Endogenous ligands have not, to date, been identified for the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed by parenchymal cells in the liver of mammals. On the basis of the rapid clearance of BSA bearing multiple chemically coupled sialic acid (Sia)␣2,6GalNAc␤1,4GlcNAc␤1,2Man tetrasaccharides (SiaGGnM-BSA) from the circulation, and the ability of the ASGP-R hepatic lectin-1 subunit to bind SiaGGnM-BSA, we previously proposed that glycoproteins modified with structures terminating with Sia␣2,6GalNAc may represent previously unrecognized examples of endogenous ligands for this receptor. Here, we have taken a genetic approach using wild-type and ASGP-R-deficient mice to determine that the ASGP-R in vivo does indeed account for the rapid clearance of glycoconjugates terminating with Sia␣2,6GalNAc. We have also determined that the ASGP-R is able to bind core-substituted oligosaccharides with the terminal sequence Sia␣2,6Gal␤1,4GlcNAc but not those with the terminal Sia␣2,3Gal␤1,4GlcNAc. We propose that glycoproteins bearing terminals Sia␣2,6GalNAc and Sia␣2,6Gal are endogenous ligands for the ASGP-R, and that the ASGP-R helps to regulate the relative concentration of serum glycoproteins bearing ␣2,6-linked Sia.clearance ͉ galactose ͉ N-acetylgalactosamine ͉ hepatic lectin ͉ serum glycoproteins

The asialoglycoprotein receptor (ASGP-R) is an abundant, carbohydrate-specific, endocytic receptor expressed by parenchymal cells of the liver. We recently demonstrated that the ASGP-R mediates the clearance of glycoproteins bearing Sia␣2,6GalNAc as well as those bearing terminal Gal or GalNAc. We now report that glycoproteins such as haptoglobin, serum amyloid protein (SAP), and carboxylesterase that bear oligosaccharides with terminal Sia␣2,6Gal are elevated in the plasma of ASGP-R-deficient mice. Because of their abundance in plasma, glycoproteins bearing terminal Sia␣2,6Gal will saturate the ASGP-R and compete with each other on the basis of their relative affinities for the ASGP-R and their relative abundance. We propose that the ASGP-R mediates the clearance of glycoproteins that bear oligosaccharides terminating with Sia␣2,6Gal and thereby helps maintain the relative concentrations of these glycoproteins in the blood. The asialoglycoprotein receptor (ASGP-R)3 was initially identified and characterized by Ashwell and co-workers (1, 2) on the basis of its ability to rapidly remove glycoproteins bearing oligosaccharides terminating with ␤1,4-linked Gal from the circulation. The ASGP-R has been extensively characterized since its initial discovery; however, its biologic function in vivo has remained unclear. This endocytic receptor is highly abundant with 500,000 receptors expressed at the plasma membrane of hepatocytes (3-5) and is rapidly internalized (3, 6). The abundance of the ASGP-R and its rapid rate of internalization in combination with the large number of hepatocytes that are present in the liver, 1.35 ϫ 10 8 /g of liver (7,8), results in an enormous potential capacity to remove glycoproteins from the circulation. Until recently, mice that have had either subunit of the ASGP-R ablated, subunit 1 ASGP-R1(Ϫ/Ϫ) or subunit 2 ASGP-R2(Ϫ/Ϫ), have not been reported to have altered levels of circulating glycoproteins in their blood or to have a physiologic phenotype (9, 10). However, Grewal et al. (11) have reported that the ASGP-R plays a role in von Willebrand factor homeostasis and promotes thrombocytopenia during Steptococcus pneumoniae sepsis by removing platelets that have had their surface sialic acid removed by the bacterial neuraminidase.We recently established that glycoproteins bearing Asnlinked oligosaccharides terminating with the sequence Sia␣2,6GalNAc␤1,4GlcNAc are recognized by the ASGP-R and rapidly removed from the blood (12, 13). Glycoproteins bearing terminal Sia␣2,6GalNAc␤1,4GlcNAc are the first examples of endogenous glycoproteins that can be recognized by the ASGP-R without further modification; i.e. removal of terminal Sia. Glycoproteins bearing these structures, for example the prolactin-like proteins (14), glycodelin (15), urokinase (16), and glycoprotein hormones (17), are not highly abundant, suggesting that the ASGP-R recognizes and clears additional more abundant glycoproteins. The most likely candidates are glycoproteins bearing Asn-linked oligosaccharides that terminate with...

