Hiraoka Miki, Abe Akira, Shayman James A
Nephrology Division, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0676, USA.
J Biol Chem. 2002 Mar 22;277(12):10090-9. doi: 10.1074/jbc.M111977200. Epub 2002 Jan 14.
Recently, a novel enzyme, 1-O-acylceramide synthase (ACS), was purified and characterized from bovine brain. This enzyme has both calcium-independent phospholipase A(2) and transacylase activities. The discovery of this enzyme led us to propose a new pathway for ceramide metabolism in which the sn-2-acyl group of either phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl group of ceramide. In this study, the partial amino acid sequences from the purified enzyme revealed that the enzyme contains amino acid sequences identical to those of human lecithin:cholesterol acyltransferase-like lysophospholipase (LLPL). The coding sequences of the mouse, bovine, and human genes were obtained from the respective kidney cDNAs by PCR. The open reading frames of LLPL were cloned into pcDNA3 to generate carboxyl-terminally tagged proteins. The expression of mouse LLPL in COS-7 cells demonstrated that transfected cells had higher transacylase and phospholipase A(2) activities than did non-transfected cells. Immunoprecipitation confirmed that LLPL had ACS activity. There were no significant lecithin:cholesterol acyltransferase and lysophospholipase activities in the mouse LLPL-transfected cells under either acidic or neutral conditions. Amino acid sequences from cDNAs of mouse, human, and bovine LLPLs demonstrated a signal peptide cleavage site, one lipase motif (AXSXG), and several N-linked glycosylation sites in each LLPL molecule. The replacement of serine with alanine in the lipase motif of mouse LLPL resulted in elimination of enzyme activity, indicating that the serine residue is part of the catalytic site. Deglycosylation of mouse, human, and bovine LLPLs yielded core proteins with a molecular mass of 42 kDa without change in enzyme activities. LLPL was post-translationally modified by signal peptide cleavage and N-linked glycosylation, and each mature LLPL had the same size core protein. Subcellular fractionation demonstrated that ACS activity co-localized with N-acetylglucosaminidase. Therefore, LLPL encodes a novel lysosomal enzyme, ACS.
最近，一种新型酶，1-O-酰基神经酰胺合酶（ACS），从牛脑中被纯化并鉴定。这种酶同时具有不依赖钙的磷脂酶A(2)和转酰基酶活性。该酶的发现促使我们提出一种新的神经酰胺代谢途径，即磷脂酰乙醇胺或磷脂酰胆碱的sn-2-酰基被转移至神经酰胺的1-羟基上。在本研究中，纯化酶的部分氨基酸序列显示该酶含有与人卵磷脂：胆固醇酰基转移酶样溶血磷脂酶（LLPL）相同的氨基酸序列。通过PCR从各自的肾脏cDNA中获得了小鼠、牛和人类基因的编码序列。将LLPL的开放阅读框克隆到pcDNA3中以产生羧基末端标记的蛋白质。小鼠LLPL在COS-7细胞中的表达表明，转染细胞比未转染细胞具有更高的转酰基酶和磷脂酶A(2)活性。免疫沉淀证实LLPL具有ACS活性。在酸性或中性条件下，小鼠LLPL转染细胞中均未检测到显著的卵磷脂：胆固醇酰基转移酶和溶血磷脂酶活性。小鼠、人类和牛LLPL的cDNA氨基酸序列显示每个LLPL分子中有一个信号肽切割位点、一个脂肪酶基序（AXSXG）和几个N-连接糖基化位点。小鼠LLPL脂肪酶基序中的丝氨酸被丙氨酸取代导致酶活性丧失，表明丝氨酸残基是催化位点的一部分。小鼠、人类和牛LLPL的去糖基化产生了分子量为42 kDa的核心蛋白，且酶活性未改变。LLPL通过信号肽切割和N-连接糖基化进行翻译后修饰，每个成熟的LLPL具有相同大小的核心蛋白。亚细胞分级分离表明ACS活性与N-乙酰葡糖胺酶共定位。因此，LLPL编码一种新型溶酶体酶，ACS。
