Structural basis for recognition of phosphodiester-containing lysosomal enzymes by the cation-independent mannose 6-phosphate receptor

Abstract

Mannose 6-phosphate (Man-6-P)-dependent trafficking is vital for normal development. The biogenesis of lysosomes, a major cellular site of protein, carbohydrate, and lipid catabolism, depends on the 300-kDa cation-independent Man-6-P receptor (CI-MPR) that transports newly synthesized acid hydrolases from the Golgi. The CI-MPR recognizes lysosomal enzymes bearing the Man-6-P modification, which arises by the addition of GlcNAc-1-phosphate to mannose residues and subsequent removal of GlcNAc by the uncovering enzyme (UCE). The CI-MPR also recognizes lysosomal enzymes that elude UCE maturation and instead display the Man-P-GlcNAc phosphodiester. This ability of the CI-MPR to target phosphodiester-containing enzymes ensures lysosomal delivery when UCE activity is deficient. The extracellular region of the CI-MPR is comprised of 15 repetitive domains and contains three distinct Man-6-P binding sites located in domains 3, 5, and 9, with only domain 5 exhibiting a marked preference for phosphodiester-containing lysosomal enzymes. To determine how the CI-MPR recognizes phosphodiesters, the structure of domain 5 was determined by NMR spectroscopy. Although domain 5 contains only three of the four disulfide bonds found in the other seven domains whose structures have been determined to date, it adopts the same fold consisting of a flattened β-barrel. Structure determination of domain 5 bound to N-acetylglucosaminyl 6-phosphomethylmannoside, along with mutagenesis studies, revealed the residues involved in diester recognition, including Y679. These results show the mechanism by which the CI-MPR recognizes Man-P-GlcNAc-containing ligands and provides new avenues to investigate the role of phosphodiester-containing lysosomal enzymes in the biogenesis of lysosomes.carbohydrate recognition | lectin | lysosome biogenesis | protein targeting L ysosomes are acidified organelles that serve as the terminal degradative compartment of the phagocytic, autophagocytic, and endocytic pathways. The diverse degradative capacity of this organelle depends on a repertoire of over 60 different soluble acid hydrolases (e.g., proteases, glycosidases, nucleases, lipases, and phosphatases) and involves the continual delivery of these newly synthesized hydrolytic enzymes to the lysosome (reviewed in ref.

Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

“…Trp-409 of GII␤ is boxed in green. presence of a carbohydrate ligand (35,37). These structures allowed us to identify four conserved residues (Gln, Arg, Glu, and Tyr), which interact with the 2-, 3-, and 4-hydroxyl groups of the mannose ring, that are essential for Man-6-P recognition by the MRH domains of MPRs.…”

“…Determination of Binding Affinities by Heteronuclear NMR Spectroscopy-NMR studies in which 15 N-labeled GII␤ MRH domain was titrated with increasing concentrations of ligand (Man-6-P (Sigma), Man␣1,2Man (Dextra Laboratories), or Man 9 GlcNAc purified from soybean agglutinin were carried out as described previously (35).…”

Molecular and Structural Basis for Sugar Recognition by Mannose 6-Phosphate Receptor Homology Domain-Containing Lectins and Proteins

“…Unlike ERAD lectins, the MRH domain has higher affinity for α1,2-linked mannobiose structure on the D3 arm (39). The MRH domain of Glc-II-β does not possess a WW motif, but instead has a tyrosine residue conserved among MPRs (49)(50)(51)54,55). Like Glc-II-β, GlcNAcPT also has the corresponding tyrosine residue in the MRH domain and shows a similar substrate specificity for α1,2-linked mannosyl ligands (35).…”

“…To date, many three-dimensional structures of MRH domains of MPRs have been determined (49)(50)(51)(52)(53)(54)(55); all showed structurally similar MRH domains with a P-type lectin fold (Fig. 5).…”

“…The CD-MPR uses three residues (Asp, Asn, and His) to coordinate the phosphate group of M6P (49,50,54), whereas domain 3 of the CI-MPR uses serine residue with an ordered water molecule (51). In CI-MPR domain 5, a characteristic tyrosine residue is responsible for GlcNAc-P-Man phosphodiester recognition (55). Although CD-MPR also has a corresponding tyrosine residue, it is unable to bind phosphodiesters possibly due to the architecture of its binding pocket.…”