Faculty Collaboration Database

Identification of a fourth mannose 6-phosphate binding site in the cation-independent mannose 6-phosphate receptor. Glycobiology 2015 Jun;25(6):591-606

Date

01/13/2015

Pubmed ID

25573276

Pubmed Central ID

PMC4410830

DOI

10.1093/glycob/cwv001

Scopus ID

2-s2.0-84930944628 (requires institutional sign-in at Scopus site)   24 Citations

Abstract

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in lysosome biogenesis by targeting ∼ 60 different phosphomannosyl-containing acid hydrolases to the lysosome. This type I membrane glycoprotein has a large extracellular region comprised of 15 homologous domains. Two mannose 6-phosphate (M6P) binding sites have been mapped to domains 3 and 9, whereas domain 5 binds preferentially to the phosphodiester, M6P-N-acetylglucosamine (GlcNAc). A structure-based sequence alignment predicts that the C-terminal domain 15 contains three out of the four conserved residues identified as essential for carbohydrate recognition by domains 3, 5 and 9 of the CI-MPR, but lacks two cysteine residues that are predicted to form a disulfide bond. To determine whether domain 15 of the CI-MPR has lectin activity and to probe its carbohydrate-binding specificity, truncated forms of the CI-MPR were tested for binding to acid hydrolases with defined N-glycans in surface plasmon resonance analyses, and used to interrogate a phosphorylated glycan microarray. The results show that a construct encoding domains 14-15 binds both M6P and M6P-GlcNAc with similar affinity (Kd = 13 and 17 μM, respectively). Site-directed mutagenesis studies demonstrate the essential role of the conserved Tyr residue in domain 15 for phosphomannosyl binding. A structural model of domain 15 was generated that predicted an Arg residue to be in the binding pocket and mutagenesis studies confirmed its important role in carbohydrate binding. Together, these results show that the CI-MPR contains a fourth carbohydrate-recognition site capable of binding both phosphomonoesters and phosphodiesters.

Author List

Olson LJ, Castonguay AC, Lasanajak Y, Peterson FC, Cummings RD, Smith DF, Dahms NM

Authors

Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of Wisconsin
Linda J. Olson PhD Assistant Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Francis C. Peterson 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
Cations
Cattle
Hydrolases
Mannosephosphates
Microarray Analysis
Models, Molecular
Receptor, IGF Type 2
Surface Plasmon Resonance