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J Biol Chem, Vol. 274, Issue 50, 35400-35406, December 10, 1999

Identification of Amino Acid Residues That Determine pH Dependence of Ligand Binding to the Asialoglycoprotein Receptor during Endocytosis^*

Stephanie Wragg and Kurt Drickamer

From the Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

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 pH 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 His²⁵⁶, Asp²⁶⁶, and Arg²⁷⁰ singly and in combination indicate that these residues reduce the affinity of the CRD for Ca²⁺, so that ligands are released at physiological 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 His²⁵⁶ and Asp²⁶⁶ 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.

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^* This work was supported by Grants 041845 and 054508 from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Wellcome Principal Research Fellow. To whom all correspondence should be addressed: Dept. of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom. Tel.: 44-1865-275727; Fax: 44-1865-275339; E-mail: kd@glycob.ox.ac.uk.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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