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.
Kramer
Werner
W
Aventis Pharma Deutschland GmbH, ein Unternehmen der sanofi-aventis-Gruppe, D-65926 Frankfurt am Main, Germany. Werner.Kramer@aventis.com
Girbig
Frank
F
Corsiero
Daniel
D
Pfenninger
Anja
A
Frick
Wendelin
W
Jähne
Gerhard
G
Rhein
Matthias
M
Wendler
Wolfgang
W
Lottspeich
Friedrich
F
Hochleitner
Elisabeth O
EO
Orsó
Evelyn
E
Schmitz
Gerd
G
D000042:Absorption / Q000187:drug effects; D000818:Animals; D000924:Anticholesteremic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001384:Azetidines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D018826:CD13 Antigens / Q000378:metabolism*; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D002846:Chromatography, Affinity; D003106:Colon / Q000166:cytology; D020895:Enterocytes / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D000069438:Ezetimibe; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007421:Intestine, Small / Q000166:cytology; D007668:Kidney / Q000166:cytology; D008823:Micelles; D008871:Microvilli / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008970:Molecular Weight; D019920:Photoaffinity Labels; D011485:Protein Binding; D011817:Rabbits; D021241:Spectrometry, Mass, Electrospray Ionization; D013270:Stomach / Q000166:cytology
D000924:Anticholesteremic Agents; D001384:Azetidines; D002352:Carrier Proteins; D008823:Micelles; D019920:Photoaffinity Labels; D002784:Cholesterol; D006026:Glycoside Hydrolases; C020668:glycanase; D018826:CD13 Antigens; D000069438:Ezetimibe