Degradation of the extracellular matrix proteoglycans begins in the cytosol, where they are proteolyzed to GAGs such as HS, chondroitin sulfate, dermatan sulfate, and keratan sulfate. The proteolyzed GAGs are further degraded to monosaccharides and free sulfate residues within the lysosomes in GAG-specific multi-enzyme degradation pathways. Dysfunction, often inherited, of the enzymes involved in GAG degradation causes abnormal accumulation of the partial degradation products within the lysosomes, resulting in mucopolysaccharidoses (MPSs), a collective term for the group of rare autosomal recessive lysosomal storage disorders characterized by the accumulation of partially degraded GAGs (Coutinho et al., 2012; Huizing and Gahl, 2020; Klein et al., 1978; Platt et al., 2018; Ruivo et al., 2009). HS is a GAG made of repeating units of N-acetylglucosamine and glucuronic acid, and impairment of enzymes in the HS degradation pathway causes mucopolysaccharidosis III (MPS III), or Sanfilippo’s syndrome that is characterized by abnormal storage of HS degradation pathway intermediates within the lysosomes in all organs and excretion of these intermediates in the urine. The onset of the symptoms of MPS III, which include organomegaly, abnormal joint mobility, compromised cardiac function, degeneration of vision and hearing, and dementia, often begins in juveniles and ends in premature death. Currently, no therapies for MPS III are available (Coutinho et al., 2012; Huizing and Gahl, 2020; McBride and Flanigan, 2021; Pshezhetsky et al., 2018).

Depending on the dysfunctional enzyme, MPS III is classified into four subtypes – MPS IIIA-IIID. Of these four MPS III subtypes, MPS IIIC (prevalence rate of 1 in 1.4 million) is caused by the dysfunction of heparan-α-glucosaminide N-acetyltransferase (HGSNAT, EC 2.3.1.78). HGSNAT is the only enzyme of the GAG pathway that is not a hydrolase. It catalyzes the only known biosynthetic reaction of the GAG degradation pathway within the lysosome, that is, the acetyl-CoA (ACO) mediated N-acetylation of the terminal non-reducing amino group of α-D-glucosamine (Fan et al., 2006; Hrebícek et al., 2006; Huizing and Gahl, 2020; Klein et al., 1978; Nagel et al., 2019; Pshezhetsky et al., 2018). Acetylation of the terminal α-D-glucosamine group is essential for subsequent HS degradation within the lysosomes. HGSNAT is not homologous to any known proteins, including acetyl-CoA binding proteins, N-acetyltransferases, and other lysosomal proteins. HGSNAT mRNA has two translation start sites (M1 and M29), yielding two functionally active isoforms (73 kDa and 70 kDa) of HGSNAT, both targeted to the lysosomal membrane (Durand et al., 2010; Fan et al., 2011). In this study, we use isoform 2 of HGSNAT, which is produced as a 635 amino acid protein containing a 30 amino acid N-terminal signal peptide, a ~110 amino acid long luminal domain, and 11 transmembrane helices (TMs). HGSNAT is believed to be expressed in the cell as an immature precursor protein that localizes as an N-glycosylated dimer on the lysosomal membrane (Durand et al., 2010; Fan et al., 2011). The localization of HGSNAT happens via the adaptor protein-mediated pathway, aided by the lysosomal targeting motifs [DE]-XXXL[LI] (²⁰⁴ETDRLI²⁰⁹) and YXXØ (⁶²⁴YILYRKK⁶³⁰) present towards the N- and C-terminus of HGSNAT, respectively (Rudnik and Damme, 2021; Schwake et al., 2013). Deleting the C-terminal lysosomal sorting signal in HGSNAT retains the protein in the plasma membrane (Bonifacino and Traub, 2003; Durand et al., 2010). Once targeted to the lysosomal membrane, HGSNAT, like a few other lysosomal membrane proteins, is proteolyzed by unidentified acid proteases in the lysosomal lumen into two unequal fragments - a smaller luminal N-terminal α-HGSNAT and a larger transmembrane C-terminal β-HGSNAT that continue to co-localize despite the proteolysis (Durand et al., 2010; Fan et al., 2011; Rudnik and Damme, 2021; Steenhuis et al., 2012). Upon proteolytic maturation, it is believed that HGSNAT is assembled as a hetero oligomer of α- & β-HGSNAT chains (Durand et al., 2010; Feldhammer et al., 2009b). However, the essentiality of proteolytic cleavage and oligomerization for HGSNAT activity remains debated as endoplasmic reticulum (ER) retained monomeric HGSNAT was also shown to be active (Durand et al., 2010; Fan et al., 2011).

