Wang-Sik Lee

Murine UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase Lacking the γ-Subunit Retains Substantial Activity toward Acid Hydrolases

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on acid hydrolases. The transferase exists as anα2β2γ2 hexameric complex with the α- and β-subunits derived from a single precursor molecule. The catalytic function of the transferase is attributed to the α- and β-subunits, whereas the γ-subunit is believed to be involved in the recognition of a conformation-dependent protein determinant common to acid hydrolases. Using knock-out mice with mutations in either the α/β gene or the γ gene, we show that disruption of the α/β gene completely abolishes phosphorylation of high mannose oligosaccharides on acid hydrolases whereas knock-out of the γ gene results in only a partial loss of phosphorylation. These findings demonstrate that the α/β-subunits, in addition to their catalytic function, have some ability to recognize acid hydrolases as specific substrates. This process is enhanced by the γ-subunit.

Human mannose 6-phosphate-uncovering enzyme is synthesized as a proenzyme that is activated by the endoprotease furin.

N-Acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase, also known as “uncovering” enzyme (UCE), is localized in the trans-Golgi network, where it removes a covering N-acetylglucosamine from the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Here we show that UCE is synthesized as an inactive proenzyme that is activated by the endoprotease furin, which cleaves an RARLPR↓D sequence to release a 24-amino acid propiece. As furin is localized in the trans-Golgi network, newly synthesized UCE is inactive until it reaches this terminal Golgi compartment. LoVo cells (derived from a human colon adenocarcinoma) lack furin activity and have extremely low UCE activity. Addition of furin to LoVo cell extracts restores UCE activity to normal levels, demonstrating that the UCE proenzyme is stable in this cell type. LoVo cells secrete acid hydrolases with phosphomannose diesters as a consequence of the deficient UCE activity. This demonstrates for the first time that UCE is the only enzyme in these cells capable of efficiently uncovering phosphomannose diesters. UCE also hydrolyzes UDP-GlcNAc, a sugar donor for Golgi N-acetylglucosaminyltransferases. The fact that UCE is not activated until it reaches the trans-Golgi network may ensure that the pool of UDP-GlcNAc in the Golgi stack is not depleted, thereby maintaining proper oligosaccharide assembly.

Functions of the α, β, and γ Subunits of UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α2β2γ2 hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the γ subunit along with kinetic studies of recombinant α2β2γ2 and α2β2 forms of the transferase, we have explored the function of the α/β and γ subunits. The findings demonstrate that the α/β subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the α/β subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the γ subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the α/β subunits toward a subset of the acid hydrolases, the γ subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the γ subunit binds and presents the high mannose glycans of the acceptor to the α/β catalytic site in a favorable manner.

Comparative Pathology of Murine Mucolipidosis Types II and IIIC

UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an α2β2γ2 hexameric enzyme that catalyzes the first step in the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. In humans, mutations in the gene encoding the α/β subunit precursor give rise to mucolipidosis II (MLII), whereas mutations in the gene encoding the γ subunit cause the less severe mucolipidosis IIIC (MLIIIC). In this study we describe the phenotypic, histologic, and serum lysosomal enzyme abnormalities in knockout mice lacking the γ subunit and compare these findings to those of mice lacking the α/β subunits and humans with MLII and MLIIIC. We found that both lines of mutant mice had elevated levels of serum lysosomal enzymes and cytoplasmic alterations in secretory cells of several exocrine glands; however, lesions in γ-subunit deficient (Gnptab -/-) mice were milder and more restricted in distribution than in α/β-subunit deficient (Gnptab -/-) mice. We found that onset, extent, and severity of lesions that developed in these two different knockouts correlated with measured lysosomal enzyme activity; with a more rapid, widespread, and severe storage disease phenotype developing in Gnptab -/- mice. In contrast to mice deficient in the α/β subunits, the mice lacking the γ subunits were of normal size, lacked cartilage defects, and did not develop retinal degeneration. The milder disease in the γ-subunit deficient mice correlated with residual synthesis of the mannose 6-phosphate recognition marker. Of significance, neither strain of mutant mice developed cytoplasmic vacuolar inclusions in fibrocytes or mesenchymal cells (I-cells), the characteristic lesion associated with the prominent skeletal and connective tissue abnormalities in humans with MLII and MLIII. Instead, the predominant lesions in both lines of mice were found in the secretory epithelial cells of several exocrine glands, including the pancreas, and the parotid, submandibular salivary, nasal, lacrimal, bulbourethral, and gastric glands. The absence of retinal and chondrocyte lesions in Gnptab -/- mice might be attributed to residual β-glucuronidase activity. We conclude that mice lacking either α/β or γ subunits displayed clinical and pathologic features that differed substantially from those reported in humans having mutations in orthologous genes.

Analysis of mannose 6-phosphate uncovering enzyme mutations associated with persistent stuttering

Background: Three mutations in the NAGPA gene encoding uncovering enzyme (UCE) have been found in some individuals with persistent stuttering. Results: All three mutations lead to lower UCE activity. Conclusion: NAGPA mutations present in individuals with persistent stuttering have negative effects on the enzyme. Significance: These findings extend the genetic data implicating NAGPA mutations in the persistent stuttering phenotype. GlcNAc-1-phosphodiester-N-acetylglucosaminidase (“uncovering enzyme” (UCE); EC 3.1.4.45) is a Golgi enzyme that mediates the second step in the synthesis of the mannose 6-phosphate lysosomal targeting signal on acid hydrolases. Recently, three mutations (two missense and one deletion/frameshift) in the NAGPA gene that encodes UCE have been identified in individuals with persistent stuttering. We now demonstrate that each mutation leads to lower cellular UCE activity. The p.R328C mutation impairs folding in the endoplasmic reticulum, resulting in degradation of a significant portion by the proteasomal system. The p.H84Q mutation also impairs folding and, in addition, decreases the specific activity of the enzyme that folds sufficiently to traffic to the Golgi. The p.F513SfsX113 frameshift mutation adds 113 amino acids to the C terminus of the cytoplasmic tail of the protein, including a VWLL sequence that causes rapid degradation via the proteasomal system. These biochemical findings extend the genetic data implicating mutations in the NAGPA gene in the persistent stuttering phenotype.

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

The Golgi enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2 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 non-lysosomal 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 α 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 γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ 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.

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

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

The 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.