Identification of Residues Essential for Carbohydrate Recognition by the Insulin-like Growth Factor II/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: Maintenance Of the Scd-mpr Binding Pocket In The Absence Of mentioning

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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“…9A), as equivalent mutations in the MRH domain of the CI-MPR had been shown to abolish mannose binding without altering folding (15,25). Preliminary experiments showed that the ␥ mutant was well expressed, interacted with the ␣/␤ subunits in the Ni-NTA pulldown assay, and localized to the Golgi in a process that required coexpression of ␣/␤ (Fig.…”

Section: Rescue Of Acid Hydrolase Phosphorylation In Gnptg ϫ/ϫmentioning

confidence: 99%

Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes

Meel¹,

Lee²,

Liu³

et al. 2016

The Golgi enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an ␣ 2 ␤ 2 ␥ 2 hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous nonlysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the ␣ subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the ␥-binding site on the ␣ subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the ␥ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif.Correct targeting of newly synthesized acid hydrolases to lysosomes is essential for this organelle to maintain its function of degrading intracellular and endocytosed material. In higher eukaryotes, this process is mediated by the mannose 6-phosphate (Man-6-P) 5 recognition system whereby the newly synthesized acid hydrolases acquire Man-6-P residues in the Golgi that serve as high affinity ligands for binding to Man-6-P receptors (MPRs) in the trans-Golgi network and subsequent transport to the endo-lysosomal system (1). The initial and most critical step in the generation of the Man-6-P tag is mediated by the Golgi enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase). This enzyme binds selectively to conformation-dependent protein determinants in the 60 or so lysosomal acid hydrolases and transfers GlcNAc-1-P from UDP-GlcNAc to mannose residues on high mannose-type N-linked glycans of the hydrolases (2). The GlcNAc is subsequently excised by a second Golgi enzyme ("uncovering enzyme") to generate the high affinity Man-6-P ligand (3).GlcNAc-1-phosphotransferase is an ␣ 2 ␤ 2 ␥ 2 hexamer that is encoded by two genes (4 -7). The GNPTAB gene encodes the ␣ and ␤ subunits, whereas the GNPTG gene encodes the ␥ subunit. Enzyme kinetic studies have indicated that the ␣ and ␤ subunits specifically bind lysosomal acid hydrolases and mediate the catalytic function of the enzyme (8, 9). The ␥ subunit enhances the rate of GlcNAc-P transfer to a subset of the acid hydrolases without substantially altering the binding to these acceptors. Consistent with this, analysis of the level of mannose phosphorylation of the acid hydrolases in the brain of mice lacking the ␥ subunit, as estimated by the extent of binding to a cation-independent (CI)-MPR affinity resin, indicated that about one-third of the acid hydrol...

“…These residues are also conserved in the two high affinity binding sites (i.e. domains 3 and 9) of all CI-MPRs sequenced to date, and substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 of the CD-MPR (31) or their corresponding residues in domains 3 and 9 of the CI-MPR (21) results in a decrease in the affinity of the receptor for a lysosomal enzyme by Ͼ1000-fold. The crystal structures of the CD-MPR (13,27) and domains 1-3 of the CI-MPR (28,29) confirm the importance of these residues by demonstrating that their location is within hydrogen bonding distance of the hydroxyl groups of the mannose ring.…”

Section: Discussionmentioning

confidence: 99%

“…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%

“…Sequence analysis of each of the 15 domains of the CI-MPR demonstrated significant amino acid identity to each other (16 -38%) and to the ϳ150-residue extracytoplasmic region of the CD-MPR (14 -28%) (15). In contrast to the CD-MPR, which binds one molecule of Man-6-P/polypeptide (16), the CI-MPR contains two high affinity (nanomolar) Man-6-P-binding sites, which have been mapped to domains 1-3 and 9 (17)(18)(19), with essential residues localized to domains 3 and 9 (20,21). The CI-MPR, unlike the CD-MPR, also interacts with a number of non-Man-6-P-containing molecules that include insulin-like growth fac-tor II, plasminogen, the urokinase-type plasminogen activator receptor, and retinoic acid.…”

mentioning

confidence: 99%

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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.