Structural Insights into the Mechanism of pH-dependent Ligand Binding and Release by the Cation-dependent Mannose 6-Phosphate Receptor

Abstract

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a key component of the lysosomal enzyme targeting system that binds newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and transports them to endosomal compartments. The interaction between the MPRs and its ligands is pH-dependent; the homodimeric CD-MPR binds lysosomal enzymes optimally in the pH environment of the trans Golgi network (pH ϳ 6.5) and releases its cargo in acidic endosomal compartments (<pH 5.5) and at the cell surface. In addition, CD-MPR binding affinities are modulated by divalent cations. Our previous crystallographic studies have shown that at pH 6.5, the CD-MPR bound to Man-6-P adopts a significantly different quaternary conformation than the CD-MPR in a ligand-unbound state, a feature unique among known lectin structures. To determine whether different pH conditions elicit conformational changes in the receptor that alters ligand binding affinities, we have obtained additional crystal structures representative of the various environments encountered by the receptor including: 1) the CD-MPR bound at pH 6.5 (i.e. trans Golgi network) to a high affinity ligand (the terminally phosphorylated trisaccharide P-Man(␣1,2)Man(␣1,2)Man-O-(CH 2 ) 8 COOMe), 2) the CD-MPR at pH 4.8 in an unbound state (i.e. endosome), and 3) the CD-MPR at pH 7.4 (i.e. cell surface). A detailed comparison of the available CD-MPR structures reveals the positional invariability of specific binding pocket residues and implicates intermonomer contact(s), as well as the protonation state of Man-6-P, as regulators of pH-dependent carbohydrate binding.The two members of the P-type lectin family, the 46-kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) 3 and the 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR), function to direct the delivery of ϳ50 different newly synthesized soluble lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) on their N-linked oligosaccharides to the lysosome. Both MPRs display optimal ligand binding at pH ϳ6.5 and no detectable binding below pH ϳ5, which is consistent with their function of binding their cargo in the trans Golgi network and subsequently releasing their ligands in the acidic environment of endosomes. The lysosomal enzymes are packaged into lysosomes, whereas the MPRs recycle back to the trans Golgi network to retrieve additional hydrolytic enzymes from the secretory pathway (1-3) or move to the cell surface where the CI-MPR, but not the CD-MPR, binds and internalizes lysosomal enzymes (4, 5). In vitro binding studies support these observations, demonstrating that the CI-MPR retains phosphomannosyl binding capabilities at neutral pH, whereas the CD-MPR displays a bell-shaped pH-sensitive binding profile, with optimum binding occurring at pH ϳ6.4 and little or no binding observed at pH values above pH 7.5 or below pH 5.5 (6, 7). Cells treated with reagents that raise the pH of organelles of the endosomal-lysosomal system exhibit decreased sorting of lysosomal enzymes to lyso...

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