Human Α-L-Iduronidase Uses Its Own N-Glycan As a Substrate-Binding and Catalytic Module

Human α-L-iduronidase uses its own N-glycan as a substrate-binding and catalytic module

Nobuo Maitaa,b,1, Takahiro Tsukimurac, Takako Taniguchib, Seiji Saitod, Kazuki Ohnoe, Hisaaki Taniguchib, and Hitoshi Sakurabaf,g

aLaboratory of X-Ray Crystallography, Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan; bDivision of Disease Proteomics, Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan; cDepartment of Functional Bioanalysis, Meiji Pharmaceutical University, Tokyo 204-8588, Japan; dDepartment of Medical Management and Informatics, Hokkaido Information University, Hokkaido 069-8585, Japan; eResearch Section, NPO for the Promotion of Research on Intellectual Property Tokyo, Tokyo 100-0005, Japan; fDepartment of Analytical Biochemistry, Meiji Pharmaceutical University, Tokyo 204-8588, Japan; and gDepartment of Clinical Genetics, Meiji Pharmaceutical University, Tokyo 204-8588, Japan

Edited by Elizabeth F. Neufeld, David Geffen School of Medicine at University of California, Los Angeles, CA, and approved July 25, 2013 (received for review April 12, 2013) N-glycosylation is a major posttranslational modification that Aldurazyme) was developed for enzyme replacement therapy endows proteins with various functions. It is established that (12), which is widely used for MPS I treatment. N-glycans are essential for the correct folding and stability of some IDUA belongs to glycoside hydrolase (GH) family 39 in the enzymes; however, the actual effects of N-glycans on their activ- CAZy database (13). To date, crystal structures of the bacterial ities are poorly understood. Here, we show that human α-L-idur- GH39 β-xylosidase (XynB) have been reported (14, 15). In ad- onidase (hIDUA), of which a dysfunction causes accumulation of dition, a homology model of hIDUA constructed from Ther- dermatan/heparan sulfate leading to mucopolysaccharidosis type I, moanaerobacterium saccharolyticum XynB (PDB ID code 1PX8) uses its own N-glycan as a substrate binding and catalytic module. has been reported (16). As the sequence homology between Structural analysis revealed that the mannose residue of the N-gly- hIDUA and T. saccharolyticum XynB is quite low (28.4% simi- can attached to N372 constituted a part of the substrate-binding larity), the reliability of the model is not high. Recently, the pocket and interacted directly with a substrate. A deglycosylation crystal structures of apo-hIDUA, expressed in a plant seed, were study showed that enzyme activity was highly correlated with the solved. Nevertheless, the structure of hIDUA expressed in N-glycan attached to N372. The kinetics of native and deglycosy- mammalian cells is strongly required for an insight into the basis lated hIDUA suggested that the N-glycan is also involved in catalytic of MPS I and the development of new therapies. In this study, we processes. Our study demonstrates a previously unrecognized func- explored the functions of the N-glycans in hIDUA. We found tion of N-glycans. that the deglycosylation of hIDUA with endoglycosidase H (Endo H), but not peptide-N-glycosidase F (PNGase F), reduces X-ray crystallography | N-linked glycan | glycoside hydrolase family 39 the enzyme’s activity. Concanavalin A (ConA) pull-down assay suggested that PNGase F–resistant N-glycans are essential for sparagine-linked protein glycosylation, one of the major the enzyme activity of hIDUA. We also solved the crystal posttranslational modifications in eukaryotes, causes linking structures of hIDUA alone and in a complex with IdoA: they A N of an oligosaccharide chain to the Nδ atom of an asparagine in revealed that the -glycan attached at N372 makes up one side the N-glycosylation signal (Asn-Xaa-Ser/Thr, Xaa can be any of the substrate-binding pocket and directly interacts with the amino acid except proline). N-glycosylation endows proteins with IdoA. Further, we found that the enzyme activity showed high N several abilities including immune recognition, ligand-receptor correlation with the amount of -glycan at N372. The kinetics N binding, and cell signaling, trafficking, folding, and stability (1– of native and deglycosylated hIDUA implied that the -glycan fi 3). For some lysosomal enzymes such as glucocerebrosidase (4) is also involved in the catalytic process. Our nding indi- N and α-galactosidase A (5), the deglycosylation reduces the enzymes’ cates a previously unrecognized function of -glycans in the activities, presumably through a reduction in protein stability. enzyme activity. However, the precise relationships between N-glycans and en- Results zyme activities remain unknown (2, 6). Activity of hIDUA Is Reduced on Endo H but Not PNGase F Treatment. α-L-Iduronidase (IDUA; EC 3.2.1.76) is a lysosomal enzyme, To examine the influence of N-glycans on hIDUA activity, we and deficient activity of IDUA causes accumulation of glyco- carried out a deglycosylation study. Aldurazyme was digested saminoglycans in lysosomes leading to mucopolysaccharidosis with PNGase F or Endo H overnight and then subjected to the type I (MPS I) (7). Human IDUA is translated as 653 amino enzyme assay with 4-methylumbelliferyl α-L-iduronide (17) as the N acids and -glycosylated at six potential sites (N110, N190, substrate. A small amount of each digested sample was subjected N336, N372, N415, and N451), and then its N-terminal 26 resi- to ConA-Sepharose pull-down assaying to detect the residual dues are removed and it is processed to the mature form in N-glycans. The PNGase F–treated hIDUA showed no defect in lysosomes (8, 9). IDUA hydrolyses the glycosidic bond between enzyme activity, and some N-glycans showed PNGase F resistance the terminal L-iduronic acid (IdoA) and the second sugar of N- (Fig. 1B, lanes 10 and 12). These glycans became sensitive to acetylgalactosamine (GalNAc)-4-sulfate/N-sulfo-D-glucosamine (GlcNS)-6-sulfate, which are the major components of dermatan/ heparan sulfate (Fig. 1A). Thus, a defect of IDUA leads to excess Author contributions: N.M. and H.S. designed research; N.M., T. Tsukimura, and T. Taniguchi storage of dermatan/heparan sulfate and causes a systemic dis- performed research; N.M., T. Tsukimura, T. Taniguchi, S.S., K.O., and H.T. analyzed data; and N.M., H.T., and H.S. wrote the paper. order, MPS I, involving progressive mental retardation, gross The authors declare no conflict of interest. facial features, an enlarged and deformed skull, a small stature, This article is a PNAS Direct Submission. corneal opacities, hepatosplenomegaly, valvular heart defects, Data deposition: The atomic coordinates and structure factors have been deposited in the thick skin, joint contractures, and hernias (10). The prevalence of Protein Data Bank, www.pdb.org (PDB ID codes 3W81 and 3W82). MPS I in England and Wales from 1981 to 2003 was 1.07 cases 1To whom correspondence should be addressed. E-mail: [email protected]. per 100,000 births (11). A recombinant human IDUA (hIDUA) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. expressed in Chinese hamster ovary (CHO) cells (marketed as 1073/pnas.1306939110/-/DCSupplemental.

