Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase.
Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase.
复制标题
通过胎盘碱性磷酸酶 Asp-484 的位点特异性诱变确定磷脂酰肌醇-聚糖锚定膜蛋白的切割/附着位点的选择性。
DOI：
10.1073/pnas.87.1.157
复制
发表时间：
1990
影响因子：
11.1
通讯作者：
Sidney Udenfriend
中科院分区：
文献类型：
--
作者：
R. Micanovic;L. Gerber;Joel P. Berger;K. Kodukula;Sidney Udenfriend
现在已知许多蛋白质通过附着在其 COOH 末端的磷脂酰肌醇聚糖 (PI-G) 部分锚定在质膜上。胎盘碱性磷酸酶 (PLAP) 已被用作研究 PI-G 尾蛋白 COOH 末端加工机制的模型。 pre-pro-PLAP 的 COOH 末端结构域提供了一个加工信号，在此过程中，大部分疏水性 29 个残基 COOH 末端肽被去除，并且 PI-G 部分被添加到新暴露的 Asp-484 末端。该切割/附着位点经历了几乎饱和诱变，并且确定了各种突变PLAP形式的酶活性、COOH末端加工和细胞定位。用甘氨酸、丙氨酸、半胱氨酸、天冬酰胺或丝氨酸（I 类）取代 Asp-484 产生带有 PI-G 尾且具有酶活性的蛋白质。然而，并非所有 I 类突变蛋白都具有相同程度的 PI-G 尾部。在 484 位具有其他取代基（苏氨酸、脯氨酸、甲硫氨酸、缬氨酸、亮氨酸、酪氨酸、色氨酸、赖氨酸、谷氨酸和谷氨酰胺；II 类）的前原 PLAP 以及 I 类氨基酸均得到表达，但几乎没有或没有加工成 PI-G 尾形式，并且后者表现出非常低的酶活性。 II 类突变体产生的大部分 PLAP 蛋白和 I 类突变体产生的一些 PLAP 蛋白被隔离在细胞内，显然在内质网 (ER) 中。最有可能的是，残基 484 处的某些氨基酸是优选的，因为它们为推定的“转酰胺基化”酶产生更好的底物。至少在转染的 COS 细胞中，即使对于首选底物，翻译后 PI-G 尾加工也无法完成。显然，PI-G 尾部是内质网转运和 PLAP 酶活性所必需的。未转酰胺基化的蛋白质显然以非活性构象保留在内质网中。
Many proteins are now known to be anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety that is attached to their COOH termini. Placental alkaline phosphatase (PLAP) has been used as a model for investigating mechanisms involved in the COOH-terminal processing of PI-G-tailed proteins. The COOH-terminal domain of pre-pro-PLAP provides a signal for processing during which a largely hydrophobic 29-residue COOH-terminal peptide is removed, and the PI-G moiety is added to the newly exposed Asp-484 terminus. This cleavage/attachment site was subjected to an almost saturation mutagenesis, and the enzymatic activities, COOH-terminal processing, and cellular localizations of the various mutant PLAP forms were determined. Substitution of Asp-484 by glycine, alanine, cysteine, asparagine, or serine (category I) resulted in PI-G-tailed and enzymatically active proteins. However, not all category I mutant proteins were PI-G tailed to the same extent. Pre-pro-PLAP with other substituents at position 484 (threonine, proline, methionine, valine, leucine, tyrosine, tryptophan, lysine, glutamic acid, and glutamine; category II) were expressed, as well as the category I amino acids, but there was little or no processing to the PI-G-tailed form, and this latter group exhibited very low enzyme activity. The bulk of the PLAP protein produced by category II mutants and some produced by category I mutants were sequestered within the cell, apparently in the endoplasmic reticulum (ER). Most likely, certain amino acids at residue 484 are preferred because they yield better substrates for the putative "transamidating" enzyme. In transfected COS cells, at least, posttranslational PI-G-tail processing does not go to completion even for preferred substrates. Apparently PI-G tailing is a requisite for transport from the ER and for PLAP enzyme activity. Proteins that are not transamidated are apparently retained in the ER in an inactive conformation.