Man‐6‐P‐GlcNAc recognition by domain 5 of the cation‐independent mannose 6‐phosphate receptor

Abstract

The 300kDa cation‐independent mannose 6‐phosphate receptor (CI‐MPR) targets newly synthesized acid hydrolases displaying mannose 6‐phosphate (Man‐6‐P) modified glycans to lysosomes. The extracellular region of the CI‐MPR is comprised of 15 domains and contains two high and one low affinity Man‐6‐P binding sites (localized to domains 1‐3, 9, and 5 respectively) which bind through four conserved residues (Gln, Glu, Tyr, Arg). Unlike domains 1‐3 and 9, domain 5 exhibits a higher affinity for phosphodiester‐containing lysosomal enzymes than Man‐6‐P. The goal of this study is to determine the residues of domain 5 that are responsible for phosphodiester recognition. Domain 5 containing single amino acid substitutions of residues in the binding pocket were expressed in Pichia pastoris. Surface plasmon resonance analyses revealed that the Y679F mutant exhibited similar affinities to lysosomal enzymes, acid α‐glucosidase (GAA) monoester and GAA diester, as wild‐type domain 5. In contrast, Y679L displayed a 10‐fold decreased binding affinity to GAA diester and no detectable binding to GAA monoester. Furthermore, substitution at position 653 (W653K/T) resulted in no detectable binding to either ligand. Taken together, Y679 and W653 are important for the architecture of the binding pocket of domain 5 and its specificity for diester.