Jost B, Duluc I, Richardson M, Lathe R, Freund J N
Institut National de la Santé et de la Recherche Médicale, Unité 381, 3 avenue Molière, 67200 Strasbourg, France.
Biochem J. 1997 Oct 1;327 ( Pt 1)(Pt 1):95-103. doi: 10.1042/bj3270095.
Lactase-phlorizin hydrolase (LPH), a major digestive enzyme in the small intestine of newborns, is synthesized as a high-molecular-mass precursor comprising four tandemly repeated domains. Proteolytic cleavage of the precursor liberates the pro segment (LPHalpha) corresponding to domains I and II and devoid of known enzymic function. The mature enzyme (LPHbeta) comprises domains III and IV and is anchored in the brush border membrane via a C-terminal hydrophobic segment. To analyse the roles of the different domains of LPHalpha and LPHbeta, and the interactions between them, we have engineered a series of modified derivatives of the rat LPH precursor. These were expressed in cultured cells under the control of a cytomegalovirus promoter. The results show that recombinant LPHbeta harbouring both domains III and IV produces lactase activity. Neither domain III nor IV is alone sufficient to generate active enzyme, although the corresponding proteins are transport-competent. Tandem duplication of domains III or IV did not restore lactase activity, demonstrating the separate roles of both domains within LPHbeta. Further, the development of lactase activity did not require LPHalpha; however, LPHalpha potentiated the production of active LPHbeta but the individual LPHalpha subdomains I and II were unable to do so. Lactase activity and targeting required the C-terminal transmembrane anchor of LPH; this requirement was terminal transmembrane anchor or LPH; this requirement was not satisfied by the signal/anchor region of another digestive enzyme: sucrase-isomaltase. On the basis of this study we suggest that multiple levels of intramolecular interactions occur within the LPH precursor to produce the mature enzyme, and that the repeat domains of the precursor have distinct and specific functions in protein processing, substrate recognition and catalysis. We propose a functional model of LPHbeta in which substrate is channelled from an entry point located within domain II to the active site located in domain IV.
乳糖酶 - 根皮苷水解酶（LPH）是新生儿小肠中的一种主要消化酶，它作为一种高分子量前体被合成，该前体包含四个串联重复结构域。前体的蛋白水解切割释放出对应于结构域I和II的前体片段（LPHα），该片段缺乏已知的酶功能。成熟酶（LPHβ）包含结构域III和IV，并通过C末端疏水片段锚定在刷状缘膜中。为了分析LPHα和LPHβ不同结构域的作用以及它们之间的相互作用，我们构建了一系列大鼠LPH前体的修饰衍生物。这些衍生物在巨细胞病毒启动子的控制下在培养细胞中表达。结果表明，同时包含结构域III和IV的重组LPHβ产生乳糖酶活性。尽管相应的蛋白质具有转运能力，但结构域III和IV单独都不足以产生活性酶。结构域III或IV的串联重复并不能恢复乳糖酶活性，这表明LPHβ中这两个结构域具有各自独立的作用。此外，乳糖酶活性的发展不需要LPHα；然而，LPHα增强了活性LPHβ的产生，但LPHα的单个亚结构域I和II则无法做到这一点。乳糖酶活性和靶向需要LPH的C末端跨膜锚定；另一种消化酶蔗糖酶 - 异麦芽糖酶的信号/锚定区域不能满足这一要求。基于这项研究，我们认为LPH前体内发生了多层次的分子内相互作用以产生成熟酶，并且前体的重复结构域在蛋白质加工、底物识别和催化中具有独特而特定的功能。我们提出了一个LPHβ的功能模型，其中底物从位于结构域II内的入口点被引导至位于结构域IV内的活性位点。
