Cloning of a human acid sphingomyelinase cDNA with a new mutation that renders the enzyme inactive.
Cloning of a human acid sphingomyelinase cDNA with a new mutation that renders the enzyme inactive.
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克隆人类酸性鞘磷脂酶 cDNA，其中包含使该酶失活的新突变。
DOI：
10.1093/oxfordjournals.jbchem.a124131
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发表时间：
1993
影响因子：
2.7
通讯作者：
Chan,WY
中科院分区：
文献类型：
--
作者：
Ida,H;Rennert,OM;Eto,Y;Chan,WY
首先用人工合成的寡核苷酸探针筛选λ gt11胎盘cDNA文库，然后用部分cDNA进行筛选，获得了编码人酸性鞘磷脂酶的cDNA。全长cDNA hPSM55由2376个核苷酸组成，其中5‘未翻译序列122个核苷酸，开放阅读框1884个核苷酸，编码627个氨基酸的蛋白，3’未翻译区370个碱基。hPSM55与Schuchman等人报道的psm - lfl几乎相同。Chem266, 8531-8539, 1991)，除了信号肽中有6个碱基对的缺失，这表明缬氨酸和亮氨酸残基可能在36和37位之间被删除，以及463t -到c -的过渡，这表明155精氨酸可能被胱氨酸取代。该cDNA在COS-7细胞和中国仓鼠卵巢细胞中均有表达。转染后，两种细胞系的酸性鞘磷脂酶活性均未升高。然而，在hPSM55中，一个碱基的改变（463C到T）的修正导致了酸性鞘磷脂酶活性的增加。这些结果表明，核苷酸463c向T的突变在酸性鞘磷脂酶的催化活性中起重要作用。
A cDNA encoding human acid sphingomyelinase was initially obtained by screening a placental cDNA library in λ gt11 with a synthetic oligonucleotide probe and subsequently with partial cDNA. The full-length cDNA, hPSM55, comprised 2, 376 nucleotides, with a 5' untranslated sequence of 122 nucleotides, an open reading frame of 1, 884 nucleotides encoding a protein of 627 amino acids, and a 3' untranslated region of 370 bases. hPSM55 was almost identical to pASM-lFL reported by Schuchman et al. (J. Biol. Chem266, 8531–8539, 1991) except for a 6 base pair deletion in the signal peptide, which indicated the possible removal of valine and leucine residues between positions 36 and 37, and a463T- to C-transition, which indicated a possible substitution of155arginine for cystine. This cDNA was expressed in both COS-7 cells and Chinese hamster ovary cells. There was no increase in acid sphingomyelinase activity in either cell line following transfection. However, the correction of a single base change,463C to T, in hPSM55 caused increased acid sphingomyelinase activity in transfectants. These results suggest that the mutation of nucleotide463C to T plays an important role in the catalytic activity of acid sphingomyelinase.