C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal
C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal
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DOI：
10.1042/bj3360367
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发表时间：
1998-12-01
影响因子：
4.1
通讯作者：
Van Veldhoven, PP
中科院分区：
文献类型：
--
作者：
Amery, L;Brees, C;Van Veldhoven, PP
人 D-天冬氨酸氧化酶（一种被认为在突触释放的 D-天冬氨酸失活中发挥作用的酶）的 C 末端（Ser-Asn-Leu；SNL）功能作为过氧化物酶体靶向信号（PTSI）在体内和体外进行了研究。细菌表达的人 D-天冬氨酸氧化酶被证明与人 PTS1 结合蛋白过氧化物蛋白 5 (PEX5p) 相互作用。通过氧化酶的羧肽酶处理，结合逐渐消除，并被含有 Ser-Lys-Leu (SKL) 的肽竞争性抑制。用编码由 PKSNL（氧化酶的 C 端五肽）延伸的绿色荧光蛋白（GFP）的质粒转染小鼠成纤维细胞后，点状荧光图案很明显。间接免疫荧光显示，修饰后的 GFP 与过氧化物酶体硫解酶共定位。在转染缺乏 PEX5p 受体的成纤维细胞时，GFP-PKSNL 染色为胞质。通过 PGSNL 延伸的 GFP 的过氧化物酶体导入（用甘氨酸替换带正电的倒数第四个氨基酸）似乎比 GFP-PKSNL 慢，而通过 PKSNG 的延伸消除了修饰的 GFP 的导入。总而言之，这些结果表明，SNL（一种不符合目前在哺乳动物系统中定义的 PTSI 共识的三肽）在哺乳动物系统中充当功能性 PTS1，并且基于这项工作和其他组的共识序列必须扩展到 (S/A/C/K/N)-(K/R/H/Q/N/S)-L。
The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human D-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released D-aspartate, as a peroxisome-targeting signal (PTSI) was investigated in vivo and in vitro. Bacterially expressed human D-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP-PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP-PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTSI consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.