Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors.

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M(r) 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.

Aminopeptidase N (CD13) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane.

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.

Intestinal cholesterol absorption: identification of different binding proteins for cholesterol and cholesterol absorption inhibitors in the enterocyte brush border membrane.

Absorption of cholesterol from the intestine is a central part of body cholesterol homeostasis. The molecular mechanisms of intestinal cholesterol absorption and the proteins mediating membrane transport are not known. We therefore aimed to identify the proteins involved in intestinal cholesterol absorption across the luminal brush border membrane of small intestinal enterocytes. By photoaffinity labeling using photoreactive derivatives of cholesterol and 2-azetidinone cholesterol absorption inhibitors, an 80-kDa and a 145-kDa integral membrane protein were identified as specific binding proteins for cholesterol and cholesterol absorption inhibitors, respectively, in the brush border membrane of small intestinal enterocytes. The 80-kDa cholesterol-binding protein did not interact with cholesterol absorption inhibitors and vice versa; cholesterol or plant sterols did not interfere with the 145-kDa molecular target for cholesterol absorption inhibitors. Both proteins showed an identical tissue distribution and were exclusively found at the anatomical sites of cholesterol absorption-duodenum, jejunum and ileum. Neither stomach, cecum, colon, rectum, kidney, liver nor fat tissue expressed the 80- or 145-kDa binding proteins for cholesterol and cholesterol absorption inhibitors. Both proteins are different from the hitherto described candidate proteins for the intestinal cholesterol transporter,-SR-BI, ABC G5/ABC G8 or ABC A1. Our data strongly suggest that intestinal cholesterol absorption is not facilitated by a single transporter protein but occurs by a complex machinery. Two specific binding proteins for cholesterol (80 kDa) and cholesterol absorption inhibitors (145 kDa) of the enterocyte brush border membrane are probable protein constituents of the mechanism responsible for the intestinal absorption of cholesterol.

Synthesis of a biotin-tagged photoaffinity probe of 2-azetidinone cholesterol absorption inhibitors.

The design and synthesis of a biotin-tagged photoreactive analogue C-4 of the cholesterol absorption inhibitor Ezetimibe is described. Photoaffinity labeling of intestinal brush border membrane vesicles with C-4 and subsequent streptavidin-biotin chromatography leads to selective extraction of a 145 kDa integral membrane protein as the molecular target for cholesterol absorption inhibitors.