So far, over 70 unique mutations in the HGSNAT (TMEM76) gene have been identified. These mutations span the entire sequence and include deletions, nonsense mutations, splice-site variants, and silent and missense mutations (Canals et al., 2011; Fan et al., 2006; Fedele and Hopwood, 2010; Feldhammer et al., 2009a; Feldhammer et al., 2009b; Hrebícek et al., 2006; Huizing and Gahl, 2020). The majority of the mutations that cause MPS IIIC are missense mutations that result in HGSNAT folding and localization defects, making them ideal targets for pharmacochaperone therapy. Site-specific inhibitors of HGSNAT activity have been explored as agents to rescue the misfolded conformation of certain missense variants, thereby restoring the partial N-acetyltransferase activity (Coutinho et al., 2012; Fedele and Hopwood, 2010; Feldhammer et al., 2009b; Pan et al., 2022). Despite its clinical relevance, the structure of HGSNAT and the mechanism of N-acetyltransferase activity are poorly understood. Here, using single-particle cryo-EM, we report the high-resolution structure of full-length HGSNAT in a complex with acetyl-CoA. This is the first structure of a member of the transmembrane acyl transferase (TmAT) superfamily (classes 9.B.97 and 9.B.169 in the Transporter Classification Database (TCDB)) (Saier et al., 2021). The HGSNAT-acetyl-CoA complex structure presented here provides a high-resolution snapshot of the first step in HGSNAT catalyzed acetyltransferase reaction, and reveals critical cofactor binding amino acids in the HGSNAT active site. In addition, our structure also provides molecular insights into the impact of MPS IIIC-causing mutations on acetyl-CoA binding and the overall architecture of the protein.

Direct involvement of acetyl-CoA in the heparan sulfate degradation was shown in the early 1980 s, by incubating the extracted intact lysosomes with radiolabeled acetyl-CoA and by subsequently monitoring the incorporation of radiolabeled acetate into lysosomal HS (Rome and Crain, 1981; Rome et al., 1983). Using purified lysosomal membranes, Rome and colleagues showed that the acetyl-CoA-dependent N-acetyltransferase activity, which is required for acetylation of HS, resides on the lysosomal membrane. In these studies, they also suggested that the HS acetylation reaction occurs in two steps - acetylation of the lysosomal membranes and transfer of acetyl from the membranes to HS. By performing acetylation and acetyltransferase reactions at different pH, and in the presence of different amino acid modification reagents, they showed that the acetylation process is optimal at pH 7, acetyltransferase activity is optimal at pH 5.5, and the amino acid on the lysosomal membrane that gets acetylated is a histidine. They proposed that the N-acetyltransferase activity follows a Ping Pong Bi Bi mechanism of reaction, where acetyl-CoA from the cytosol acetylates the active site histidine of the N-acetyltransferase located on the lysosomal membrane, inducing a conformational change in the transmembrane enzyme that enables the access of active site histidine from the luminal side (Figure 5D). Due to the change in pH of the active site cavity, the acetyl-histidine interaction is destabilized, and the acetyl group is transferred to the α-D-glucosaminide on the luminal side (Bame and Rome, 1985; Bame and Rome, 1986a; Bame and Rome, 1986b). Pshezhetsky and colleagues showed that acetylation of lysosomal membranes expressing N-acetyltransferase activity occurred even in the absence of the acetyl group acceptor, suggesting the formation of an acetylated enzyme intermediate (Ausseil et al., 2006; Durand et al., 2010). Here, we purified HGSNAT at a pH favorable for acetyl-CoA binding, but not acetyl transfer reaction. In our structure, we find that the HGSNAT ACOS is more accessible via the lumen as opposed to the cytosol and that the His269 of LL1 is not acetylated. Instead, we find an acetyl-CoA molecule in the ACOS, hydrogen bonded to N258 of TM2, a residue within a close vicinity (~5 Å) of H269 (Figure 3 and Figure 3—figure supplement 1). We believe N258 holds onto acetyl-CoA and aids H269 in the acetyltransferase reaction, at low pH and in the availability of acetyl group acceptor.

