Structural and Mechanistic Insight Into N-Glycan Processing by Endo-Α-Mannosidase

Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase

Andrew J. Thompsona, Rohan J. Williamsb, Zalihe Hakkib, Dominic S. Alonzic, Tom Wennekesd, Tracey M. Glostera, Kriangsak Songsrirotea,e, Jane E. Thomas-Oatesa,e, Tanja M. Wrodniggf, Josef Spreitzf, Arnold E. Stützf, Terry D. Buttersc, Spencer J. Williamsb,1, and Gideon J. Daviesa,1

aDepartment of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom; bSchool of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia; cOxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom; dLaboratory of Organic Chemistry, Wageningen University, 6703 HB, Wageningen, The Netherlands; eCentre of Excellence in Mass Spectrometry, University of York, Heslington, York YO10 5DD, United Kingdom; and fInstitute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria

Edited by Chi-Huey Wong, Academia Sinica, Taipei, Taiwan, and approved November 28, 2011 (received for review August 9, 2011)

N-linked glycans play key roles in protein folding, stability, and glycans are cotranslationally transferred en bloc by the multipro- function. Biosynthetic modification of N-linked glycans, within tein complex oligosaccharyltransferase from the glycophospholi- the endoplasmic reticulum, features sequential trimming and read- pid precursor, Glc3Man9GlcNAc2-diphospho-dolichol, to Asn ornment steps. One unusual enzyme, endo-α-mannosidase, cleaves residues within nascent polypeptide chains (Fig. S1A). The initial mannoside linkages internally within an N-linked glycan chain, processing steps of this 14-sugar glycan commence while the short circuiting the classical N-glycan biosynthetic pathway. Here, unfolded glycoprotein remains attached to the ribosome, and using two bacterial orthologs, we present the first structural and involves the sequential removal of the terminal α-1,2-glucose by mechanistic dissection of endo-α-mannosidase. Structures solved glucosidase I and removal of both the subsequent and final α-1,3- – ðβ∕αÞ at resolutions 1.7 2.1 Å reveal a 8 barrel fold in which the glucose moieties by glucosidase II, a procedure normally required catalytic center is present in a long substrate-binding groove, for the formation of mature N-glycans (1, 5, 10) (Fig. S1B). Under consistent with cleavage within the N-glycan chain. Enzymatic normal conditions, the final α-1,3-glucose residue represents a

