Structural insights into GlcNAc-1-phosphotransferase that directs lysosomal protein transport

GlcNAc-1-phosphotransferase (GNPT) catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GNPT cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GNPT remains unclear. Mammalian GNPTs are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from GNPTAB. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog (DmGNPTAB). Four conserved regions located far apart in the sequence fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase (UGGT). Comparison with UGGT revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. DmGNPTAB forms an evolutionarily conserved homodimer, and perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GNPT function and related diseases.


Introduction 1
Protein phosphorylation is universally present as a regulatory strategy in eukaryotic cells. 2 Glycan phosphorylation, though not as abundant, also plays essential roles in modulating 3 cellular processes, particularly within the secretory compartments. For example, two novel 4 secretory pathway glycan kinases have been recently characterized: the proteoglycan xylose  A canonical glycan phosphorylation event involves ~60 secretory proteins that are 10 destined for lysosomes. Similar to other secretory molecules, these lysosomal proteins are first 11 synthesized in the endoplasmic reticulum and then traverse through the Golgi network. At the 12 Golgi apparatus, these proteins are "phosphorylated" on a terminal mannose residue in their 13 N-linked glycans, resulting in the formation of a mannose 6-phosphate (M6P) tag that is 14 recognized by two specific M6P receptors to direct their lysosomal transport. Interestingly, 15 this essential modification is not performed by an ATP-dependent kinase but is generated by 16 the sequential action of two enzymes: first, the N-acetylglucosamine-1-phosphotransferase 17 (GNPT) catalyzes the addition of an N-acetylglucosamine-1-phosphate (GlcNAc-1-P) group 18 to the terminal mannose, and the GlcNAc-1-phosphodiester α-N-acetylglucosaminidase 19 (NAGPA) then removes the GlcNAc moiety to uncover M6P (Varki and Kornfeld, 2015). 20 Mammalian GNPTs are large protein complexes that comprise two α-subunits, two β- and assessed their potential impacts. Together, our results advance the understanding of GNPT 1 and related human diseases. Cryo-EM structure of Drosophila melanogaster GNPT 5 We sought to investigate the structural basis of GNPT function. Despite intensive 6 attempts, we were unable to obtain the structure of the GNPT complex and could not 7 determine the structure of the α/β subcomplex or the GNPTAB precursor. Drosophila 8 melanogaster has a GNPTAB homolog (DmGNPTAB) but lacks a discernable gene encoding four CRs and a similar CR3-CR4 spacer but has shorter CR1-CR2 and CR2-CR3 spacers, only 12 one Notch repeat, and lacks the DMAP interaction domain and S1P cleavage site ( Figure 1A, 13 Figure S1). We obtained the luminal portion of DmGNPTAB and analyzed its structure by 14 cryo-EM ( Figure 1B, Figure S2). The structure was determined at an overall resolution of 3.5 15 Å (Table S1). The center region displayed high resolutions, which allowed us to build the 16 structural model de novo. Approximately half of the protein molecule, including all four CRs 17 and the CR3-CR4 spacer, could be confidently placed. The amino and carboxyl termini are 18 located on the same side of a monomer, which sheds light on the topology of the full-length 19 protein on the Golgi membrane ( Figure 1C). The rest of the molecule, particularly the CR2-20 CR3 spacer including the Notch repeat, displayed weak densities and was thus not modeled. parallel manner to form a β-sheet, whereas the two helices are situated on each side of the 1 sheet. CR3 consists of one strand and three helices. The single CR3 strand runs antiparallel to 2 the four strands described above, and the three helices bundle together with the short helix in 3 CR2. CR4 features a single helix, which packs against the long helix in CR2. The small 4 domain is encoded by the CR3-CR4 spacer, including the calcium-binding EF hand motif, and 5 features a helix bundle to mediate dimerization. 6 The four Stealth CRs, which are located far apart in the sequence, fold into a single  DmGNPTAB has a similar surface pocket, which is formed by a group of conserved residues, 24 including Thr69 from CR1; Ser156, Ile159, Glu160, Tyr175, Asn177, Asp178, and Asp179 TdUGGT, whereas Cys572 appears to take the position of TdUGGT-Asp1427. It is thus likely 10 that these residues are also involved in the binding to a divalent cation that assists in 11 positioning the UDP-GlcNAc in DmGNPTAB.

