Identification of amino acid residues that determine pH dependence of ligand binding to the asialoglycoprotein receptor during endocytosis

The rat hepatic asialoglycoprotein receptor mediates clearance of galactose- and N-acetylgalactosamine-terminated glycoproteins by endocytosis, binding ligands through a C-type, Ca2+ dependent carbohydrate-recognition domain (CRD) at extracellular pH and releasing them at lower PEI in endosomes. At physiological Ca2+ concentrations, the midpoint for ligand release from the CRD of the major subunit of the receptor is pH 7.1. In contrast, the midpoint is pH 5.0 for a galactose-binding derivative of the homologous C-type CRD of serum mannose-binding protein, which would thus not efficiently release ligand at an endosomal pH of 5.4. Site-directed mutagenesis of the CRD from the major subunit of the asialoglycoprotein receptor has been used to identify residues that are essential for efficient release of ligand at endosomal pH, The effects of changes to residues His(256), Asp(266) and Arg(270) singly and in combination indicate that these residues reduce the affinity of the CRD for Ca2+, so that ligands are released at physioloscal Ca2+ concentrations. The proximity of these three residues to the ligand-binding site at Ca2+ site 2 of the domain suggests that they form a pH-sensitive switch for Ca2+ and ligand binding. Introduction of histidine and aspartic acid residues into the mannose-binding protein CRD at positions equivalent to His(256) and Asp(266) raises the pH for half-maximal binding of ligand to 6.1. The results, as well as sequence comparisons with other C-type CRDs, confirm the importance of these residues in conferring appropriate pH dependence in this family of domains.