Mutational analysis of the binding site residues of the bovine cation-dependent mannose 6-phosphate receptors

被引:22
作者
Olson, LJ [1 ]
Hancock, MK [1 ]
Dix, D [1 ]
Kim, JJP [1 ]
Dahms, NM [1 ]
机构
[1] Med Coll Wisconsin, Dept Biochem, Milwaukee, WI 53226 USA
关键词
D O I
10.1074/jbc.274.52.36905
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-l 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-lll, 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.
引用
收藏
页码:36905 / 36911
页数:7
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