cleavage of authentic Glc1∕3Man9GlcNAc2 yields Glc1∕3-Man. Using checkpoint in protein folding and quality control because mono- the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was glucosylated immature N-glycans with a terminal α-Glc-1,3-α- shown to act with retention of anomeric configuration. Complexes Man-1,2-α-Man chain are ligands for the molecular chaperones with the established endo-α-mannosidase inhibitor α-Glc-1,3-deox- calnexin (CNX) and calreticulin (CRT) (11). The fate of newly ymannonojirimycin and a newly developed inhibitor, α-Glc-1,3- synthesized glycoproteins is determined by a molecular inspec- isofagomine, and with the reducing-end product α-1,2-mannobiose tion process that monitors their folding state. Correctly folded structurally define the −2toþ2 subsites of the enzyme. These proteins exit the CNX/CRT cycle with removal of the final α- structural and mechanistic data provide a foundation upon which 1,3-glucose from the Man9GlcNAc2 polysaccharide followed by to develop new enzyme inhibitors targeting the hijacking of α-mannosidase I cleavage of a mannose residue from the first N-glycan synthesis in viral disease and cancer. branch and translocation to the Golgi apparatus. Misfolded proteins undergo cycles of deglucosylation and reglucosylation, 3D structure ∣ enzyme inhibition ∣ enzyme mechanism ∣ glycobiology ∣ catalyzed by luminal UDP-glucose-dependent glycoprotein glyco- glycosidase syltransferase. Terminally misfolded proteins are extracted from the folding cycle in a process termed ER-associated degradation -linked glycans are present on the majority of eukaryotic and are retrotranslocated to the cytosol, where they are ubiqui- tinylated and proteasomally degraded. Nproteins and direct their folding and influence their stability. α These polysaccharide decorations play important roles in pro- Endo- -mannosidase (classified into Carbohydrate-Active cesses such as protein folding, targeting, antigenicity, and lectin Enzymes Database family GH99; refs. 12 and 13; www.cazy.org) interactions, with defects leading to cellular dysfunction (1). Aber- provides a glucosidase I and II independent pathway for the maturation of N-glycans (14). Endo-α-mannosidase hydrolyzes rant N-glycan composition, through either incorrect or incomplete α processing, is associated with various conditions, including Alzhei- the -1,2-mannosidic bond between the glucose-substituted man- mer’s disease, congenital disorders of glycosylation, viral infection, nose and the remainder of the N-glycan, and acts on the struc- and metastatic cancer progression (2–4). Alteration of N-glycan tures Glc1–3Man9GlcNAc2 as well as structures that have been trimmed by mannosidases in the 6′-pentamannosyl branch, BIOCHEMISTRY biosynthesis in cancerous cells and by human pathogenic viruses releasing Glc1–3-1,3-α-Man oligosaccharides (15) (Fig. S1C). renders the various proteins involved in their biosynthesis and Despite growing insight into the biosynthetic function and subcel- modification therapeutic targets. N-glycans are built-up and then “biosynthetically” degraded in an orchestrated process involving synthetic enzymes (glycosyltransferases) and catabolic enzymes Author contributions: A.J.T., R.J.W., T.D.B., S.J.W., and G.J.D. designed research; A.J.T., R.J. (glycoside hydrolases) (5) (Fig. S1). Efforts to control diseases W., Z.H., D.S.A., T.W., T.M.G., and K.S., performed research; R.J.W., Z.H., T.M.W., J.S., A.E.S., of glycoprotein biosynthesis have largely focused on the use of and T.D.B. contributed new reagents/analytic tools; A.J.T., R.J.W., J.E.T.-O., T.D.B., S.J.W., and G.J.D. analyzed data; and A.J.T., S.J.W., and G.J.D. wrote the paper. inhibitors of the “biosynthetic” trimming glycoside hydrolases in- CHEMISTRY The authors declare no conflict of interest. volved in the early stages of N-glycan remodeling, such as gluco- sidases I and II. However, the failure of these inhibitors to provide This article is a PNAS Direct Submission. an effective treatment of these conditions likely occurs, in part, Freely available online through the PNAS open access option. because of a unique enzyme, endo-α-mannosidase, which cleaves Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4acy, 4acz, 4ad0, 4ad1, 4ad2, 4ad3, 4ad4, mannoside linkages internally, within the first branch of an N-gly- and 4ad5). – can chain (6 8). 