Structures of the mannose-6-phosphate pathway enzyme, GlcNAc-1-phosphotransferase

Abstract

The mannose-6-phosphate (M6P) pathway is responsible for the transport of hydrolytic enzymes to lysosomes. N-acetylglucosamine-1-phosphotransferase (GNPT) catalyzes the first step of tagging these hydrolases with M6P, which when recognized by receptors in the Golgi diverts them to lysosomes. Genetic defects in the GNPT subunits, GNPTAB and GNPTG, cause the lysosomal storage diseases mucolipidosis types II and III. To better understand its function, we determined partial three-dimensional structures of the GNPT complex. The catalytic domain contains a deep cavity for binding of uridine diphosphate-N-acetylglucosamine, and the surrounding residues point to a one-step transfer mechanism. An isolated structure of the gamma subunit of GNPT reveals that it can bind to mannose-containing glycans in different configurations, suggesting that it may play a role in directing glycans into the active site. These findings may facilitate the development of therapies for lysosomal storage diseases.

Keywords: GlcNAc-1-phosphotransferase; lysosome; mannose-6-phosphate.

Fig. 1.

Structure of the GNPTAB catalytic domain. (A) Domain organization of vertebrate GNPTAB. TM, transmembrane helix; cat, catalytic domain segment; RRM, RNA recognition motif-like domain; N, Notch extracellular repeats-like domain; IG, immunoglobulin domain; DMAP1, DMAP1 binding-like domain; EF-hand, calcium-binding domain. The site-1 protease (S1P) cleavage site between the α and β subunits is marked by a red triangle. (B) Crystal structure of the zebrafish minimal construct dimer, comprising the catalytic (green, light gray) and EF-hand (orange, dark gray) domains. Calcium ions are represented by yellow spheres and the active sites by pink spheres. The N and C termini are labeled. Disulfide bonds are shown as thick yellow sticks. N-linked glycans (white sticks) are simplified for clarity. The deletions in the minimal construct are labeled (human residue numbers) and marked by green spheres. (C) Phosphotransfer reaction catalyzed by GNPTAB. MAN, mannose; GlcNAc, N-acetylglucosamine.

Fig. 2.

Structure of the GNPTAB catalytic domain with UDP-GlcNAc. (A) GNPT catalytic domain (light green) bound to UDP-GlcNAc (sticks). The two residues in dark green represent the junction point between segments 2 and 3 of the catalytic domain; the full-length protein contains a large insertion there that was removed in the minimal construct. (B) Polar and charged contacts (dashed lines) between the protein, UDP-GlcNAc (black sticks), magnesium ions (yellow spheres), and water molecules (red spheres). Distances are below 3.7 Å. Due to the complexity of the zebrafish minimal construct residue numbering and its high sequence identity to the human homolog (87%), human residue numbers are shown throughout this figure. Side chains that were functionally investigated are in bold. (C) In vitro enzymatic activity of purified hamster soluble GNPTAB–GNPTG active complex (furin-cleaved, referred to as WT) on the small molecule substrate α-methyl d-mannoside (100 mM) in presence of UDP-GlcNAc (1 mM). The precursor is not furin-cleaved. Values are the means and SDs of a representative of two experiments, each performed in triplicates. The 100% activity corresponds to 0.35/s. Sequence conservation in the 1,000 closest homologs of the GNPT catalytic domain (Stealth family) from unicellular organisms is indicated. (D) Residues lining the opening of the active site cavity and potentially interacting with the glycan substrate. (E) Proposed catalytic mechanism. (F) In vitro activity of GNPT as above on the purified lysosomal hydrolase acid ceramidase (ASAH1), a substrate of GNPT (2, 21). This protein migrates as a smeared double band on the gel due to glycan heterogeneity from recombinant expression. Tagging of its glycans by GNPT shifts the bands to a higher molecular weight.

Fig. 3.

Structure of GNPTG and its complex with glycans. (A) Domain organization of GNPTG. SP, signal peptide; MRH, mannose 6-phosphate receptor homology domain; DMAP1, DMAP1 binding-like domain. The disulfide bond between two GNPTG subunits is represented by black dots. (B) The two subunits of the crystallized GNPTG dimer are superimposed, illustrating the different orientations of their bound glycans. A loop that adopts a different angle in chain B is labeled. (C) Crystal structure of the clawed frog GNPTG dimer, comprising the MRH (yellow, light gray) and DMAP1 (pink, dark gray) domains. The MRH domain carbohydrate-binding site is occupied by N-linked glycans (green sticks) from adjacent proteins in the crystal lattice (chains B′ and A′), with the glycosylated Asn residues represented by green spheres. The corresponding glycans linked to Asn residues of the depicted MRH domains (chains A and B) are shortened as white sticks for clarity. (D) Hydrogen bonds (dashed lines) between mannose residues (green sticks) and side chains of clawed frog GNPTG in chain A (Left) and B (Right). Distances are below 3.6 Å, except for the contact with Arg97 (3.8 Å). Human residue numbers are shown throughout this figure. The types of glycosidic bonds within the glycans are indicated. (E) Superimposition of loops 1 and 2 of GNPTG, cation-dependent mannose-6-phosphate receptor (CDMPR, PDB ID 1C39) (51, 52), cation-independent mannose-6-phosphate receptor (CIMPR) domains 3 (PDB ID 1SYO) (53, 54), 5 (PDB ID 6P8I) (55, 56) and 9 (PDB ID 6Z30) (57), glucosidase 2 β subunit (PDB ID 4XQM) (58, 59), and lectin OS-9 (PDB ID 3AIH) (60). Two residues from GNPTG and one from glucosidase 2 β are labeled. (F) DMAP1 domain of GNPTG, with residues forming the hydrophobic core as white sticks and acidic side chains as red spheres.