Meikle and colleagues argued that the N-acetyltransferase reaction occurs via a random-order mechanism (Figure 5C). They demonstrated that both N-acetyl-α-D-glucosamine and COA are required for the reverse reaction, suggesting that the formation of a ternary complex of acetyl-CoA, α-D-glucosamine, and the enzyme enables the reaction to proceed in a single step without the requirement of an acetylated-enzyme intermediate. They also report two K_m values, that differ by 5–10 times, for both acetyl-CoA and glucosamine substrates, suggesting that both protomers, perhaps, bind to the substrates at varying affinity and only one of the protomers is catalyzing the acetyl transfer at any given point of time (Meikle et al., 1995). Mahuran and colleagues showed that acetylated enzyme intermediate could not be identified by affinity pulldown of purified transmembrane N-acetyltransferase after incubating it with [³H] acetyl-CoA and proposed that the formation of a stable acetylated enzyme intermediate is not required for the reaction to proceed (Fan et al., 2011). Our structure does not provide evidence of H269 acetylation (Figure 3). However, we observe that acetyl-CoA binds tightly to HGSNAT endogenously resulting in a stable HGSNAT-acetyl-CoA intermediate that stayed as an intact complex all through the affinity purification, size-exclusion chromatography, and cryo-EM sample preparation steps even without the addition of exogenous acetyl-CoA to any of the buffers (Figure 5—figure supplement 1). Perhaps, this tight endogenous binding of HGSNAT is the reason why Mahuran and colleagues could not observe the labeling of purified transmembrane N-acetyltransferase by [³H] acetyl-CoA. The structure that we obtained does not by itself settle the debate about the mechanism of HGSNAT-mediated acetylation. Our structure is in a conformation that could proceed through any of the three enzyme-catalyzed bisubtrate reaction mechanisms for acetyl transfer (Figure 5). While we were revising our manuscript, another group published structures of apo HGSNAT and HGSNAT-ACO, HGSNAT-COA-NAG complexes. All these structures were obtained at pH 8.0 in 1% GDN and revealed no drastic conformational isomerization in the protein except for localized motion in TM2 and TM3 and residues at ACOS (Xu et al., 2024). Xu and colleagues do not show if the order of binding of the substrate is crucial for catalysis, and nor do they disprove the existence of acetylated enzyme intermediate. We believe structures of HGSNAT at lysosomal luminal pH in the presence and absence of an acetyl group acceptor will demonstrate not only if the enzyme gets acetylated during the reaction but also show if there are pH-driven conformational changes within the protein. It is also likely that the choice of detergent used determines the conformational isomerization. So, obtaining structures in membrane mimetics like nanodiscs in the presence of native HGSNAT lipids could provide a clearer insight into HGSNAT catalyzed reaction mechanism.

HGSNAT is believed to be produced as a pro-protein that gets proteolytically processed into its mature form where two fragments of unequal sizes, the α-HGSNAT and β-HGSNAT, continue to stay together because of a disulfide bond mediated interaction between C123 (β6) in the N-terminal LD of the α-HGSNAT and C434 in the LL3 of the C-terminal TMD of β-HGSNAT (Durand et al., 2010). However, according to the structure, these amino acids are ~34 Å apart making such a bond impossibility in the acetyl-CoA bound HGSNAT conformation reported here (Figures 1A, D, 2B). Moreover, we notice that C434 is disulfide bonded to C415 on LL3. C123 of β6 is predicted to form a disulfide bond with C151 of β8, and we do not see density to model such a disulfide. A bond between C123 of LD and C434 of LL3 could be a possibility only if there is a drastic conformational change during that brings LD closer to the LL3. Thus, we believe that the α-HGSNAT and β-HGSNAT stay together because of a series of hydrogen bonds, dipole-dipole and hydrophobic interactions between β2-β3 turn, β4-β5 turn, LL1, LL3, and LL4 (Figure 2 and Figure 2—figure supplement 2).