1To whom correspondence may be addressed. E-mail: [email protected] or sjwill@ N-glycans are covalently bound to the side-chain nitrogen of unimelb.edu.au. asparagine (Asn) residues in the consensus Asn-Xxx-Ser/Thr (9). This article contains supporting information online at www.pnas.org/lookup/suppl/ Within the endoplasmic reticulum (ER), presynthesized 14-mer doi:10.1073/pnas.1111482109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1111482109 PNAS ∣ January 17, 2012 ∣ vol. 109 ∣ no. 3 ∣ 781–786 Downloaded by guest on October 1, 2021 lular localization of endo-α-mannosidase (14), no structures have pyranosyl-1,3-α-mannopyranosyl fluoride (Glc-ManF), was synthe- been reported and nothing is known about its catalytic mechan- sized (see SI Methods). Hydrolysis of Glc-ManF releases fluoride, ism. Endo-α-mannosidase therefore represents an important which is monitored using a fluoride ion-selective electrode. BtGH99 K k ∕K enzyme for further study, with the ultimate goal of developing shows high activity against Glc-ManF with m and cat m values approaches to treat diseases involving aberrant N-glycosylation. of 0.48 mM and 9.9 s−1 mM−1 (Fig. 1B). Although fluoride detec- We present a structural and kinetic analysis of two GH99 endo- tion is constrained to a relatively limited pH range, further obser- α-mannosidase enzymes from the enteric bacteria Bacteroides the- vation of this reaction across the pH range 5.5–7.5 revealed the taiotaomicron (BtGH99) and Bacteroides xylanisolvens (BxGH99), enzyme to be optimally active at approximately pH 7.0, consistent the first structures for any GH99 enzyme. We show that these with another bacterial homolog from Shewanella amazonensis (17). protein orthologs are endo-α-mannosidases active on glucosylated That Glc-ManF acts as a substrate although various aryl manno- N-glycans and perform catalysis with a net retention of anomeric sides do not, coupled with activity on GlcMan9GlcNAc2 and configuration. Structures of complexes obtained with two aza/imino Glc3Man9GlcNAc2, confirms BtGH99 as an endo-α-mannosidase sugar inhibitors reveal intimate details of the catalytic residues, with a requirement for a minimal α-1,3-linked disaccharide sub- allowing the proposal of a unique blueprint for catalysis and pro- strate and hence with obligate binding for catalysis in a −2 subsite viding a structural rationale for inhibition in both a mechanistic (for subsite nomenclature see ref. 18). context and ultimately for the development of therapeutic agents. Further evidence for subsite specificity and the requirement for a minimal occupancy of the −2 and −1 subsites was obtained Results through binding of two BtGH99 inhibitors. α-Glucopyranosyl-1,3- Activity and Kinetics of GH99 Endo-α-Mannosidase. B. thetaiotaomi- deoxymannojirimycin (Glc-DMJ) (Fig. 1C) is in widespread use cron and B. xylanisolvens GH99 enzymes display 42% and 41% se- as a specific endo-α-mannosidase inhibitor and was developed by quence identity, respectively, to Homo sapiens endo-α-mannosidase modification of the known exo-α-mannosidase inhibitor deoxy- (sequence alignments in Fig. S2). In order to ascertain whether mannojirimycin with an endo-α-mannosidase-targeting 1,3-linked the Bacteroides enzymes were appropriate models of the human α-glucosyl residue (19, 20). Inspired by the success of this ap- enzyme, activity on GlcMan9GlcNAc2 was studied by mass spectro- proach, a new inhibitor, α-glucosyl-1,3-isofagomine (Glc-IFG) metry. Both BtGH99 and BxGH99 catalyze the removal of a dis- (Fig. 1C), was synthesized (SI Methods) from the broad-spectrum accharide from GlcMan9GlcNAc2, with peaks observed for both azasugar inhibitor isofagomine, by introduction of a 1,3-linked reaction products, yet have no activity on the unglucosylated sub- α-glucosyl residue to capitalize upon beneficial binding interac- strate Man9GlcNAc2, indicative of conversion to Man8GlcNAc2 tions in the −2 subsite. Isothermal titration calorimetry (ITC) through release of a disaccharide, likely GlcMan (Fig. 1A). We next revealed that BtGH99 binds both Glc-DMJ and Glc-IFG, with Bt K μ D studied the GH99-catalyzed hydrolysis of a fluorescently labeled d values of 24 M and 625 nM, respectively (Fig. 2 ). The in- mammalian glycan, Glc3Man7GlcNAc2 by normal phase HPLC hibition data support the requirement for a 1,3-linked disacchar- (see SI Methods). BtGH99 shows biologically relevant activity ide occupying the −1 and −2 subsites, which is further supported K k ∕K μ against this substrate with m and cat m values of 83 Mand by analysis of crystal structures of inhibitor-enzyme complexes 2.6 s−1 mM−1, respectively (Fig. S3). These data are in good agree- (see below). K μ ment with the m value of 55 M determined for the rat liver enzyme studied by Lubas and Spiro (16). Thus, BtGH99 possesses Three-Dimensional Structures of BtGH99 and BxGH99. The crystal specific α-1,2-mannosidase activity when the −1 position manno- structure of a selenomethionine derivative of the B. thetaiotaomi- side is modified by one or more α-1,3-linked glucose residues. No cron GH99 endo-α-mannosidase enzyme was solved at a resolu- activity was observed on a range of aryl α-mannosides. To further tion of 1.70 Å (Table S1). Crystal structures of the native BtGH99 probe the activity of this enzyme, the activated substrate, α-gluco- and a second bacterial homolog BxGH99 were solved by mole-

