Moran P, Raab H, Kohr W J, Caras I W
Department of Immunobiology, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1991 Jan 15;266(2):1250-7.
The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophosphatidylinositol (GPI) membrane anchor in a process involving proteolytic removal of 17-31 COOH-terminal residues. Previous work suggested that two elements are required for anchor addition, a COOH-terminal hydrophobic domain (the GPI signal) and an element located NH2-terminal to it, postulated to be the cleavage/attachment site. Using [3H]ethanolamine (a component of the anchor) to tag the COOH terminus, we isolated and sequenced a COOH-terminal tryptic peptide, thereby identifying Ser-319 as the COOH-terminal residue attached to the GPI anchor. This indicates that a 28-residue peptide is removed during processing and localizes the cleavage/attachment site precisely to the region previously shown to be required for anchor attachment (between 10 and 20 residues NH2-terminal to the hydrophobic domain). Since DAF contains multiple cryptic cleavage/attachment sites, we used a GPI-linked human growth hormone-DAF fusion to study the structural requirements for cleavage/attachment. Our results show that while sequences immediately NH2-terminal to the attachment site are not required for anchor addition, deletion of Ser-319 abolishes both anchor attachment and transport to the cell surface. Systematic replacement of the attachment site serine with all possible amino acids indicated that alanine, aspartate, asparagine, glycine, or serine efficiently support GPI anchor attachment while valine and glutamate are partially effective. All other substitutions including cysteine (permitted at the attachment site in other GPI-anchored proteins) abolish both GPI anchor attachment and transport to the cell surface, resulting in accumulation of uncleaved fusion protein in internal compartments (endoplasmic reticulum and Golgi). These results support the general rule that the residue at the cleavage/attachment site must be small. Further, addition of a GPI anchor appears to be necessary for transport to the cell surface in transfected COS cells.
衰变加速因子（DAF）的COOH末端包含一个信号，该信号在一个涉及蛋白水解去除17 - 31个COOH末端残基的过程中指导糖基磷脂酰肌醇（GPI）膜锚的附着。先前的研究表明，添加锚需要两个元件，一个COOH末端疏水结构域（GPI信号）和位于其NH2末端的一个元件，推测为切割/附着位点。我们使用[3H]乙醇胺（锚的一个成分）标记COOH末端，分离并测序了一个COOH末端胰蛋白酶肽段，从而确定Ser - 319为附着于GPI锚的COOH末端残基。这表明在加工过程中会去除一个28个残基的肽段，并将切割/附着位点精确地定位到先前显示的锚附着所需区域（在疏水结构域NH2末端的10至20个残基之间）。由于DAF含有多个隐蔽的切割/附着位点，我们使用GPI连接的人生长激素 - DAF融合蛋白来研究切割/附着的结构要求。我们的结果表明，虽然附着位点NH2末端紧邻的序列对于添加锚不是必需的，但Ser - 319的缺失会消除锚的附着以及向细胞表面的转运。用所有可能的氨基酸系统替换附着位点的丝氨酸表明，丙氨酸、天冬氨酸、天冬酰胺、甘氨酸或丝氨酸能有效地支持GPI锚的附着，而缬氨酸和谷氨酸部分有效。所有其他替换，包括半胱氨酸（在其他GPI锚定蛋白的附着位点是允许的），都会消除GPI锚的附着以及向细胞表面的转运，导致未切割的融合蛋白在内质网和高尔基体等内部区室中积累。这些结果支持了一般规则，即切割/附着位点的残基必须较小。此外，在转染的COS细胞中，添加GPI锚似乎是转运到细胞表面所必需的。
