Aminopeptidase N (CD13) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane
Aminopeptidase N (CD13) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane
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DOI：
10.1074/jbc.m406309200
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发表时间：
2005-01-14
影响因子：
4.8
通讯作者：
Schmitz, G
中科院分区：
文献类型：
--
作者：
Kramer, W;Girbig, F;Schmitz, G
肠道胆固醇吸收是血清胆固醇水平的重要调节器。依折麦布是一种肠胆固醇吸收的特异性抑制剂，最近被引入医疗实践;然而，其作用机制仍不清楚。依折麦布既不影响胆固醇从肠腔混合胶束中的释放，也不影响胆固醇向肠细胞刷状缘膜的转移。使用膜不可渗透的依折麦布类似物，我们可以证明胆固醇吸收抑制剂与肠腔小肠上皮细胞刷状缘膜的结合足以抑制胆固醇吸收。通过用光反应性依泽替米贝类似物进行光亲和标记，将145-kDa的整合膜蛋白鉴定为肠上皮细胞刷状缘膜中胆固醇吸收抑制剂的分子靶标（克雷默，W.，Glenyk，H.，Petry，S.，豪雅，H.，Schafer，H. L.，Wendler，W.，Corsiero，D.，Girbig，F.，和Weyland，C. 04 The Dog（2000）487，293-297）。通过三种不同方法纯化145-kDa依折麦布结合蛋白，测序显示其与膜结合胞外酶氨肽酶N（（丙氨酰）氨肽酶; EC 3.4.11.2; APN;白血病抗原CD 13）相同。APN的酶活性不受依折麦布（类似物）的影响。依折麦布和不可吸收的依折麦布类似物可部分抑制融合CaCo-2细胞对混合胶束递送的胆固醇的摄取。用依折麦布预孵育融合的CaCo-2细胞导致质膜中单克隆抗体的荧光APN染色强烈减少。氨肽酶N不依赖于其酶活性，参与内吞过程，如病毒的摄取。我们的研究结果表明，依折麦布与来自小肠腔的APN的结合阻断了富含胆固醇的膜微区的内吞作用，从而限制了肠道胆固醇吸收。
Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schafer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293-297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N (( alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe ( analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption.