Purification and characterization of human intestinal neutral ceramidase
Purification and characterization of human intestinal neutral ceramidase
复制标题
DOI：
10.1016/j.biochi.2007.03.009
复制
发表时间：
2007-08-01
期刊：
影响因子：
3.9
通讯作者：
Nilsson, Ake
中科院分区：
文献类型：
--
作者：
Ohlsson, Lena;Palmberg, Carina;Nilsson, Ake
鞘脂在肠道中被鞘磷脂酶和神经酰胺酶降解为神经酰胺和鞘氨醇，其可以抑制细胞增殖并诱导细胞凋亡，从而在肠道中具有抗肿瘤作用。尽管先前的啮齿动物研究（包括基因敲除小鼠的实验）表明中性神经酰胺酶在神经酰胺消化中的作用，但人类酶从未被纯化并以其纯化形式表征。我们在这里报告的纯化和鉴定中性神经酰胺酶从人类回肠造口术内容，使用辛酰基-[C-14]鞘氨醇作为底物。经4步层析后，得到一条分子量为116 kDa的均一蛋白条带。MALDI质谱法鉴定了16种与El Bawab等人[Molecular cloning and characterization of a human mitochondrial ceramidase，J.Biol.Chem.275（2000）21508-21513]和Hwang等人[Subcellular localization of human neutral ceramidase expressed in HEK 293 cells，Biochem. Biophys.通信资源331（2005）37-42]。通过RT-PCR和5 '-RACE方法，从人十二指肠活检样品中获得了预测的中性神经酰胺酶的部分核苷酸序列，其与已知的中性/碱性神经酰胺酶的核苷酸序列同源。该酶具有中性pH最适值，并催化水解和神经酰胺的形成，而没有明显的胆盐依赖性。它被Cu 2+和Zn 2+离子以及低浓度的胆固醇抑制。该酶是一种糖蛋白，但去糖基化不影响其活性。我们的研究表明，中性神经酰胺酶在人肠道中表达，在肠腔中释放，并在人肠道中的神经酰胺代谢中起主要作用。(c)2007年，Elsevier Masson SAS。All rights reserved.
Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[C-14] sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116 kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase previously cloned by El Bawab et al. [Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513] and Hwang et al. [Subcellular localization of human neutral ceramidase expressed in HEK293 cells, Biochem. Biophys. Res. Commun. 331 (2005) 37-42]. By RT-PCR and 5'-RACE methods, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide without distinct bile salt dependence. It is inhibited by Cu2+ and Zn2+ ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut. (c) 2007 Elsevier Masson SAS. All rights reserved.