14628–14633 | PNAS | September 3, 2013 | vol. 110 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1306939110 Downloaded by guest on September 24, 2021 Fig. 1. Deglycosylation study of hIDUA. (A) Re- action scheme of dermatan sulfate hydrolysis cata- lyzed by hIDUA. (B) hIDUA was deglycosylated with PNGase F overnight under the indicated conditions (lanes 1–6), and then enzyme activities were measured (lanes 1–6, bottom; ND, not detected). Small amounts of the reaction mixture were mixed with ConA-Sepharose and washed three times, and then ConA-gel was separated by SDS/PAGE (lanes 7–12). The gel was stained with CBB. Nonidet P-40 pre- vents unfavorable aggregation, resulting in in- creasing activity. Human IDUA aggregates were produced due to the reaction mixture’s pH (20). (C) Deglycosylation analysis of hIDUA with Endo H. The procedure was the same as that in B other than the use of Endo H.

PNGase F on denaturation (Fig. 1B, lane 8). These results suggest that in the presence of Nonidet P-40, the activity increased by that some N-glycans of hIDUA are so rigid or buried, and they about twofold (Fig. 1B, lanes 3 and 4). This result is presumably cannot gain access to the catalytic site of PNGase F. We observed due to the prevention of unfavorable aggregation of hIDUA (18). BIOCHEMISTRY

Fig. 2. Crystal structure of IdoA-bound hIDUA. (A) Domain organization of hIDUA. N-glycosylation sites observed in the crystal structure are indicated as yellow hexagons. N336, another N-glycosylation site, but with no glycans, is also indicated. The disulﬁde bond between C541 and C577 is indicated as S-S below the bar. The overall structure of hIDUA complexed with IdoA (side and end views) is shown. Human IDUA (subunit A) is shown as a cartoon representation with the TIM barrel domain (pink), β-sandwich domain (green), and Ig-like domain (blue). N-glycans (yellow), IdoA (blue), and phosphate are shown as stick models. The positions of N-glycosylation sites are indicated. (B) A stereo image of the omit map of the high mannose type N-glycan attached to N372 (subunit A) in an apo-state crystal. The map is contoured at 2.5σ. N372 and catalytic glutamates (E182 and E299) are also shown. (C) Schematic drawing of the N-glycan structure at N372. The cleavage sites for Endo H and PNGase F are also indicated. Glycan linkage patterns are denoted as follows: β4, β1–4; α2, α1–2; α3, α1–3; α6, α1–6.

