Wüthrich M, Grünberg J, Hahn D, Jacob R, Radebach I, Naim H Y, Sterchi E E
Institute of Biochemistry and Molecular Biology, University of Berne, Switzerland.
Arch Biochem Biophys. 1996 Dec 1;336(1):27-34. doi: 10.1006/abbi.1996.0528.
Human lactase-phlorizin hydrolase (EC 3.2.1.23/62) is a major disaccharidase in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a single-chain precursor protein and undergoes proteolytic processing during maturation. We studied proteolytic processing of human lactase-phlorizin hydrolase in transfected COS-1, Caco-2, and MDCK cells using metabolic labeling, surface immunoprecipitation, protease sensitivity assays, and microsequencing. Furthermore, we generated mutated forms of the enzyme to alter potential proteolytic cleavage sites and expressed these in Caco-2 and COS-1 cells. Since the N-terminal amino acid of microvillus lactase-phlorizin hydrolase corresponds to Ala869 in the precursor protein, it has been speculated that processing occurs at position Arg868-Ala869. Substitution of Arg868 with isoleucine, lysine, or glutamic acid had no effect on the proteolytic processing of pro-LPH in Caco-2 cells. As in wild-type enzyme a processed 160-kDa form was generated. These data are not consistent with a primary proteolytic processing at position Arg868-Ala869. Using amino-terminal amino acid sequencing of this processed form isolated from stable transfected MDCK cells we identified the cleavage site at Arg734-Leu735. Treatment of pro-lactase-phlorizin hydrolase expressed in COS-1 and MDCK cells by trypsin yielded a 145-kDa form with an identical amino terminal as the mature microvillus enzyme isolated from intestinal mucosa (Ala869). These data provide unambiguous evidence of a two-step processing of human lactase-phlorizin hydrolase. The first cleavage occurs intracellularly after a dibasic site (Arg734-Leu735) and yields the 160-kDa intermediate form. In a second step the intermediate form inserted into the microvillus membrane is trimmed to the mature enzyme by luminal trypsin.
人乳糖酶 - 根皮苷水解酶（EC 3.2.1.23/62）是小肠上皮细胞微绒毛膜中的一种主要双糖酶。该酶作为单链前体蛋白合成，并在成熟过程中经历蛋白水解加工。我们使用代谢标记、表面免疫沉淀、蛋白酶敏感性测定和微量测序技术，研究了人乳糖酶 - 根皮苷水解酶在转染的COS - 1、Caco - 2和MDCK细胞中的蛋白水解加工过程。此外，我们生成了该酶的突变形式以改变潜在的蛋白水解切割位点，并在Caco - 2和COS - 1细胞中表达这些突变形式。由于微绒毛乳糖酶 - 根皮苷水解酶的N端氨基酸对应于前体蛋白中的Ala869，因此推测加工发生在Arg868 - Ala869位点。用异亮氨酸、赖氨酸或谷氨酸替代Arg868对Caco - 2细胞中前乳糖酶 - 根皮苷水解酶的蛋白水解加工没有影响。与野生型酶一样，产生了一种加工后的160 kDa形式。这些数据与在Arg868 - Ala869位点进行初级蛋白水解加工不一致。通过对从稳定转染的MDCK细胞中分离出的这种加工形式进行N端氨基酸测序，我们确定了切割位点在Arg734 - Leu735。用胰蛋白酶处理在COS - 1和MDCK细胞中表达的前乳糖酶 - 根皮苷水解酶，产生了一种145 kDa的形式，其N端与从肠黏膜分离出的成熟微绒毛酶（Ala869）相同。这些数据提供了人乳糖酶 - 根皮苷水解酶两步加工的明确证据。第一次切割发生在细胞内的一个双碱性位点（Arg734 - Leu735）之后，产生160 kDa的中间形式。在第二步中，插入微绒毛膜的中间形式被腔内胰蛋白酶修剪成成熟酶。