The protease that cleaves HGSNAT into α-HGSNAT and β-HGSNAT is unknown, and the site for proteolysis is unclear. Fan et al, proposed that the site of proteolysis is between amino acids N144 and G145 in the LD (Fan et al., 2011). N144-G145 is located on the β7-β8 turn of the LD, a region poorly resolved in our structure (Figures 1A and 2A). Although proteolysis at this position will cleave the LD into two parts (β1–7 and β8), the LD should remain attached to TMD because of the extensive network of interactions along the LD-TMD interface (Figure 2B and Figure 2—figure supplement 2). Durand et al, proposed that the site of proteolysis is between the end of the luminal domain and the beginning of LL1 (Durand et al., 2010). Sequence-based protease site prediction in Procleave and Prosper servers, using the sequence between β8 of LD and LL1, suggests multiple potential sites of proteolysis, for example, L154-A155 at the C-term of β8, S162-N163 in the loop connecting LD to TM1, F170-L171 at the N-term of TM1, L187-S188, and W231-R232 at the N- and C-term of the CL1 (Li et al., 2020; Song et al., 2012). It is hard to speculate which of these are most probable, as the lysosomes are rich in aspartic, serine, and cysteine proteases, including intra-membrane proteases that are involved in protein degradation and proprotein processing. In addition, some matrix metalloproteinases have also been shown to act in intracellular compartments and have been implicated in MPS diseases (Batzios et al., 2012; Jobin et al., 2017; Müller et al., 2012; Schröder and Saftig, 2016; Turk et al., 2000). The recombinant HGSNAT that we produced is a non-proteolyzed form of HGSNAT, as we see a band corresponding to full-length HGSNAT in our SDS-PAGE analysis and a peak corresponding to an intact full-length dimer in our FSEC analysis. HGSNAT is encoded by 18 exons, where the first 6 exons encode an N-terminal LD & TM1, which are only present in the metazoans. The remaining 10 TMs and the C-terminus are well conserved in archaea, bacteria, and plants (Fan et al., 2006; Hrebícek et al., 2006). Based on the evolutionary sequence and structure conservation and conformational flexibility observed in CL1, we speculate that LD & TM1 together form α-HGSNAT and TMs 2–11 form β-HGSNAT (Figures 2A and 4A, and Figure 2—figure supplement 1A).

There are no known homologs of the LD, and even the strand arrangement in two sheets appears to be rare (Koch et al., 1992). A structure-based search in the Dali server lists hits with <15% identity, with immunoglobulin-like & transthyretin folds as top hits (Supplementary file 1; Beale et al., 2015; Felisberto-Rodrigues et al., 2011; Parker et al., 2021; Rajasekar et al., 2019). Unlike the LD, a typical immunoglobulin-like fold is a disulfide-containing β-sandwich made of 7–9 β-strands arranged in two anti-parallel β-sheets, with a conserved core formed by four strands β2, β3, β5, & β6 (Figure 2—figure supplement 1C and D). In the Ig-like fold, the β2 and β5 strands are in one sheet, and the β3 and β6 strands are in the second one, with β2 and β6 being linked by a disulfide bond. The remaining 3–5 strands comprise the remaining variable region of the Ig-like fold (Bork et al., 1994; Chidyausiku et al., 2022). Although the % identity of LD and the transthyretin fold containing hits is <15%, the strand composition of the two sheets that make the β-sandwich is identical. However, a typical transthyretin fold contains a conserved short α-helix between β5 & β6 and often does not contain disulfide bonds (Figure 2—figure supplement 1C and D; Hörnberg et al., 2000). Another β-sandwich that shows a similar secondary structure assignment as LD is the type-II C2 domain, where the top sheet is made of β1, β4, β7, & β8 strands while the bottom sheet is made of β2, β3, β5, & β6 (Hirano et al., 2019; Kretsinger et al., 2013). However, both sheets in the type-II C2 domain are anti-parallel, and the arrangement of the top strand is β4-β1-β8-β7. In LD, one sheet is anti-parallel, and the other is a mixed sheet where the arrangement in the top sheet is β4-β1-β7-β8 (Figure 2—figure supplement 1C and D). Because of these non-trivial dissimilarities of LD with three major types of β-sandwiches, we believe that LD is a new fold of β-sandwich that could be best described as a ‘transthyretin-like’ fold. In fact, two proteins in the top hits from a structure-based similarity search on the Dali server show the same sheet arrangement as LD (Supplementary file 1 and Figure 2—figure supplement 1D). One of these proteins is AlgF (PDB ID: 6CZT), an adaptor protein from Pseudomonas believed to be involved in O-acetylation of alginate exopolysaccharides and the other is an SPH (self-incompatibility protein homolog) domain (PDB ID: 6G7G) that is described as transthyretin-like and is believed to be a plant secreted protein involved in cell death (Figure 2—figure supplement 1D).

