Aceto J, Kieber-Emmons T, Cines D B
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Biochemistry. 1999 Jan 19;38(3):992-1001. doi: 10.1021/bi9810914.
Proteins linked to cell membranes by a glycosylphosphatidylinositol (GPI) anchor must first undergo cleavage by a putative transamidase between the omega and omega + 1 positions within a proposed small amino acid (SAD) domain in the carboxy terminus of the nascent polypeptide. The requirements for such processing, defined in an engineered placental alkaline phosphatase construct (miniPLAP), suggest the SAD domain functions as an autonomous unit within the context of an otherwise permissive carboxy-terminal sequence with only certain amino acids tolerated at the omega, omega + 1, and omega + 2 positions. To test whether this hypothesis could be generalized, we engineered a chimeric molecule containing the extracellular domain of miniPLAP and the carboxy-terminal portion of the urokinase receptor (MP/uPAR) into which various amino acid substitutions were introduced. The variant proteins were translated and metabolically labeled in vitro using a cell-free translation system that contains the enzymatic machinery required for carboxy-terminal processing and GPI anchor addition. The results of this study indicate that the SAD domain functions as an independent, but not an autonomous, unit. The requirements for processing in miniPLAP and MP/uPAR differed markedly in some respects, in part due to the influence of the amino acid at the omega + 4 position which both modified cleavage between the omega and omega + 1 positions and permitted a second cleavage site to be generated in some cases. In addition, substitution of bulky hydrophobic amino acids in series at the omega + 2 and omega + 3 positions inhibited carboxy-terminal processing in a dose-dependent manner, suggesting the presence of a critical docking site adjacent to the cleavage site. These results suggest the carboxy-terminal transamidase recognizes a more extended structure similar to the mechanism proposed for serine proteases. Further, the data provide a potential means for isolating the transamidase.
通过糖基磷脂酰肌醇（GPI）锚定连接到细胞膜的蛋白质，必须首先在新生多肽羧基末端一个假定的小氨基酸（SAD）结构域内的ω和ω + 1位置之间，经过一种假定的转酰胺酶的切割。在一种工程化的胎盘碱性磷酸酶构建体（miniPLAP）中确定的这种加工要求表明，SAD结构域在一个其他方面宽松的羧基末端序列的背景下作为一个自主单元发挥作用，在ω、ω + 1和ω + 2位置仅容忍某些氨基酸。为了测试这个假设是否可以推广，我们构建了一个嵌合分子，它包含miniPLAP的细胞外结构域和尿激酶受体（MP/uPAR）的羧基末端部分，并在其中引入了各种氨基酸替代。使用包含羧基末端加工和GPI锚定添加所需酶机制的无细胞翻译系统，在体外翻译并代谢标记这些变体蛋白。这项研究的结果表明，SAD结构域作为一个独立但非自主的单元发挥作用。miniPLAP和MP/uPAR中的加工要求在某些方面有显著差异，部分原因是ω + 4位置的氨基酸的影响，它既改变了ω和ω + 1位置之间的切割，又在某些情况下允许产生第二个切割位点。此外，在ω + 2和ω + 3位置串联取代大的疏水氨基酸以剂量依赖的方式抑制羧基末端加工，表明在切割位点附近存在一个关键的对接位点。这些结果表明羧基末端转酰胺酶识别一种更扩展的结构，类似于为丝氨酸蛋白酶提出的机制。此外，这些数据提供了一种分离转酰胺酶的潜在方法。
