Faculty Collaboration Database

Recognition of Dictyostelium discoideum lysosomal enzymes is conferred by the amino-terminal carbohydrate binding site of the insulin-like growth factor II/mannose 6-phosphate receptor. Biochemistry 2000 Mar 07;39(9):2243-53

Date

03/01/2000

Pubmed ID

10694390

DOI

10.1021/bi992226o

Scopus ID

2-s2.0-0034056050 (requires institutional sign-in at Scopus site)   37 Citations

Abstract

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains 15 repeating domains; the two carbohydrate recognition domains (CRDs) have been localized to domains 1-3 and 7-9, and the high-affinity IGF-II binding site maps to domain 11. To characterize the carbohydrate binding properties of the IGF-II/MPR, regions of the receptor encompassing the individual CRDs were produced in a baculovirus expression system. Characterization of the recombinant proteins revealed that the pH optimum for carbohydrate binding is significantly more acidic for the carboxyl-terminal CRD than for the amino-terminal CRD (i.e., pH 6.4-6.5 vs 6.9). Equilibrium binding studies demonstrated that the two CRDs exhibit a similar affinity for Man-6-P. Furthermore, substitution of the conserved arginine residue in domain 3 (R435) or in domain 9 (R1334) with alanine resulted in a similar >1000-fold decrease in the affinity for the lysosomal enzyme, beta-glucuronidase. In contrast, the two CRDs differ dramatically in their ability to recognize the distinctive modifications (i.e., mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes: the amino-terminal CRD binds mannose 6-sulfate and Man-6-P methyl ester with a 14-55-fold higher affinity than the carboxyl-terminal CRD. Taken together, these results demonstrate that the IGF-II/MPR contains two functionally distinct CRDs.

Author List

Marron-Terada PG, Hancock MK, Haskins DJ, Dahms NM

Author

Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Binding Sites
Chromatography, Affinity
Dictyostelium
Esters
Genetic Vectors
Glucuronidase
Humans
Lysosomes
Mannosephosphates
Mutagenesis, Insertional
Oligosaccharides
Peptide Fragments
Protein Binding
Protein Structure, Tertiary
Receptor, IGF Type 2
Recombinant Proteins
Sepharose
Sequence Deletion