Carbohydrate recognition by the mannose-6-phosphate receptors

Abstract

The two P-type lectins, the 46kDa cation-dependent mannose-6-phosphate (Man-6-P) receptor (CD-MPR), and the 300kDa cation-independent Man-6-P receptor (CI-MPR), are the founding members of the growing family of mannose-6-phosphate receptor homology (MRH) proteins. A major cellular function of the MPRs is to transport Man-6-P-containing acid hydrolases from the Golgi to endosomal/lysosomal compartments. Recent advances in the structural analyses of both CD-MPR and CI-MPR have revealed the structural basis for phosphomannosyl recognition by these receptors and provided insights into how the receptors load and unload their cargo. A surprising finding is that the CD-MPR is dynamic, with at least two stable quaternary states, the open (ligand-bound) and closed (ligand-free) conformations, similar to those of hemoglobin. Ligand binding stabilizes the open conformation; changes in the pH of the environment at the cell surface and in endosomal compartments weaken the ligand-receptor interaction and/or weaken the electrostatic interactions at the subunit interface, resulting in the closed conformation.

Figure 1

Lysosomal enzyme trafficking. Movements of lysosomal enzymes and MPRs between the various intracellular compartments and the cell surface are shown. Phosphorylation of mannose residues on N-linked oligosaccharides occurs in two steps (see text). The five potential sites of phosphorylation are indicated in pink letters. Lysosomal enzymes that acquire the Man-6-P tag in early Golgi compartments bind specifically to MPRs in the Golgi. The resulting receptor-lysosomal enzyme complex is transported from the trans Golgi network (TGN) to an early endosomal compartment (step 3) and to an acidified late endosomal compartment where the low pH of the compartment causes dissociation of the complex. Lysosomal enzymes that are not phosphorylated (•), contain a phosphomonoester (•-P), or contain a diester with N-acetyl glucosamine are shown (•-P-). The dimeric CD-MPR is depicted as two pink balls and the CI-MPR is shown as 15 repeating balls. The three Man-6-P binding domains of the CI-MPR are depicted as pink balls.

Figure 2

Schematic representation of the CD-MPR (left) and CI-MPR (right). The MPRs are transmembrane glycoproteins. Various post-translational modifications are indicated, including palmitoylation and phosphorylation. The CD-MPR is shown as a dimer with each subunit having one Man-6-P binding site. The 15 repeating domains of the CI-MPR are numbered sequentially from the N-terminus to C-terminus. Domains 3, 5, and 9 bind Man-6-P with domain 5 preferentially binding to phosphodiesters. Domain 1 is known to bind to urokinase plasminogen activator receptor (uPAR) and plasminogen (Plg). Domain 11 binds IGF-II and Domain 13 has a fibronectin II insert.

Figure 3

A collage of structures of the CD-MPR and CI-MPR. A) Dimer of the CD-MPR. β-Strands are numbered from N- to C- terminus and loops between strands are labeled in alphabetic order. B) Structure of domains 1–3 of CI-MPR, with bound Man-6-P in domain 3. C) Structure of domains 11–14 of the CI-MPR. N- and C-termini are indicated. FNII denotes the fibronectin II insert in domain 13. D) Superposition of the structures of the CD-MPR (monomer, purple) and domains of 3 (green) and 11 (gold) of the CI-MPR, demonstrating that they all have a similar polypeptide fold. For clarity, the structures of domains 1, 2, 12, 13, and 14, whose structures have been determined to have the same fold, are not included in the overlay. The ligand binding site is indicated with an arrow.

Figure 4

Conservation of the Man-6-P binding site and essential residues for carbohydrate binding. A) Superposition of the Man-6-P binding sites of the CD-MPR (purple) and domain 3 of the CI-MPR (green). The architecture of both binding pockets is essentially the same with the exception of loop D (dark blue, CD-MPR; grey, domain 3), which is shorter in domain 3. B) A schematic drawing showing interactions between Man-6-P and residues in the CD-MPR and their homologous residues in domains 3, 5, and 9 of the CI-MPR. Dotted lines indicate potential hydrogen bonds. Mutational studies have shown that the four residues shown in purple are essential for Man-6-P binding and that mutation of the residues shown in blue partially decreased Man-6-P binding affinity. The two residues in grey have not been tested. C) Sequence alignment of domains 3, 5, 9 and 11 of the CI-MPR and the extracytosolic domain of the CD-MPR. The conserved cysteines are highlighted in yellow and the four residues that are essential for Man-6-P binding are highlighted in red. Residues that are within hydrogen bonding distance of Man-6-P in the crystal structures of CD-MPR and domains 1–3 of the CI-MPR, but found to be not essential for Man-6-P binding are boxed in red. Secondary structural elements are indicated with arrows and the single α-helix found in the CD-MPR is indicated with a green cylinder. Residues involved in IGF-II binding in domain 11 of the CI-MPR are boxed in blue.

Figure 5

Comparison of the ligand-bound and ligand-free structures of CD-MPR. A) Superposition of the monomer structures of the bound (red) and unbound (blue) structures. Note the difference in the conformations of loop D. B) The dimer structures, with the same color scheme as in (A). The molecule is scissoring along its molecular two-fold axis (z-axis) and twisting along the x-axis. The ligand, Man-6-P, is shown with ball-and-sticks. C) Superposition of the ligand binding sites of the bound (red) and unbound (blue) structures. Loop D in the bound structure forms the side of the binding pocket, while in the unbound structure loop D folds down and occupies the Man-6-P binding site. Mn⁺² ion in the bound state is denoted as a red ball. The movements of E133 and R135 are indicated (curved arrows). Inter-subunit interfaces are shown, including the N-terminus and loop D of the bound (panel D) and unbound (panel E) structures. Electrostatic interactions found between the two subunits in the bound structure are disrupted in the unbound structure, resulting in a weaker dimer interface.