13
Human GNPTAB mutants are functionally defective 14 We sought to validate the functional importance of some of these residues in human  Figure 2D). In contrast, N406A, D408A, and C1149A displayed reduced 2 activities compared with the WT protein, and D408A appeared to be completely inactive.

3
These results demonstrate the importance of these residues for GNPT function, corroborating 4 our structural analyses.

6
A conserved dimeric architecture 7 Mammalian GNPTs are α2β2γ2 hexameric complexes, and the construction of the 8 hexamer remains poorly understood. In our structure, two DmGNPTAB molecules form a 9 homodimer that resembles two fishes nestling against each other in a head-to-tail orientation 10 ( Figure 1B). The dimer is mainly mediated by the CR3-CR4 spacer and CR4, whose 11 counterparts both reside in the β-subunit of human GNPT ( Figure 1A). A few residues in CR2 12 also contribute to dimer formation. The dimer interface buries ~1,500-Å 2 solvent-accessible 13 surfaces from each molecule and involves a number of invariant residues ( Figure S1), which 14 suggests that the dimerization mechanism observed in this study is likely generally conserved 15 in the GNPTAB family. 16 To verify the functional relevance of the dimer, we generated two human GNPTAB of GNPTAB dimers in the cells. In contrast, the interactions between G1 or G2 and WT 26 GNPTAB were markedly reduced, which suggested that these two mutants exhibit decreased 27 abilities to dimerize with the WT protein. Furthermore, unlike WT GNPTAB, these two 1 mutants were not efficiently processed because the ~48-kDa band that corresponds to the β-2 subunit was not observed after their expression ( Figure 3B). Importantly, these mutations 3 could also not fully rescue the maturation of CatB in GNPTAB -/cells ( Figure 2D). Together, 4 these results demonstrated that proper dimer formation is important for the processing of 5 GNPTAB and for the activity of the GNPT holoenzyme. . We also showed that D408A was unable to restore the maturation of residues. Arg105 forms a bidentate interaction with Glu607 to support Pro601, which is 1 involved in dimerization. Ser170 likely forms hydrogen bond interactions with Asp65 and 2 Arg115, and the corresponding human residues, Asp76 and Arg344, are also mutated in some 3 patients ( Figure 4A). Similarly, some of the other mutations also lead to structural disturbance 4 of human GNPT, as can be rationalized by the DmGNPTAB structure.

5
Human GNPT is an α2β2γ2 hexamer. The DmGNPTAB structure reveals a dimeric 6 framework that sheds light on the assembly mechanism of human GNPT. The CR3-CR4 7 spacer and CR4 play dominant roles in mediating the formation of the DmGNPTAB 8 homodimer, which likely reflects how the β2 dimer is formed in GNPT because most of the 9 residues involved in the dimer interface are conserved ( Figure S1) and because mutations of 10 two of these residues reduced human GNPTAB dimerization ( Figure 3B). In addition to the 11 interactions between β-subunits, Cys70 in human GNPTAB is involved in disulfide-linked  vertebrates to ensure the proper transport of lysosomal proteins. 6 In summary, we have elucidated the structure of DmGNPT, and our findings offer a more    Table S1.            The asterisk indicates the S1P cleavage site in human GNPTAB.   panel. Human GNPTAB residues N406, D408, and C1149 that correspond to N177, D179, 6 and C572 in DmGNPTAB are highlighted red.            G. Cryo-EM density maps around select DmGNPTAB residues described in the manuscript.  C. Asn177, Asp179, and Cys572 in DmGNPTAB appear to align with Asp1294, Asp1296, 7 and Asp1427 in TdUGGT, which coordinate a Ca 2+ ion to position UDP-Glc.