Maita et al. PNAS | September 3, 2013 | vol. 110 | no. 36 | 14629 Downloaded by guest on September 24, 2021 Table 1. N-glycan structures observed in the IDUA crystal structures Subunit Position apo-IDUA holo-IDUA

Subunit A N110 GlcNAc GlcNAc N190 Not observed Not observed N336 Not observed Not observed N372 (GlcNAc)2(Man)8 (GlcNAc)2(Man)8 N415 (GlcNAc)2 (GlcNAc)2 N451 Not observed Not observed Subunit B N110 GlcNAc GlcNAc N190 (GlcNAc)2(Fuc)(Man)2(GlcNAc)(Gal) (GlcNAc)(Fuc) N336 Not observed Not observed N372 (GlcNAc)2(Man)7* (GlcNAc)2(Man)7* N415 (GlcNAc)2 (GlcNAc)2 N451 GlcNAc Not observed

*Man9 was not observed.

On the other hand, on Endo H digestion, the hIDUA activity was a protein through many polar- and water-mediated contacts (Fig. reduced to 7% (Fig. 1C, lane 5). Endo H–resistant N-glycans were S3) including the side chains of H58, W306, S307, and Q370 and also observed (Fig. 1C, lanes 8 and 10); this is in agreement with the backbone carbonyls of P54, L56, and H356. In addition, the previous study showing that hIDUA expressed in CHO cells a hydrophobic interaction between Y355 and Man3 was ob- N carries a complex type of -glycans (8). These results suggest that served. The structural characteristics of N-glycan at N372 were N high-mannose and/or hybrid type -glycans have a rigid confor- very similar in both subunits. A similar N-glycan interaction with mation and affect the activity of hIDUA. an enzyme has been reported for Trichoderma reesei β-galacto- N Overall Structure of hIDUA. To determine the structural basis of sidase. In the structure of the latter, the tip of the -glycan at- the relationships between the N-glycans and enzyme activity of tached at N930 comes near the active site; however, it seems too hIDUA, we solved the crystal structures of apo- and IdoA-bound far for any direct interactions with a substrate (21). hIDUA at 2.3- and 2.76-Å resolution, respectively (19) (Fig. 2A; Table S1). We could build an almost full-length hIDUA (residues 27–642). There are two hIDUA subunits per asymmetric unit. Judging from the small buried surface area (548.3 Å2 against the total accessible surface area of 25,127 Å2) and the results of gel filtration analysis, these two subunits are unlikely to represent a functional dimer (20). hIDUA consists of three domains: residues 42–396 form a classic (β/α)8 triosephosphate isomerase (TIM) barrel fold, residues 27–42 and 397–545 form a β-sandwich domain with a short helix–loop–helix (482–508), and residues 546–642 form an Ig(Ig)-like domain. The latter two domains are linked through a disulfide bridge between C541 and C577. The β-sandwich and Ig-like domains are attached to the first, seventh, and eighth α-helices of the TIM barrel. A β-hairpin (β12–β13) is inserted between the eighth β-strand and the eighth α-helix of the TIM barrel, which includes N-glycosylated N372 (Fig. 2A). The topologies of the TIM barrel and β-sandwich domains of hIDUA are almost identical to that of XynB, which belongs to the same GH family 39 (12, 13). However, XynB has a shorter amino acid length than hIDUA and lacks the C-terminal Ig-like domain (Fig. S1). We observed at least five N-glycans in the electron density map other than that at N336 (Table 1). The loop region including N336 exhibited slightly poor electron density, and we could not place any sugars there. Although only one or two sugars were detected in most of the N-glycans, we observed long oligosaccharide chains at N190 (subunit B) and N372 (subunits A and B). N190 has complex type oligosaccharides, as previously predicted (8). The GlcNAc3Man2Gal1Fuc1 structure (Fig. S2) was visible at N190 of subunit B. This oligosaccharide interacts with the 590–592 loop region of symmetry-related subunit B. The most remarkable feature is the N-glycan attached to Fig. 3. Substrate binding site of hIDUA for IdoA. (A) Molecular surface N372, which is of the high-mannose type (8), and we clearly representation around the substrate-binding pocket of hIDUA (subunit B). Mannose and basic residues are colored yellow and blue, respectively. The observed a GlcNAc2Man8 oligosaccharide chain in subunit A B N IdoA molecule and phosphate are drawn as stick models. An omit map of the (Fig. 2 ). The -linked oligosaccharide chain is tightly bound to IdoA (contour at 3σ) is also shown. (B) Details of the interactions between the surface of the TIM barrel. The tip of mannose residue (Man7) the IdoA and hIDUA (subunit B) are shown as a stereo image. The hydrogen reaches the active site, and constitutes a part of the substrate- bonds between IdoA and other related residues are indicated by black and binding pocket (Fig. 2B). The N-glycan at N372 interacts with cyan dashed lines, respectively.

