The asialoglycoprotein receptor (ASGPr) on hepatocytes plays a role in the clearance of desialylated proteins from the serum. Although its sugar preference (N-acetylgalactosamine (GalNAc) > > galactose) and the effects of ligand valency (tetraantennary > triantennary > > diantennary > > monoantennary) and sugar spacing (20 Å > > 10 Å > > 4 Å) are well documented, the effect of particle size on recognition and uptake of ligands by the receptor is poorly defined. In the present study, we assessed the maximum ligand size that still allows effective processing by the ASGPr of mouse hepatocytes in vivo and in vitro. Hereto, we synthesized a novel glycolipid, which possesses a highly hydrophobic steroid moiety for stable incorporation into liposomes, and a triantennary GalNAc 3 -terminated cluster glycoside with a high nanomolar affinity (2 nM) for the ASGPr. Incorporation of the glycolipid into small (30 nm) [ 3 H]cholesteryl oleate-labeled long circulating liposomes (1-50%, w/w) caused a concentration-dependent increase in particle clearance that was liver-specific (reaching 85 ؎ 7% of the injected dose at 30 min after injection) and mediated by the ASGPr on hepatocytes, as shown by competition studies with asialoorosomucoid in vivo. By using glycolipid-laden liposomes of various sizes between 30 and 90 nm, it was demonstrated that particles with a diameter of >70 nm could no longer be recognized and processed by the ASGPr in vivo. This threshold size for effective uptake was not related to the physical barrier raised by the fenestrated sinusoidal endothelium, which shields hepatocytes from the circulation, because similar results were obtained by studying the uptake of liposomes on isolated mouse hepatocytes in vitro. From these data we conclude that in addition to the species, valency, and orientation of sugar residues, size is also an important determinant for effective recognition and processing of substrates by the ASGPr. Therefore, these data have important implications for the design of ASGPr-specific carriers that are aimed at hepatocyte-directed delivery of drugs and genes.The hepatic asialoglycoprotein receptor (ASGPr) 1 is a C-type (Ca 2ϩ -dependent) lectin that is expressed on the surface of hepatocytes (1) and plays a role in the clearance (endocytosis and lysosomal degradation) of desialylated proteins from the serum (2, 3) as has been shown for cellular fibronectin (4) and all IgA2 allotypes (5). The human functional receptor is a noncovalent heterotetramer composed of two homologous type II membrane polypeptides with 55% sequence identity, generally called HL-1 (hepatic lectin 1) and HL-2, at a 2:2 stoichiometry (6). The ASGPr binds glycoproteins with either nonreducing terminal ␤-D-galactose (Gal) or N-acetylgalactosamine (GalNAc) residues, at which the affinity for GalNAc is approximately 50-fold higher than for Gal (7-9). From studies using mice that are deficient in either the subunit HL-1 (10) or HL-2 (11), it is evident that both polypeptides are necessary for efficient clearance of asialoglyco...

Objective-Interventions that promote liver-directed cholesterol flux can suppress atherosclerosis, as demonstrated for scavenger receptor-BI overexpression in hypercholesterolemic mice. In analogy, we speculate that increasing lipoprotein flux to the liver via the asialoglycoprotein receptor (ASGPr) may be of therapeutic value in hypercholesterolemia. Methods and Results-A bifunctional glycolipid (LCO-Tyr-GalNAc 3 ) with a high-nanomolar affinity for the ASGPr (inhibition constant 2.1Ϯ0.2 nmol/L) was synthesized that showed rapid association with lipoproteins on incubation with serum. Prior incubation of LCO-Tyr-GalNAc 3 with radiolabeled low-density lipoprotein or high-density lipoprotein (0.5 g/g of protein) resulted in a dramatic induction of the liver uptake of these lipoproteins when injected intravenously into mice (70Ϯ3% and 78Ϯ1%, respectively, of the injected dose at 10 minutes of low-density lipoprotein and high-density lipoprotein), as mediated by the ASGPr on hepatocytes. Intravenously injected LCO-Tyr-GalNAc 3 quantitatively incorporated into serum lipoproteins and evoked a strong and persistent (Ն48 hour) cholesterol-lowering effect in normolipidemic mice (37Ϯ2% at 6 hours) and hyperlipidemic apoE Ϫ/Ϫ mice (32Ϯ2% at 6 hours). The glycolipid was also effective on subcutaneous administration. Key Words: cholesterol-lowering drugs Ⅲ hyperlipoproteinemia Ⅲ lipoproteins Ⅲ receptors Ⅲ transgenic models C linical data indicate a positive correlation between lowdensity lipoprotein (LDL) cholesterol levels and the occurrence of arteriosclerosis, 1,2 whereas a strong inverse correlation has been demonstrated between high-density lipoprotein (HDL) cholesterol levels and cardiovascular disease. 3,4 Current therapies for hypercholesterolemia are mainly focused on enhancing the hepatic clearance and catabolism of LDL and very low-density lipoprotein (VLDL) by the induction of LDL receptors (LDLrs) in the liver through inhibition of cholesterol synthesis by 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors ("statins") or stimulation of biliary cholesterol excretion with bile-acid sequestrants ("resins"), 5 leaving HDL levels relatively unaffected. However, recent findings indicate that the antiatherogenic capacity of HDL is not determined by its steady-state level in the serum, per se, but rather by its kinetics and functionality, which governs the cholesterol flux from peripheral cells to the liver ("reverse cholesterol transport"), 6 -10 as reviewed by Von Eckardstein and Assmann. 11 Permanent overexpression of the hepatic HDL receptor (scavenger receptor BI [SR-BI]) in heterozygous LDLr-deficient mice on a high-fat/ high-cholesterol diet, resulted in a strong reduction in plasma HDL cholesterol (90%) and atherosclerotic lesion area. 9 Even temporary stimulation of reverse cholesterol transport by transient hepatic overexpression of SR-BI in these mice, which resulted in a transient lowering of plasma HDL cholesterol (55%) and apolipoprotein AI (40%) levels, led to a significant decrease in lesion forma...

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