Closely Related Mammals Have Distinct Asialoglycoprotein Receptor Carbohydrate Specificities

Abstract

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...

From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

“…In our case, the positional effect of sialylation is revealed by comparatively analyzing the two sets of sialylated N-glycans. Historically, following its discovery the hepatic asialoglycoprotein receptor has been shown to tolerate a2,6-sialylation as branch-end modification, with species-dependent differences (Park and Baenziger, 2004;Park et al, 2005). Marked differences in serum clearance and hepatic content were thus detected between N-glycans with a2,3-or a2,6-sialylation, and an influence of core substitution for a2,6-sialylated N-glycans was noted ( Figure 5, middle and bottom).…”

Multiple Hepatic Receptors Cooperate to Eliminate Secretory Mucins Aberrantly Entering the Bloodstream: Are Circulating Cancer Mucins the “Tip of the Iceberg”?

“…Both types of receptors form homo-oligomer/heterooligomer, permitting recognition of a broad range of glycans containing terminal hGal, hGalNAc, or aGalNAc (39, 50). The ASGPR was also recently reported to recognize Siaa2-6GalNAch1-2Man (51).…”

Role of sialic acid for platelet life span: exposure of β-galactose results in the rapid clearance of platelets from the circulation by asialoglycoprotein receptor–expressing liver macrophages and hepatocytes

“…The ASGPR mediates the binding and removal of glycoconjugates having exposed ␤Gal and GalNAc, 32 although recognition of SA␣2,6GalNAc and SA␣2,6Gal has also been reported. 33,34 The binding affinity of the ASGPR subtypes on hepatocytes or macrophages depends on ligand valency, spatial arrangement, and size. 35,36 Small ligands (Ͻ 70 nm) are thought to be preferentially internalized via the hepatocyte ASGPR-mediated endocytic pathway.…”