Oda K, Cheng J, Saku T, Takami N, Sohda M, Misumi Y, Ikehara Y, Millán J L
Department of Biochemistry, Niigata University School of Dentistry, Japan.
Biochem J. 1994 Jul 15;301 ( Pt 2)(Pt 2):577-83. doi: 10.1042/bj3010577.
Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.
胎盘碱性磷酸酶（PLAP）最初作为一种带有C末端延伸的前体（proPLAP）进行合成。我们构建了一个重组cDNA，其编码一种嵌合蛋白（αGL-PLAP），该嵌合蛋白由大鼠α2u球蛋白（αGL）和PLAP的C末端延伸部分组成。在转染了αGL-PLAP cDNA的COS-1细胞中表达了两种分子形式（25 kDa和22 kDa）。只有22 kDa的形式同时被[3H]硬脂酸和[3H]乙醇胺标记。用磷脂酰肌醇特异性磷脂酶C消化后，22 kDa的形式被释放到培养基中，表明这种形式通过糖基磷脂酰肌醇（GPI）锚定在细胞表面。针对proPLAP的C末端九肽产生的特异性IgG沉淀了25 kDa的形式，但没有沉淀22 kDa的形式，这表明25 kDa的形式是保留C末端前肽的前体。当在COS-1细胞中表达一种突变的αGL-PLAP，其中在假定的切割/附着位点天冬氨酸残基被色氨酸取代时，通过免疫荧光显微镜判断，25 kDa的前体是细胞内唯一发现的形式，并保留在内质网中。用编码αGL-PLAP野生型和突变型形式的mRNA进行的体外翻译表明，C末端前肽从野生型嵌合蛋白上被切割下来，但没有从突变型蛋白上被切割下来。这产生了带有GPI锚的22 kDa形式，表明GPI与αGL-PLAP的天冬氨酸残基（Asp159）共价连接。综上所述，这些结果表明PLAP的C末端前肽在体外和培养细胞的体内发挥信号作用，使αGL成为一种GPI连接的膜蛋白，并且本研究构建的嵌合蛋白可能有助于阐明在内质网中发生的前肽切割和GPI附着的潜在机制。
