Mammalian Lass6 and its related family members regulate synthesis of specific ceramides
Mammalian Lass6 and its related family members regulate synthesis of specific ceramides
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DOI：
10.1042/bj20050291
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发表时间：
2005-08-15
影响因子：
4.1
通讯作者：
Igarashi, Y
中科院分区：
文献类型：
--
作者：
Mizutani, Y;Kihara, A;Igarashi, Y
Lass家族成员在真核生物中高度保守，在神经酰胺合成中起作用。在小鼠中，至少有5个Lass家族成员，分别是Lass1、Lass2、Lass4、Lass5和迄今未被鉴定的Lass6。为了研究每个Lass成员在神经酰胺合成中的具体作用，我们克隆了这五个小鼠蛋白。在培养的细胞中，任何一种Lass蛋白的过量产生都会导致细胞神经酰胺的增加，但产生的神经酰胺种类不同。过量生产的Lass1优先增加c -18:0-神经酰胺水平，过量生产的Lass2和Lass4增加较长神经酰胺如C-22:0-和C-24:0-神经酰胺水平。Lass5和Lass6产生较短的神经酰胺种类（C-14:0-和c -16:0-神经酰胺）；然而，它们对饱和/不饱和脂肪酰基辅酶a的底物偏好不同。除了底物偏好的差异外，我们还通过Northern blotting证明了Lass家族成员在组织之间的表达差异。此外，我们发现Lass蛋白在糖基化方面存在差异。在5个成员中，只有Lass2、Lass5和Lass6在其n端Asn残基上进行了n糖基化。一些Lass蛋白的n -糖基化的发生提供了拓扑上的见解，表明Lass家族成员的n端可能面向内质网膜的管腔侧。此外，基于蛋白酶K消化实验，我们证明了Lass6的c端面向膜的细胞质侧。根据这些数据，我们提出了Lass家族成员中保守Lag1基序的拓扑结构，即n端区域面向内质网膜的管腔侧，c端区域面向胞浆侧。
The Lass (longevity-assurance homologue) family members, which are highly conserved among eukaryotes, function in ceramide synthesis. In the mouse, there are at least five Lass family members, Lass1, Lass2, Lass4, Lass5 and the hitherto uncharacterized Lass6. To investigate specific roles for each Lass member in ceramide synthesis, we cloned these five mouse proteins. Overproduction of any Lass protein in cultured cells resulted in an increase in cellular ceramide, but the ceramide species produced varied. Overproduction of Lass1 increased C-18:0-ceramide levels preferentially, and overproduction of Lass2 and Lass4 increased levels of longer ceramides such as C-22:0- and C-24:0- ceramides. Lass5 and Lass6 produced shorter ceramide species (C-14:0- and C-16:0-ceramides); however, their substrate preferences towards saturated/unsaturated fatty acyl-CoA differed. In addition to differences in substrate preferences, we also demonstrated by Northern blotting that Lass family members are differentially expressed among tissues. Additionally, we found that Lass proteins differ with regard to glycosylation. Of the five members, only Lass2, Lass5 and Lass6 were N-glycosylated, each at their N-terminal Asn residue. The occurrence of N-glycosylation of some Lass proteins provides topological insight, indicating that the N-termini of Lass family members probably face the luminal side of the endoplasmic reticulum membrane. Furthermore, based on a proteinase K digestion assay, we demonstrated that the C-terminus of Lass6 faces the cytosolic side of the membrane. From these data we propose topology for the conserved Lag1 motif in Lass family members, namely that the N-terminal region faces the luminal side and the C-terminal region the cytosolic side of the endoplasmic reticulum membrane.