Carboxyl-terminal disulfide bond of acid sphingomyelinase is critical for its secretion and enzymatic function.
Carboxyl-terminal disulfide bond of acid sphingomyelinase is critical for its secretion and enzymatic function.
复制标题
酸性鞘磷脂酶的羧基末端二硫键对其分泌和酶功能至关重要。
作者：
Ching Yin Lee;Taku Tamura;N. Rabah;D. Lee;I. Ruel;A. Hafiane;I. Iatan;Dana Nyholt;F. Laporte;C. Lazure;I. Wada;L. Krimbou;J. Genest
人酸性鞘磷脂酶（ASM, EC 3.1.4.12）是一种由鞘磷脂磷酸二酯酶1 （SMPD-1）基因编码的溶酶体和分泌蛋白，可催化鞘磷脂（SM）降解为神经酰胺和磷胆碱。我们检查了其羧基端（氨基酸462-629）的结构功能特性，其中包含了SMPD-1基因中发现的大约1/3的突变。我们创建了4个自然突变体（DeltaR608、R496L、G577A和Y537H）和5个连续羧基末端缺失突变体（N620、N590、N570、N510和N490）。将His/ v5标记的野生型和突变型重组ASM瞬时转染中国仓鼠卵巢细胞，发现所有突变体均正常表达。然而，除了最小的缺失突变体N620保留了所有翻译后修饰外，没有发现它们能够分泌到培养基中。此外，只有N620保持了功能完整性（野生型活性100%）；所有其他突变体完全失去了催化SM水解的能力。重要的是，细胞表面生物素化表明，突变型DeltaR608转染的CHO细胞和来自复合杂合型尼曼-匹克病B （NPD-B）患者（DeltaR608和R441X）的成纤维细胞在质膜上的移位存在缺陷。此外，我们证明了与野生型溶酶体定位相比，DeltaR608和N590被困在内质网（ER）质量控制检查点。有趣的是，虽然野生型ASM的稳态泛素化水平很低，但通过s5a亲和层析可以纯化大量lys63连接的多泛素化DeltaR608和N590，这表明在羧基末端突变体中存在重要的错误折叠。总之，我们提供的证据表明，ASM的羧基端对其蛋白质结构至关重要，这反过来又决定了酶的功能和分泌。
The human acid sphingomyelinase (ASM, EC 3.1.4.12), a lysosomal and secretory protein coded by the sphingomyelin phosphodiesterase 1 (SMPD-1) gene, catalyzes the degradation of sphingomyelin (SM) to ceramide and phosphorylcholine. We examined the structural-functional properties of its carboxyl-terminus (amino acids 462-629), which harbors approximately 1/3 of all mutations discovered in the SMPD-1 gene. We created four naturally occurring mutants (DeltaR608, R496L, G577A, and Y537H) and five serial carboxyl-terminal deletion mutants (N620, N590, N570, N510, and N490). Transient transfection of the His/V5-tagged wild-type and mutant recombinant ASM in Chinese hamster ovary cells showed that all the mutants were normally expressed. Nonetheless, none of them, except the smallest deletion mutant N620 that preserved all post-translational modifications, were found capable of secretion to the medium. Furthermore, only the N620 conserved functional integrity (100% activity of the wild type); all other mutants completely lost the ability to catalyze SM hydrolysis. Importantly, cell surface biotinylation revealed that mutant DeltaR608 transfected CHO cells and fibroblasts from a compound heterozygous Niemann-Pick disease type B (NPD-B) patient (DeltaR608 and R441X) have defective translocation to the plasma membrane. Furthermore, we demonstrated that the DeltaR608 and N590 were trapped in the endoplasmic reticulum (ER) quality control checkpoint in contrast to the wild-type lysosomal localization. Interestingly, while the steady-state levels of ubiquitination were minimal for the wild-type ASM, a significant amount of Lys63-linked polyubiquitinated DeltaR608 and N590 could be purified by S5a-affinity chromatography, indicating an important misfolding in the carboxyl-terminal mutants. Altogether, we provide evidence that the carboxyl-terminus of the ASM is crucial for its protein structure, which in turns dictates the enzymatic function and secretion.
影响因子：
9.8
作者：
Simonaro, CM;Desnick, RJ;Schuchman, EH
通讯作者：
Schuchman, EH