Abstract
The rat hepatic asialoglycoprotein receptor mediates clearance of galactose- and N-acetylgalactosamine-terminated glycoproteins by endocytosis, binding ligands through a C-type, Ca(2+)-dependent carbohydrate-recognition domain (CRD) at extracellular pH and releasing them at lower pH in endosomes. At physiological Ca(2+) 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 Ca(2+), so that ligands are released at physiological Ca(2+) concentrations. The proximity of these three residues to the ligand-binding site at Ca(2+) site 2 of the domain suggests that they form a pH-sensitive switch for Ca(2+) 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.
Highlights
Sylated forms of a second, homologous polypeptide [3]
PH-dependent Binding of Ligands to C-type carbohydrate-recognition domain (CRD)—A solidphase binding assay was used to analyze the pH dependence of ligand binding to the isolated CRD of rat hepatic lectin 1 (RHL-1) (Fig. 1)
At a Ca2ϩ concentration near to that found in extracellular fluids (1 mM), ligand binding to the isolated CRD decreases as the pH goes from extracellular, pH 7.3, to endosomal, pH 5.4
Summary
Materials—Restriction enzymes were purchased from New England BioLabs. Gal34-BSA was obtained from E-Y Laboratories and was iodinated by the chloramine-T method [11]. Aliquots (50 l) of 125I-Gal34-BSA (1 g/ml) in 2 ϫ incubation buffer (10 mM NaCl, 2 mM CaCl2, 0.5% BSA) were mixed with equal volumes of 2 ϫ pH buffer (50 mM sodium acetate in the pH 4.5– 6.5 range, 50 mM MES in the pH 6 –7.5 range, and 50 mM Tris-Cl in the pH 6 – 8 range, with all solutions containing 2.5 M NaCl and 4% BSA) and transferred to the Immulon-4 wells. For the determination of Ca2ϩ dependence of neoglycoprotein ligand binding, Immulon-4 wells were coated with protein and incubated at 4 °C overnight. For the determination of Ca2ϩ affinity, aliquots of CaCl2 were added to tubes containing 1.5 ml of a 1 M protein solution in 50 mM sodium acetate, pH 5.5– 6.2, 50 mM MES, pH 6.2–7.5, or 50 mM Tris-HCl, pH 6.7–7.8, with all solutions containing 100 mM NaCl. All buffers were made with distilled water passed over a Chelex-100 column.
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