The asialoglycoprotein-receptor (ASGP-R) located on liver parenchymal cells was originally identified and characterized on the basis of its ability to bind glycoproteins bearing terminal galactose (Gal) or N-acetylgalactosamine (GalNAc); however, endogenous ligands for the ASGP-R have not to date been definitively identified. We have determined that the rat ASGP-R specifically binds oligosaccharides terminating with the sequence Sia␣2,6GalNAc␤1,4GlcNAc␤1,2Man. Bovine serum albumin chemically modified with 10 -15 tetrasaccharides with the sequence Sia␣2,6GalNAc␤1,4GlcNAc␤1,2Man is cleared from the blood of the rat with a half-life of <1 min by a receptor located in the liver. We have isolated the receptor and identified it as the ASGP-R. Furthermore, we have determined that subunit 1 of the ASGP-R accounts for the binding of terminal Sia␣2,6GalNAc␤. Based on the newly defined specificity of the rat ASGP-R we hypothesize that glycoproteins bearing structures that are selectively modified with terminal Sia␣2, 6GalNAc␤ and are released into the blood may be endogenous ligands for the rat ASGP-R.The rapid clearance of glycoproteins from the blood following removal of sialic acid (Sia) 1 residues and exposure of underlying galactose (Gal) residues was first reported by Ashwell and Morell in the early 1970s (1, 2) and led to the discovery of the asialoglycoprotein-receptor (ASGP-R). The specificity and biochemical features of this endocytic receptor have been extensively investigated since that time (3, 4). Although it is clear that the ASGP-R receptor binds oligosaccharides with terminal ␤-linked N-acetylgalactosamine (GalNAc) or Gal and can mediate the rapid clearance of glycoproteins bearing these terminal sugars from the circulation, endogenous ligands for this abundant receptor have not yet been identified.We first described N-linked oligosaccharides containing ␤1,4-linked GalNAc on lutropin (LH) and other members of the glycoprotein hormone family of glycoproteins (5, 6). The GalNAc is found almost exclusively in the form of GalNAc-4-SO 4 , reflecting the sequential action of a protein-specific ␤1,4-Nacetylgalactosaminyltransferse (␤1,4GalNAcT) and a GalNAc-4-sulfotransferase (GalNAc-4-ST1) (7,8). The terminal GalNAc-4-SO 4 is recognized by a receptor in hepatic endothelial cells that regulates the circulatory half-life of LH following its stimulated release into the blood (9 -12). The control of circulatory half-life is important for regulating estrogen production in vivo during implantation of the embryo (13). We subsequently described the presence of N-linked oligosaccharides terminating with Sia␣2,6GalNAc␤ on prolactin/growth (PLP) hormone family members that are synthesized by rat placenta spongiotrophoblasts between mid gestation and birth (14).

We recently reported that the rat asialoglycoprotein receptor binds oligosaccharides terminating with sialic acid (Sia) ␣2,6GalNAc. Despite a high percentage of identical amino acids in their sequences, orthologues of the asialoglycoprotein receptor (ASGP-R) in different mammals differ in their specificity for terminal Sia␣2,6GalNAc. The recombinant subunit 1 of the ASGP-R from the rat (RHL-1 or rat hepatic lectin) and the mouse (MHL-1 or mouse hepatic lectin), which differ at only 12 positions in the amino acid sequence of their carbohydrate recognition domains, binds Sia␣2,6GalNAc␤1,4GlcNAc␤1,2Man-bovine serum albumin and GalNAc␤1,4GlcNAc␤1,2Man-bovine serum albumin in ratios of 16:1.0 and 1.0:1.0, respectively. Mutagenesis was used to show that amino acids both in the immediate vicinity of the proposed binding site for terminal GalNAc and on the ␣2 helix that is distant from the binding site contribute to the specificity for terminal Sia␣2,6GalNAc. Thus, multiple amino acid sequence alterations in two key locations contribute to the difference in specificity observed for the rat and mouse ASGP-Rs. We hypothesize that the altered specificity of ASPG-R orthologues in such evolutionarily closely related species reflects rapidly changing requirements for recognition of endogenous or exogenous oligosaccharides in vivo.The hepatic asialoglycoprotein receptor (ASGP-R) 1 identified by Van Den Hamer et al. (1) was the first mammalian lectin to be described. A characteristic of the receptor is its ability to rapidly remove glycoproteins from the circulation that have been treated with neuraminidase or mild acid (2). Rapid clearance reflects the specificity of the ASGP-R for terminal ␤-linked galactose (Gal) or N-acetylgalactosamine (GalNAc) residues that are exposed by the removal of terminal sialic acid (Sia). The ASGP-R was subsequently shown to be a hetero-oligomer consisting of two highly homologous subunits, hepatic lectin subunits 1 and 2 (HL-1 and HL-2, respectively) (3-5). Whereas both subunits are required for the endocytosis of ligands, the carbohydrate binding activity is associated predominantly with HL-1 (6, 7). Prior structural and functional studies of the ASGP-R have included the crystallization of human and the generation of mice with genetically ablated HL-2 (9) and HL-1 (10). These studies have been informative but have not revealed the identity of endogenous ligands that may be recognized by the ASGP-R in vivo.We recently reported that, in the rat, the ASGP-R mediates the rapid clearance of bovine serum albumin bearing multiple chemically coupled tetrasaccharides with the sequence Sia␣2,6GalNAc␤1,4GlcNAc␤1,2Man (Sia␣2,6GGnM-bovine serum albumin (BSA)) (11). Because the prolactin-like hormones bearing N-linked oligosaccharides terminating with the sequence Sia␣2,6GalNAc␤1,4GlcNAc are synthesized during the last third of pregnancy in the placenta of the rat (12), they may represent the first examples of endogenous ligands for the ASGP-R. Although rare, the terminal sequence Sia␣2,6GalNAc␤1,4GlcNAc h...