Mutational analysis of the binding site residues of the bovine cation-dependent mannose 6-phosphate receptor. J Biol Chem 1999 Dec 24;274(52):36905-11
Date
12/22/1999Pubmed ID
10601243DOI
10.1074/jbc.274.52.36905Scopus ID
2-s2.0-0033601324 (requires institutional sign-in at Scopus site) 29 CitationsAbstract
Mannose 6-phosphate receptors (MPRs) deliver soluble acid hydrolases to the lysosome in higher eukaryotic cells. The two MPRs, the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/cation-independent MPR, carry out this process by binding with high affinity to mannose 6-phosphate residues found on the N-linked oligosaccharides of their ligands. To elucidate the key amino acids involved in conveying this carbohydrate specificity, site-directed mutagenesis studies were conducted on the extracytoplasmic domain of the bovine CD-MPR. Single amino acid substitutions of the residues that form the binding pocket were generated, and the mutant constructs were expressed in transiently transfected COS-1 cells. Following metabolic labeling, mutant CD-MPRs were tested for their ability to bind pentamannosyl phosphate-containing affinity columns. Of the eight amino acids mutated, four (Gln-66, Arg-111, Glu-133, and Tyr-143) were found to be essential for ligand binding. In addition, mutation of the single histidine residue, His-105, within the binding site diminished the binding of the receptor to ligand, but did not eliminate the ability of the CD-MPR to release ligand under acidic conditions.
Author List
Olson LJ, Hancock MK, Dix D, Kim JJ, Dahms NMAuthors
Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of WisconsinLinda J. Olson PhD Assistant Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAnimals
Binding Sites
COS Cells
Cattle
Hydrogen-Ion Concentration
Molecular Sequence Data
Mutagenesis, Site-Directed
Receptor, IGF Type 2
Structure-Activity Relationship