Mutational Analysis of the Binding Site Residues of the Bovine Cation-dependent Mannose 6-Phosphate Receptor

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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 (

Section: Resultssupporting

confidence: 79%

Section: Resultsmentioning

confidence: 99%

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

Olson

Hindsgaul

Dahms

et al. 2008

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“…Four conserved residues (Gln-66, Arg-111, Glu-133, and Tyr-143) of the CD-MPR have been shown previously to be essential for Man-6-P recognition (31). These residues are also conserved in the two high affinity binding sites (i.e.…”

Section: Discussionmentioning

confidence: 93%

“…Interestingly, this study also revealed that domain 5 exhibits a comparable level of sequence identity to the CD-MPR as observed for domains 3 and 9. Our crystal structures of the CD-MPR (13,27) and domains 1-3 of the CI-MPR (28,29) plus mutagenesis studies (21,30,31) have identified conserved residues that are essential for Man-6-P binding. To evaluate the possibility that domain 5 binds carbohydrate, we performed a structure-based sequence alignment to compare domain 5 with the CD-MPR and domains 3 and 9 of the CI-MPR (see Fig.…”

mentioning

confidence: 99%

Identification of a Low Affinity Mannose 6-Phosphate-binding Site in Domain 5 of the Cation-independent Mannose 6-Phosphate Receptor

Reddy

Chai

Childs

et al. 2004

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The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity (ϳ1 nM) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was ϳ300-fold lower (K i ‫؍‬ 5.3 mM) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme ␤-glucuronidase was also much lower (K d ‫؍‬ 54 M) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.

“…results in only partial inhibition of carbohydrate binding (16). In the bound state, these seven residues are all within hydrogenbonding distance of the terminal sugar moiety (Fig.…”

Section: Resultsmentioning

confidence: 99%

Twists and Turns of the Cation-dependent Mannose 6-Phosphate Receptor

Olson

Zhang

Dahms

et al. 2002

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Mannose 6-phosphate receptors (MPRs) participate in the biogenesis of lysosomes in higher eukaryotes by transporting soluble acid hydrolases from the transGolgi network to late endosomal compartments. The receptors release their ligands into the acidic environment of the late endosome and then return to the transGolgi network to repeat the process. However, the mechanism that facilitates ligand binding and dissociation upon changes in pH is not known. We report the crystal structure of the extracytoplasmic domain of the homodimeric cation-dependent MPR in a ligand-free form at pH 6.5. A comparison of the ligand-bound and ligand-free structures reveals a significant change in quaternary structure as well as a reorganization of the binding pocket, with the most prominent change being the relocation of a loop (residues Glu 134 -Cys 141 ). The movements involved in the bound-to-free transition of the cation-dependent MPR are reminiscent of those of the oxy-to-deoxy hemoglobin transition. These results allow us to propose a mechanism by which the receptor regulates its ligand binding upon changes in pH; the pK a of Glu 133 appears to be responsible for ligand release in the acidic environment of the late endosomal compartment, and the pK a values of the sugar phosphate and His 105 are accountable for its inability to bind ligand at the cell surface where the pH is about 7.4. P-type lectins function as an essential part of the degradative pathway of higher eukaryotic organisms. A well characterized function of this receptor family is its ability to target newly synthesized acid hydrolases to the lysosome. During their transport through the Golgi complex, acid hydrolases acquire a mannose 6-phosphate (Man-6-P) 1 recognition marker on their N-linked oligosaccharides that serves as a high affinity ligand for the receptors. The resulting receptor-acid hydrolase complex is transported to late endosomal compartments. Upon encountering the lower pH of this prelysosomal compartment, the complex dissociates. The hydrolytic enzymes are then packaged into lysosomes, and the uncomplexed receptors move back to the Golgi where the process is repeated numerous times for a single receptor (1-3).The 46-kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) and the 300-kDa insulin-like growth factor II/cation-independent mannose 6-phosphate receptor (IGFII/CI-MPR) are the two known members of the P-type lectin family, both of which are type I membrane glycoproteins. Carbohydrate-binding proteins have historically been divided into two subgroups. The group I carbohydrate-binding proteins, which include periplasmic transport proteins and some enzymes, typically envelop their ligands in deep pockets. In contrast, group II carbohydrate-binding proteins, which include lectins, typically bind their ligands in shallow clefts on the protein surface (4). Our recent crystallographic studies of the CD-MPR in complex with ligands containing phosphomannosyl residues reveal that this P-type lectin is unusual with respect to the architecture of i...