14630 | www.pnas.org/cgi/doi/10.1073/pnas.1306939110 Maita et al. Downloaded by guest on September 24, 2021 Fig. 4. Characterization of N-glycosylation at N372. (A) LC-MS profile of the native 369–383 peptide obtained on trypsin digestion of hIDUA. The spectra of the trivalent-ionized glycosylated (Upper) and deglycosylated (Lower) peptides are shown. The peak corresponding to m/z = 674.7 is magnified 100 times for clarity. (B) LC-MS profile of the Endo H–treated 369–383 peptide obtained on trypsin digestion of hIDUA, with the same representation as in A. The 1,050– 1,300 range of the horizontal axis is magnified 1,000 times for clarity. (C) Ratio of the residual N-glycans during Endo H treatment determined by MS. The

data were calculated using the peak area of peptide-GlcNAc2Man7–9 or peptide-GlcNAc of triply and quadruply charged ions. The enzyme activities of Endo H–treated (+Endo H) and nontreated (−Endo H) hIDUA are also plotted. (D) Michaelis-Menten plots for Endo H–treated (+Endo H, 48 h) and nontreated (−Endo H) hIDUA. The data are means of three repeated experiments ± SD. (Inset) Kinetic parameters of Endo H–treated and nontreated hIDUA. BIOCHEMISTRY

Very recently, two crystal structures of hIDUA were released H91, D349, and Man7. In addition, the O2 atom interacts with in the Protein Data Bank with the space groups of R3 (PDB ID H91, N181, and E299, a catalytic glutamate. The O1 atom in- code 4JXO) and P21 (PDB ID code 4JXP). The structures also teracts with Man7 and E182, another catalytic glutamate. The contain a high-mannose type of N-glycan chain at N372; how- holo-hIDUA structure also provides us with information about ever, they have six sugars at most and lack Man7, possibly owing the substrate specificity of the enzyme. For example, β-D-glucuronic to the plant seed expression system used (22). acid, an epimer of IdoA, may have less affinity than IdoA for the binding site, as the hydrogen bonding between O5 and K264 N-Glycan Is Involved in the Substrate Interaction. In the IdoA-bound Nζ found in IdoA (3.18 Å) will be lost in β-D-glucuronic acid hIDUA structure, we clearly observed the electron density of the (3.94 Å; Fig. S4B). IdoA molecule at the center of the TIM barrel (Figs. 2A and 3A). The O1 atom of IdoA faces the open side of the binding There is little structural difference between the apo and IdoA- pocket, suggesting that downstream of the dermatan/heparan bound forms (rmsd = 0.253 Å, over 580 Cα atoms); only the side sulfate chain stretches out of this side (Fig. 3A; Fig. S5). We chain of D187 is flipped toward the active site, which forms hy- observed a phosphate ion between the side chains of H185 and drogen bonds between the Oδ2 atom and the backbone oxygen H226. A putative hIDUA and dermatan sulfate complex model and Nδ atom of N181 (Fig. S4A). This change presumably yields suggests that the sulfate moiety of IdoA-2-sulfate at the +2 po- a tight hydrogen network between the O2 atom of IdoA and Nδ sition overlaps the phosphate (Fig. S5). Thus, H185 and H226 of N181 (Fig. 3B). presumably interact with the sulfate moiety of the substrate There are 19 polar contacts between IdoA and the protein and sugar chain. 2 more contacts between IdoA and Man7 of the N-glycan (Fig. 3B; Table S2). The two oxygen atoms of the carboxyl group of Amount of Glycan at N372 Correlates with the Enzyme Activity. Be- IdoA interact with the backbone amide between G305 and W306 cause our structural study indicated that the N-glycan at N372 and the side chains of K264 and R363. The O4 atom of IdoA interacts with an IdoA molecule, we focused on N372. Aldur- interacts with D349 and R363. The O3 atom also interacts with azyme was incubated with Endo H for varying times at 37 °C and