Fig. 1. Activity, kinetics, and inhibition of B. thetaiotaomicron GH99 endo-α-mannosidase. (A) MALDI-TOF analysis of BtGH99 action on GlcMan9GlcNAc2 (m∕z 2,600.0); (Upper) without enzyme and (Lower) with BtGH99 to yield a Hex8GlcNAc2 product consistent with removal of Glc α-1,3-Man (the enzyme has no activity on Man9GlcNAc2). (B) Michaelis–Menten kinetics of BtGH99 on the bespoke substrate Glc-Man fluoride (shown in inset). (C) The endo-α-mannosidase inhibitors Glc-DMJ (synthesis in ref. 19) and Glc-IFG (synthesis in this work). (D) Isothermal titration calorimetry of Glc-IFG binding to BtGH99.

782 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1111482109 Thompson et al. Downloaded by guest on October 1, 2021 attack at the anomeric carbon of the intermediate. Kinetic analysis of three BtGH99 active-center variants, E154A, E329A, and E332A (E156, E333, and E336 in BxGH99), reveals substantial decreases in catalytic activity (Table S2). Both the E154A and E329A variants show near zero activity. E332A meanwhile shows reduced activity with observed rate constants indicating an approximate 50-fold decrease in catalytic activity with respect to wild type using the activated Glc-ManF substrate. Against the natural substrate, Glc3Man7GlcNAc2, the same mutant shows zero activity compared to wild type, under matching experimental conditions. In order to gain insight into the functions of the different components of the catalytic apparatus, 3D structures were Fig. 2. Three-dimensional structure and conservation in GH99 endo-α- determined with a bacterial endo-α-mannosidase in complex mannosidases. BxGH99 in complex with Glc-IFG and α-1,2-mannobiose as a with inhibitors and the truncated reducing-end product, α-1,2- ribbon (A) and (B) a surface representation colored by sequence conservation mannobiose. (40) using the partial GH99 alignment as shown in Fig. S2. Active Site Structure and Catalysis. The apo-BtGH99 crystal form cular replacement at resolutions of 2.00 and 1.90 Å, respectively. presented a major obstacle to the observation of enzyme-ligand Together, the different crystal forms and asymmetric unit con- complexes, owing to a large loop region (residues 64–79) project- “ ” tents yield 10 crystallographically independent views of the ing into the active-center of another molecule of BtGH99 forming structure. The three-dimensional structures of these bacterial an artefactual crystallographic dimer. Despite considerable effort, α endo- -mannosidase enzymes reveal a single-domain protein attempts to obtain a form of BtGH99 amenable to complex forma- ðβ∕αÞ adopting a 8 barrel fold with a central open cleft formed tion proved unsuccessful. A second bacterial ortholog, B. xylanisol- from extended loop regions linking the secondary structural vens α A endo- -mannosidase, was therefore selected for structural motifs comprising the catalytic active site, Fig. 2 . These loop characterization and proved amenable to complex formation, regions adopt different conformations in the various structures allowing structural determination of binary complexes with determined, and given the extended and branching nature of Glc-DMJ and Glc-IFG, and ternary complexes with each inhibitor the N-glycan substrate(s), it appears that these large flexible loops and the reducing-end product α-1,2-mannobiose (Fig. 3). are capable of forming stabilizing interactions with the substrate, α Imino and aza sugars have proved instrumental reagents in positioning the scissile -1,2-mannosidic bond so as to promote the glycosidase field (23, 24) both for structural and mechanistic catalysis within the active site. Perhaps unsurprisingly given the dissection and as templates for therapeutic agents. Deoxynojiri- unique enzymatic activity assigned to GH99, this loop-adorned mycin-type iminosugars possess a basic nitrogen in place of the barrel motif appears unique, with both bacterial GH99 orthologs endocyclic ring oxygen and are mimics of an oxocarbenium-ion exhibiting no significant structural and sequence similarity to bearing positive charge on the ring oxygen position. Isofagomine- glycoside hydrolases from other families. Analysis against known type azasugars possess a basic nitrogen at the pseudoanomeric structural motifs using the Dali server (21) reveals weak second- position, and can be considered mimics of a glycosyl cation with ary structure matches, the closest being endo-β-1,4-mannanase Trichoderma reesei charge located on the anomeric carbon. These compounds are from (Protein Data Bank ID code 1QNR, likely to prove effective inhibitors and high-affinity ligands of Z ¼ 15.9, rmsd 3.1 Å across 233 Cα positions, sequence the various GH99 endo-α-mannosidase orthologs. The complexes identity ¼ 8%). of BxGH99 with Glc-DMJ (25) and Glc-IFG unveil the ligand– The GH99 active center, located at the terminus of a solvent- protein interactions within the −1 and −2 subsites (18) (Fig. 3). accessible channel near the center of the barrel fold, possesses a cluster of carboxylate side chains likely to play a role in mannosidic bond hydrolysis. In order to help assign potential catalytic function to these residues, a time-course of BtGH99-catalyzed hydrolysis of Glc-ManF was studied by 1H NMR spectroscopy. Enzymatic hydrolysis of the α-linked C–F bond resulted in the rapid appearance of a product, shown by 2D NOESY analysis to be α-Glc-1,3-α-mannose, thus indicating that GH99-catalyzed substrate hydrolysis occurs with a net retention of anomeric configuration (Fig. S4). Over several hours, the initial product was BIOCHEMISTRY observed to undergo mutarotation, isomerizing to an equilibrium mixture of both α- and β-anomers at the reducing-end mannose. Classical retaining glycoside hydrolases operate through a two- step, double displacement reaction in which a covalent intermediate is formed and then hydrolyzed, via oxocarbenium-ion-like transition states (glycosidase mechanisms recently reviewed in ref. 22). In most cases, this displacement reaction is achieved by an enzymatic nucleophile such as a carboxylate (aspartate or CHEMISTRY glutamate) or, as for certain sialidases, a tyrosine residue. In some 2-acetamidoglycosidases (including families GH18, 20, 25, 56, 84, and 85), the nucleophile is the 2-acetamido group of the substrate, which acts in a “neighboring group” participation reaction. For GH99, we initially assumed that the catalytic apparatus of the Fig. 3. Electron density and ligand binding to GH99 endo-α-mannosidase. enzyme comprised a catalytic nucleophile acting to form the A–C represent binding of (A) Glc-DMJ, (B) Glc-IFG, (C) Glc-DMJ/α-1,2- covalent glycosyl-enzyme intermediate and a catalytic acid/base, mannobiose (in divergent stereo). Figures shown are REFMAC maximum- σ 2F − F first functioning as a general acid to assist leaving group depar- likelihood/ A weighted o c syntheses contoured between 0.26 and 3 ture and then as a general base to activate water for nucleophilic 0.32 electrons per Å .

