title: Identification of a fourth mannose 6-phosphate binding site in the cation-independent mannose 6-phosphate receptor.
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.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Olson
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Alicia C
authors.initials: AC
authors.lastname: Castonguay
authors.affiliation: National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lasanajak
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Francis C
authors.initials: FC
authors.lastname: Peterson
authors.affiliation: National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
authors.affiliation: National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: David F
authors.initials: DF
authors.lastname: Smith
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA ndahms@mcw.edu.
authors.forename: Nancy M
authors.initials: NM
authors.lastname: Dahms
mesh_terms: D000818:Animals; D001665:Binding Sites; D002412:Cations; D002417:Cattle; D006867:Hydrolases / Q000378:metabolism; D008360:Mannosephosphates / Q000378:metabolism*; D046228:Microarray Analysis; D008958:Models, Molecular; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D002412:Cations; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D006867:Hydrolases
keywords: glycan microarray; lectin; lysosomal enzymes; lysosome; mannose 6-phosphate receptor