Maita et al. PNAS | September 3, 2013 | vol. 110 | no. 36 | 14631 Downloaded by guest on September 24, 2021 Fig. 5. The loop inserted between β8andα8 (348–384) is highly conserved. (A) The loop inserted between β8 and α8 (348–384) is colored magenta. N372 is drawn as a stick model. (B) Alignment of sequences corresponding to the loop inserted between β11 and α10 (348–384) of human IDUA. Sequences were aligned using Clustal X ver.2 (34), and colored with ESPript (35). The names of the species and protein IDs are given in the legend to Fig. S8. N372 and T374 are indicated by pink arrowheads.

subjected to enzyme assay. We reduced the amount of Endo H to classical GH-A clan, the O1 atom of IdoA is the target for hy- see the effect of partial deglycosylation. The activity of hIDUA drolysis, and the carboxyl group of E182 would act as a proton gradually decreased in an incubation time-dependent manner, donor to O1 (24, 25). We could observe the interaction between the activity being almost completely lost after 48 h (Fig. 4C). The the O1 atom of IdoA and the O3 atom of Man7 in subunit B B fl kcat values of the native and deglycosylated (+Endo H, 48 h) (Fig. 3 ; Table S2). Thus, Man7 may somehow in uence pro- −1 hIDUA were 210 ± 3 and 9.50 ± 0.04 s , and the Km values were tonation of the O1 atom of IdoA. 290 ± 10 and 180 ± 0.4 μM, respectively (Fig. 4D). To determine The results of a molecular phylogenetic analysis of IDUA whether the activity reduction was due to unfolding of the pro- orthologs suggest the importance of the N-glycan at N372. tein, we measured circular dichroism spectra of the native and Multiple sequence alignment showed that in all of the IDUAs, deglycosylated hIDUA. The spectra were almost identical, sug- i.e., the Ciona to human ones, the positions corresponding to gesting that no aggregation or denaturation had occurred on N372 and T374 are conserved (Fig. 5; Fig. S8), whereas five Endo H treatment (Fig. S6). other N-glycosylation sites are not (Fig. S8). Additionally, iden- To clarify whether the Endo H treatment indeed caused tical or similar residues are clustered at the reaction center and deglycosylation at N372, we digested the Endo H–treated hIDUA along the interface of the N-glycan at N372 (Fig. S9A). These with trypsin, followed by analysis by LC-MS. As a result, we clearly findings strongly suggest not only the conservation of this as- detected triply and quadruply charged ions corresponding to the paragine residue but also N-glycosylation throughout multicel- N372-containing fragment (residues 364–383) with GlcNAc and lular animals, and thus, the N-glycosylation at N372 should play fi GlcNAc2Man7–9 (Fig. 4 A and B; Table S3). The ratio of the a signi cant role in the function of IDUA. fragment (364–383) with GlcNAc2Man7–9 to the total fragment We could observe a phosphate ion around H185, H226, and (364–383) was highly correlated with the enzyme activity (corre- R230 (Fig. 3A; Fig. S5). These residues are conserved among lation: 0.989; Fig. 4C). vertebrates (Fig. S8). The surface electrostatics showed that Furthermore, we examined the amounts of the other N-gly- there is another positively charged patch, comprising H226 and cans attached to N336, N415, and N451, which could be cleaved R263, just outside the exit to the substrate binding cleft (Fig. by Endo H (8). The hIDUA treated with Endo H was digested S9B). These positively charged residues are also highly conserved with chymotrypsin and the peptide GlcNAcs, the Endo H among vertebrates (Fig. S8). Such positively charged patches are products, were monitored (Fig. S7; Table S4). The amounts of likely to contribute to binding of the sulfated glycosaminoglycan the peptide GlcNAc at N336, N372, N415, and N451 increased in of dermatan/heparan sulfate. These results suggest that the a time-dependent manner. However, the degrees of the inverse vertebrate IDUAs have adapted to dermatan sulfate, the main correlations between the enzyme activities and the amount of component of skin. N372-GlcNAc were higher (correlation: −0.915) than those for More than 55 disease-associated missense mutations in the α IDUA fi the other N-glycosylation sites (N336, −0.616; N415, −0.615; and human -L-iduronidase ( ) gene have been identi ed (Hu- N451, −0.788) (Fig. S7). These results suggest that the N-glycan man Gene Mutation Database, www.hgmd.cf.ac.uk/). Among them, at N372, which forms a part of the substrate binding site, is in- we paid attention to the W306 to Leu (W306L) gene (26) as a volved in the enzymatic activity. Discussion Our structural and deglycosylation studies indicated that the N-glycan at N372 is essential for hIDUA activity. The deglycosylation study showed the different effects of Endo H and PNGase F (Fig. 1 B and C). The presence of PNGase F–resistant glycans was also observed in the previous study using CHO cell– expressed hIUDA (8). Furthermore, a report has described that treatment of Aldurazyme with PNGase F decreased enzyme activity by 50%, but significant activity still remained (23). The crystal structure revealed that the tightly bound oligosaccharide chain was linked to N372, which can be explained by the PNGase N k K Fig. 6. Molecular modeling of the Trp306 to Leu mutant. (A) Polar contact F resistance of the -glycan. The cat and m values of the WT between W306 and Man7. (B) Superposition of energy-minimized WT (cyan) and deglycosylated hIDUA suggested that deglycosylation and W306L (green). The side chains of W306 (Leu306), S307, and F352 are affects enzyme catalysis and substrate binding. As seen in the indicated as a stick model.

