Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles

Abstract

Recent studies have documented the importance of Niemann-Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1-/- mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1-/- mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1-/- mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1.

Gastrointestinal Inhibition of Sodium-Hydrogen Exchanger 3 Reduces Phosphorus Absorption and Protects against Vascular Calcification in CKD

“…Brush border membrane vesicles were prepared from jejnunal mucosal scrapings using a magnesium differential centrifugation method that has been previously described. 37 Twenty micrograms brush border membrane proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against NaPi2b (generated for Ardelyx, Inc.; raised in rabbits against rat NaPi2b; amino acids 9-26, NAHPNPNKFIEGASGPQSC), NHE3 (AB3085; EMD Millipore, Billerica, MA), and b-actin (A4700; Sigma-Aldrich, St. Louis, MO). Imaging and densitometric analysis of the immunoblots were performed using Quantity One software (Bio-Rad, Hercules, CA).…”

Influence of class B scavenger receptors on cholesterol flux across the brush border membrane and intestinal absorption

“…Ϫ / Ϫ mice is the same ( 21,22 ). It is now known that NPC1L1 traffi cs between the cell surface and intracellular compartments ( 15,(23)(24)(25)(26) and plays a critical role in the internalization of BBM cholesterol through clathrin-mediated endocytosis ( 27,28 ).…”

“…Given that SR-BI and CD36 can facilitate the uptake of cholesterol into the BBM ( 21,30,31,42 ), removal of either receptor from the BBM is expected to decrease the cholesterol uptake specifi c activity of intestinal segments. The in vitro BBMV uptake experiments using a defi ned cholesterol donor particle ( Table 6 ) were performed to investigate this issue.…”

Extracellular loop C of NPC1L1 is important for binding to ezetimibe

“…However, although EZE-sensitive cholesterol transport correlates well with the amount of NPC1L1 expression (7,8), it is not possible to determine whether NPC1L1 functions alone or as part of a multiprotein complex [SR-B1 (9-15), CD36 (14), CD13 (9), caveolin-1/annexin-2 (16)], facilitating the transfer of cholesterol from outside the cell to internal cholesterol pools (9,15). Furthermore, it has been speculated that EZE may inhibit cholesterol transfer by binding to some of these other targets, in addition to its inhibition of NPC1L1 (9).…”