Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol-glycan-anchored protein to a secreted protein.
Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol-glycan-anchored protein to a secreted protein.
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DOI：
10.1083/jcb.116.3.799
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发表时间：
1992-02
影响因子：
7.8
通讯作者：
Lowe, M E
中科院分区：
文献类型：
--
作者：
Lowe, M E
胎盘碱性磷酸酶（PLAP）通过磷脂酰肌醇-聚糖部分（PI-聚糖）锚定在质膜上。在从COOH-末端除去29个氨基酸后，将PI-聚糖添加到新生肽链中。通过产生具有PLAP的COOH-末端和分泌蛋白的融合蛋白以及通过特异性PLAP COOH-末端氨基酸的诱变来测试所选COOH-末端氨基酸对PI-聚糖添加信号的贡献。将编码PLAP的COOH-末端的cDNA与人凝血因子X的cDNA框内融合，并在转染的COS-1细胞中表达。通过添加PI-聚糖修饰含有PLAP COOH-末端的32个氨基酸的融合蛋白。因此，PI-聚糖修饰的信号必须存在于这些氨基酸中。接下来，检查疏水结构域和切割位点之间的区域是否存在其他决定簇。间隔区中亲水性残基的突变证明这些氨基酸对PI-聚糖添加的信号没有贡献。间隔区中氨基酸的缺失阻止了PI-聚糖的添加，这表明间隔区结构域的长度或切割位点周围的氨基酸是重要的决定因素。最后，我们证明了带电残基对疏水结构域的中断阻止了PI-聚糖的添加，并导致蛋白质分泌到培养基中。疏水结构域中的单个Leu至Arg取代将PI-聚糖锚定的膜蛋白转化为分泌蛋白的发现表明，PI-聚糖锚定蛋白相对于分泌蛋白的正确分选的必要信号存在于疏水结构域中。用带电荷的氨基酸取代疏水性氨基酸可能是产生膜结合和分泌形式的相同蛋白质的机制。
Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH- terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI-glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein.