ~（2+）配体可能有助于Ca ~（2+）的结合。

We have shown that a full length cDNA sequence of bovine beta1- 4galactosyltransferase, engineered in an Okayama-Berg expression vector, upon transfection of COS-7 cells coded for a functionally active beta1-- 4galactosyltransferase protein. The 402 residue-long protein that is expressed in the COS-7 cells has amino-terminal membrane-anchoring domain and the binding sites for UDP-galactose, N-acetylglucosamine, glucose and alpha-lactalbumin that are all intact and operative. The activity of the expressed protein is modulated by alpha-lactalbumin to change the acceptor specificity to glucose, to synthesize lactose. In the present studies deletion constructs were prepared that would code for the proteins that lacked the amino-terminal membrane-anchoring domain and up to first 70 residues of the protein equivalent to the secreted form of the enzymatically active protein. Deletion constructs, engineered in Okayama-Berg vector, upon transfection of COS-7 cells do not produce enzymatically active protein. On the other hand, when expressed in E. coli, the short form of the protein, that has first 70 amino-terminal residues deleted, is enzymatically active. The results of the amino-terminal deletion constructs suggest that the beta1-4galactosyltransferase without a missing membrane-anchoring domain when expressed within a mammalian cell is either unstable or inactivated. The role of the membrane-anchoring domain in the mammalian cells may be to target the protein to a proper site there by either preventing it from inactivation or enhancing its stability. Comparison of the sequences of Ca2+-binding loop of alpha-lactalbumin with the corresponding sequences in c-type Ca2+-binding and non-binding lysozymes show that certain conserved residues which are not directly liganded to Ca2+ may contribute to the binding of Ca2+ ion.