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

Abstract

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...

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

“…Proteolytic cleavage of this precursor at K928 to give rise to the α and β subunits is mediated by the Site-1 protease in the Golgi, and this process is essential for catalytic competency of the protein 8, 9. The α subunit contains two Notch modules and a DNA methyltransferase-associated protein (DMAP) interaction domain that mediate the specific recognition of protein determinants on the lysosomal enzyme substrates (Figure 1A) 12 . In addition, there are four so-called spacer domains in the α/β precursor whose functions are beginning to be elucidated.…”

Enzyme-specific differences in mannose phosphorylation between GlcNAc-1-phosphotransferase αβ and γ subunit deficient zebrafish support cathepsin proteases as early mediators of mucolipidosis pathology

“…Since many MRH domains act as lectins that bind both M6P modified and non-modified high mannose oligosaccharides, its presence in the γ subunit suggests that it might facilitate subunit ability to bind the N-glycans of hydrolases, thereby modulating the αβ’s recognition capability (17). It was also suggested that the MRH domain binds glycans on the α subunit, but a recent study demonstrated that interaction between the αβ and γ subunits is MRH-independent (18). …”

Mucolipidosis III GNPTG Missense Mutations Cause Misfolding of the γ Subunit of GlcNAc-1-Phosphotransferase

“…As a consequence, the lysosomes accumulate undegraded material. The α and β subunits of GlcNAc-1-phosphotransferase, encoded by the gene GNPTAB, harbor the catalytic site as well as domains that mediate the recognition of the acid hydrolases (DMAP and Notch repeats) (Kudo, et al, 2005; Qian, et al, 2013; Qian, et al, 2010; Qian, et al, 2015; van Meel, et al, 2016). Depending on the residual level of GlcNAc-1-phosphotransferase activity, mutations in GNPTAB either cause the very severe lysosomal storage disorder mucolipidosis (ML) II (MIM# 252500), or result in MLIII αβ (MIM# 252600), which has an attenuated phenotype (Kudo, et al, 2006).…”

“…GNPTG -/- HeLa cells have greatly reduced levels of many lysosomal acid hydrolases compared to the parental HeLa cells and display a lysosomal storage phenotype (van Meel, et al, 2016). In these cells, transient transfection with WT γ resulted in a marked increase in the intracellular activities of a panel of lysosomal glycosidases 3 days after transfection, with the maximum increase set to 100% rescue.…”

“…The γ subunit contains two distinct domains, a Man-6-P receptor homology (MRH) domain, which binds high mannose-type N -glycans on the lysosomal enzymes to enable their phosphorylation by the αβ subunits, and a DMAP interaction domain (van Meel, et al, 2016). The amino acid residues G106, G126 and C142 are all located in the MRH domain.…”