The function of LD remains unknown. DeepSite predicts the presence of two ligand binding sites in HGSNAT, one that overlaps with the ACOS and the other in the cavity between the two sheets of LD (Figure 3—figure supplement 1B; Jiménez et al., 2017). However, it is unclear if LD binds to a ligand and/or a metal ion. It has been shown that the N-acetyltransferase activity of HGSNAT is enhanced in the presence of anionic phospholipids (Fan et al., 2011). However, we expect lipids to bind towards the cytosolic side at the dimer interface. We see unexplained density in these regions that could be best explained as ordered lipids or digitonin (Figure 2—figure supplement 3). A recent report of a computational modeling and molecular dynamics simulation study conducted on a model of OafB, a bacterial O-antigen modifying transmembrane acetyltransferase of the ATAT family within the TmAT superfamily, showed that the periplasmic SGNH domain undergoes large conformational changes and aid in O-antigen acetylation (Newman et al., 2023). OafB has two domains – the transmembrane acetyltransferase-3 domain (AT-3) and the periplasmic SGNH domain. Although the domain organization is similar to HGSNAT, there are marked differences in the structure (Figure 2—figure supplement 1B). AT-3 domain and HGSNAT TMD are not homologous, even though they share a similar architecture to the ACOS. The LD of HGSNAT is different from the SGNH domain, in the sense that LD is a β-sandwich at the N-terminus of the protein while SGNH is a α-β-α sandwich with a single β-sheet sandwiched between two helical domains at the C-terminus of the protein. There are non-trivial differences between the predicted structures of ATAT and YeiB family members. However, it is possible that the TMDs and the extra-membranous domains function similarly. It is likely that LD of HGSNAT binds to HS, stabilizing the terminal non-reducing sugar of HS near the luminal side of ACOS for catalysis and remains to be structurally investigated. In fact, it has been shown that as the size of the acetyl group acceptor was increased in an acetyltransferase reaction from mono- to di- to tetra-saccharide, the K_m values decreased from 0.6 mM to 7 μM (Meikle et al., 1995).

The high-resolution structure of HGSNAT reported in this study heralds a new beginning for structural exploration of the TmAT superfamily. The conformation of HGSNAT observed in our cryo-EM studies does not put to rest the debate on the kind of bi-substrate reaction mechanism that HGSNAT follows to catalyze the acetyl transfer. However, our structure underscores the requirement to characterize the remaining states of the enzymatic reaction. The molecular basis we delineated for the mutation-induced dysfunction in HGSNAT will serve as a blueprint for the structure-based design of novel therapeutic modulators that could rescue the function of milder variants.

Structure coordinates and cryo-EM maps have been deposited and released under the accession codes RCSB PDB-8TU9; EMDB EMD-41620. Uncropped gel showing purification has been included as Figure1-Figure_supplement_1M-Source_Data 1 and Figure1-Figure_supplement_1M-Source_Data 2.

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Author details

1. Vikas Navratna

   1. Life Sciences Institute, University of Michigan, Ann Arbor, United States
   2. Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8599-1461

2. Arvind Kumar

   Thermo Fisher Scientific, Waltham, United States

   Contribution

   Validation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8421-8669

3. Jaimin K Rana

   1. Life Sciences Institute, University of Michigan, Ann Arbor, United States
   2. Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

   Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

4. Shyamal Mosalaganti

   1. Life Sciences Institute, University of Michigan, Ann Arbor, United States
   2. Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States
   3. Department of Biophysics, College of Literature, Science and the Arts, University of Michigan, Ann Arbor, United States

   Contribution

   Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Project administration, Writing – review and editing

   For correspondence

   mosalaga@umich.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5934-687X

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