Roy P, Chatellard C, Lemay G, Crine P, Boileau G
Département de Biochimie, Faculté de Médecine, Université de Montréal, Québec, Canada.
J Biol Chem. 1993 Feb 5;268(4):2699-704.
Rabbit neutral endopeptidase-24.11 is a type II transmembrane protein with a 27-amino acid residue positively charged NH2-terminal cytoplasmic domain, a 23-amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. In order to study the mechanism of membrane anchoring of neutral endopeptidase-24.11, we created mutants in which the cytoplasmic tail was deleted. Expression of these mutants in COS-1 cells resulted in the secretion of approximately 10-20% of the protein into the culture medium, due possibly to the cleavage of part or all of the signal peptide/membrane anchor domain by the rough endoplasmic reticulum signal peptidase. In a second set of mutants, a hydrophilic sequence (GSQNS) was inserted midway in the signal peptide/membrane anchor domain of neutral endopeptidase-24.11. When this hydrophilic sequence was introduced into the full-length neutral endopeptidase-24.11, approximately 20% of the enzyme activity was recovered in the culture medium. This proportion increased to 93% when the cytosolic tail was deleted. Sequencing of the [3H]tyrosine- or [3H]isoleucine-labeled secreted protein indicated that proteolysis, possibly by signal peptidase, occurred on the COOH-terminal side of the signal peptide/membrane anchor domain. We conclude that the efficient cleavage of the signal peptide/membrane anchor domain and secretion of the protein require both the deletion of the cytosolic domain and the presence of a hydrophilic sequence.
兔中性内肽酶-24.11是一种II型跨膜蛋白，具有一个由27个氨基酸残基组成的带正电荷的NH2末端胞质结构域、一个由23个氨基酸残基组成的疏水信号肽/膜锚定结构域，以及一个位于膜外质侧的大催化COOH末端结构域。为了研究中性内肽酶-24.11的膜锚定机制，我们构建了缺失胞质尾巴的突变体。这些突变体在COS-1细胞中的表达导致约10-20%的蛋白质分泌到培养基中，这可能是由于粗面内质网信号肽酶切割了部分或全部信号肽/膜锚定结构域。在第二组突变体中，在中性内肽酶-24.11的信号肽/膜锚定结构域中间插入了一个亲水序列（GSQNS）。当将这个亲水序列引入全长中性内肽酶-24.11时，约20%的酶活性在培养基中恢复。当缺失胞质尾巴时，这个比例增加到93%。对[3H]酪氨酸或[3H]异亮氨酸标记的分泌蛋白进行测序表明，可能由信号肽酶进行的蛋白水解发生在信号肽/膜锚定结构域的COOH末端一侧。我们得出结论，信号肽/膜锚定结构域的有效切割和蛋白质的分泌既需要胞质结构域的缺失，也需要亲水序列的存在。