14632 | www.pnas.org/cgi/doi/10.1073/pnas.1306939110 Maita et al. Downloaded by guest on September 24, 2021 possible mutation that affects the N-glycan at N372. Structural Materials and Methods N analysis revealed that W306 interacts with the -glycan at N372, The methods are described in full in SI Materials and Methods. and we examined whether this mutation would lead to a dysfunction of enzyme activity by molecular modeling and energy minimization. Samples and Chemicals. The recombinant hIDUA expressed in CHO cells The rmsd between the WT and W306L is 0.023 Å, and the locations (Aldurazyme) was purchased from Genzyme Japan. IdoA was purchased of S307 and F352 in the IDUA molecule were predicted to move from Carbosynth (United Kingdom). slightly with the amino acid substitution (Fig. 6). This finding suggests that the amino acid substitution does not affect the catalytic Crystallization, Structure Determination, and Model Refinement. We crystal- center or, if at all, just a little. The W306L mutation may cause MPS lized and solved the structure of hIDUA by the single isomorphous replacement I through the effect on the conformation of the N-glycan at N372. with anomalous scattering (SIRAS) method as described previously (19). We fi Most crystallographers remove the glycans when they try to automatically built an initial model using RESOLVE (30) and subsequently xed fi crystallize glycoproteins to improve their homogeneity. However, it by hand with COOT (31), and then re ned the apo-hIDUA structure with CNS (32) and REFMAC5 (33). the functionally important glycans tend to bind to a protein tightly and are resistant to the processing in the endoplasmic reticulum – ACKNOWLEDGMENTS. We thank the beamline staff at the Photon Factory and Golgi apparatus (21, 27 29). Our study suggests that it is and SPring-8 BL44XU for supporting the data collection under Proposals better to retain the high-mannose type glycans to not overlook the 2009G074 and 2011G135. We also thank H. Saito, C. Mizuguchi, I. Sagawa essential functions. (Tokushima University), T. Nishino (National Institute of Genetics), and M. In conclusion, we first determined the structure of hIDUA, Ariyoshi (Kyoto University) for supporting the data collection. This work was and then structural and biochemical studies revealed that the performed with a Cooperative Research Grant from the Institute for Enzyme N-glycan at N372 was used as a substrate-binding module. Fur- Research, Joint Usage/Research Center, University of Tokushima. This work was supported by Grants-in-Aid for Young Scientists (20770085) and Scien- thermore, the results of a kinetic study suggested that the fi N fi ti c Research (23570139) from Japan Society for the Promotion of Science -glycan is directly involved in enzyme catalysis. These ndings (to N.M.) and the Program for the Promotion of Fundamental Studies in will be useful not only for elucidation of the molecular basis of Health Sciences of the National Institute of Biomedical Innovation (ID 09- MPS I but also for the development of new drugs for this disease. 15) (H.S.).

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