Analysis of Mucolipidosis II/III GNPTAB Missense Mutations Identifies Domains of UDP-GlcNAc:lysosomal Enzyme GlcNAc-1-phosphotransferase Involved in Catalytic Function and Lysosomal Enzyme Recognition

Qian, Yi; Meel, Eline van; Flanagan-Steet, Heather; Yox, Alex; Steet, Richard; Kornfeld, Stuart

doi:10.1074/jbc.m114.612507

Cited by 42 publications

(68 citation statements)

References 34 publications

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“…The full cDNA sequences were confirmed by DNA sequencing. Human GNPTAB-V5/His in pcDNA6 has been described (10). The various ␣/␤ deletion constructs were made by a two-step overlap-extension PCR process wherein the native cDNA restriction fragment encoding the Notch 1-DMAP (amino acids 438 -819) sequence was swapped with a similar PCR-generated restriction fragment encoding the specific deletion.…”

Section: Methodsmentioning

confidence: 99%

“…Finally, the beads were resuspended in lysis buffer containing 10 mM Man-6-P to elute the bound proteins. Lysosomal enzyme assays were performed on the eluates as described previously (10). The enzyme activity was normalized to the total protein concentration of the different cell extracts.…”

Section: Methodsmentioning

confidence: 99%

“…Support for this hypothesis has come from studies of the consequences of missense mutations in Notch 1 and the DMAP interaction domain found in patients with the autosomal recessive lysosomal storage disorders mucolipidosis II and III (10,13). These mutations were shown to impair lysosomal enzyme phosphorylation without altering the catalytic activity of the transferase toward the simple sugar ␣-methyl D-mannoside (␣MM).…”

mentioning

confidence: 95%

“…The ␣/␤ subunits contain three identifiable domains as follows: the Stealth domain, two Notch repeat modules, and a DNA methyltransferase-associated protein (DMAP) interaction domain. The Stealth domain resembles sequences within bacterial genes that encode sugar-phosphate transferases involved in cell wall polysaccharide synthesis and has been shown to mediate the catalytic function of GlcNAc-1-phosphotransferase (8,10,11). Importantly, the bacterial genes lack the Notch repeats and the DMAP interaction domain, which has been proposed to function as a protein-protein interaction domain (12).…”

mentioning

confidence: 99%

“…In looking for clues, we examined the domain structures of the ␣, ␤, and ␥ subunits of this transferase (10). The ␣/␤ subunits contain three identifiable domains as follows: the Stealth domain, two Notch repeat modules, and a DNA methyltransferase-associated protein (DMAP) interaction domain.…”

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confidence: 99%

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

Meel¹,

Lee²,

Liu³

et al. 2016

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 95%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Meel¹,

Lee²,

Liu³

et al. 2016

Journal of Biological Chemistry

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Identification of the interaction domains between α‐ and γ‐subunits of GlcNAc‐1‐phosphotransferase

et al. 2016

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The disease-associated hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α β γ ) catalyzes the formation of mannose 6-phosphate residues on lysosomal enzymes required for efficient targeting to lysosomes. Using pull-down experiments and mutant subunits, we identified a potential loop-like region in the α-subunits comprising residues 535-588 and 645-698 involved in the binding to γ-subunits. The interaction is independent of the mannose 6-phosphate receptor homology domain but requires the N-terminal unstructured part of the γ-subunit consisting of residues 26-69. These studies provide new insights into structural requirements for the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.

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Role of spacer‐1 in the maturation and function of GlcNAc‐1‐phosphotransferase

et al. 2017

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UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-PT) is an α2β2γ2 hexamer that mediates the initial step in the formation of the mannose 6-phosphate targeting signal on newly synthesized lysosomal acid hydrolases. The GNPTAB gene encodes the 1256 amino acid long α/β precursor which is normally cleaved at K928 in the early Golgi by Site-1 protease (S1P). Here, we show that removal of the so-called `spacer-1´ domain (residues 86–322) results in cleavage almost exclusively at a second S1P consensus sequence located upstream of K928. In addition, GlcNAc-1-PT lacking spacer-1 exhibits enhanced phosphorylation of several non-lysosomal glycoproteins, while the phosphorylation of lysosomal acid hydrolases is not altered. In view of these effects on the maturation and function of GlcNAc-1-PT, we suggest renaming `spacer-1´ the `regulatory-1´ domain.

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Analysis of Mucolipidosis II/III GNPTAB Missense Mutations Identifies Domains of UDP-GlcNAc:lysosomal Enzyme GlcNAc-1-phosphotransferase Involved in Catalytic Function and Lysosomal Enzyme Recognition

Cited by 42 publications

References 34 publications

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

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

Identification of the interaction domains between α‐ and γ‐subunits of GlcNAc‐1‐phosphotransferase

Role of spacer‐1 in the maturation and function of GlcNAc‐1‐phosphotransferase

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