Berger J, Howard A D, Brink L, Gerber L, Hauber J, Cullen B R, Udenfriend S
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
J Biol Chem. 1988 Jul 15;263(20):10016-21.
Placental alkaline phosphatase (PLAP) is anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety. During processing of nascent PLAP, a 29-residue COOH-terminal peptide is cleaved out and the PI-G moiety is attached to the newly created COOH terminus of the mature protein. To investigate the structural requirements of the COOH terminus of the nascent protein for PI-G tailing and anchoring to the plasma membrane, we have transfected COS cells with wild type and mutant forms of cDNA encoding human prepro-PLAP. Utilizing a series of COOH-terminal deletion mutants of prepro-PLAP, it was found that to be PI-G-tailed the newly synthesized protein must possess an uncharged, predominantly hydrophobic amino acid sequence of a minimal length in the COOH-terminal peptide. While forms of prepro-PLAP with 17 consecutive hydrophobic residues in the terminal sequence yielded PI-G-tailed and membrane-bound products, prepro-PLAP mutants with 13 or fewer of such residues yielded hydrophilic proteins that were no longer PI-G-tailed but efficiently secreted into the medium. Studies using cassette mutants demonstrated that the precise amino sequence of the COOH-terminal region could be altered as long as minimal hydrophobicity and length was maintained.
胎盘碱性磷酸酶（PLAP）通过磷脂酰肌醇聚糖（PI - G）部分锚定在质膜上。在新生PLAP的加工过程中，一个29个残基的COOH末端肽被切割掉，PI - G部分连接到成熟蛋白新形成的COOH末端。为了研究新生蛋白COOH末端对于PI - G尾化和锚定到质膜的结构要求，我们用编码人前原PLAP的野生型和突变型cDNA转染了COS细胞。利用一系列前原PLAP的COOH末端缺失突变体，发现新合成的蛋白要进行PI - G尾化，其COOH末端肽必须具有一个最小长度的不带电荷、主要为疏水的氨基酸序列。虽然在末端序列中有17个连续疏水残基的前原PLAP形式产生了PI - G尾化且与膜结合的产物，但具有13个或更少此类残基的前原PLAP突变体产生了亲水性蛋白，这些蛋白不再进行PI - G尾化，但能有效地分泌到培养基中。使用盒式突变体的研究表明，只要保持最小的疏水性和长度，COOH末端区域的精确氨基酸序列可以改变。
