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Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor. J Biol Chem 2004 Sep 10;279(37):38658-67

Date

07/15/2004

Pubmed ID

15252023

DOI

10.1074/jbc.M407474200

Scopus ID

2-s2.0-4644360229   55 Citations

Abstract

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.

Author List

Reddy ST, Chai W, Childs RA, Page JD, Feizi T, Dahms NM

Authors

Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of Wisconsin
Suresh Kumar PhD Associate Professor in the Pathology department at Medical College of Wisconsin




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

Amino Acid Sequence
Animals
Binding Sites
Carbohydrates
Cations
Cattle
Chromatography, Affinity
Chromatography, Gel
Cross-Linking Reagents
Cytoplasm
Disulfides
Dose-Response Relationship, Drug
Glycoside Hydrolases
Glycosylation
Humans
Kinetics
Ligands
Lysosomes
Mannosephosphates
Models, Statistical
Molecular Sequence Data
Oligonucleotide Array Sequence Analysis
Oligosaccharides
Pichia
Protein Binding
Protein Structure, Tertiary
Receptor, IGF Type 2
Sequence Homology, Amino Acid
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Surface Plasmon Resonance
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a