Thompson et al. PNAS ∣ January 17, 2012 ∣ vol. 109 ∣ no. 3 ∣ 783 Downloaded by guest on October 1, 2021 Both inhibitors bind to the enzyme active site with the −1 ring Isofagomine-type azasugar inhibitors are useful crystallo- located deep within a substrate-binding pocket in an undistorted graphic probes that typically allow direct observation of the en- 4 C1 conformation. The −2 glucosyl residue projects from the zymatic nucleophile of retaining glycoside hydrolases. Upon catalytic cavity and appears solvent accessible. These subsites lie protonation, the pseudoanomeric nitrogen forms a salt bridge in the center of a substrate-binding cleft with a length of approxi- with the negatively charged nucleophile, acting as an “ionic trap” mately 40 Å. The solvent-exposed nature of the −2 subsite gluco- for the nucleophile, which during the reaction coordinate must side allows the accommodation of elongated substrates with approach within bonding distance of the anomeric carbon (23). additional glucose residues at the 3 position. Aromatic platforms All X-ray structures of retaining glycoside hydrolases with isofa- are common binding elements of carbohydrate active enzymes gomine-type compounds have, without exception, revealed the (26) and in these distal negative subsites are provided by a pair catalytic nucleophile to be located within 3 Å of the azasugar of highly conserved tryptophans at positions 48 and 126. Se- nitrogen, whereas for inverting enzymes, the nucleophilic water quence alignments reveal a high degree of sequence conservation is found less than 3 Å away from nitrogen (Table S3). Of parti- across various species in the region comprising the substrate-bind- cular interest is the absence, in the GH99 orthologs, of any en- ing cleft (Fig. 2B). Indeed, all the interactions in the −2 to þ2 sub- zymatic carboxylate at a position suitable to act as the enzymatic sites appear to be essentially invariant (Figs. S2 (alignment) and nucleophile in a conventional double displacement reaction. S5 (interactions)). That the glucoside moiety interacts with resi- Indeed, without invoking a conformational change, or reposition- ing of the −1 sugar residue, the BxGH99 complex structures show dues in the −2 subsite, and also with the putative catalytic appa- no protein residue in a position to act as a potential nucleophile; ratus, provides a structural rationale for the catalytic requirement the most closely located candidates for a classical double displa- of a sugar occupying the −2 subsite of endo-α-mannosidase. cement mechanism (shown in Fig. 4A) include the OE1 atom of Aza and imino sugar inhibitors have been used to identify the Glu333 (approximately 3.5 Å distant) or the OH of Tyr46 or catalytic apparatus of glycosidases by virtue of the interactions Tyr252 (4.0 Å distant). However, none of these residues appear of potential nucleophiles with the protonated nitrogen atoms Bx close enough to interact with the inhibitor nitrogens at either ring (25, 26). GH99 structures in complex with Glc-DMJ and position in the complexes (Movie S1). Furthermore, the observa- Glc-IFG reveal a water molecule, coordinated by Glu336 (332 in tion of identical (and static) arrangements of the active site re- Bt GH99) and poised below the pseudoanomeric nitrogen of sidues in three crystal forms of two enzymes with a total of 10 Glc-IFG and the C1 of the DMJ moiety of Glc-DMJ, in the ideal different packing arrangements provides no evidence of intrinsic position for nucleophilic attack to complete the second hydrolysis conformational flexibility within the active site. Provocatively, it is step of a classical double displacement retention mechanism. well known that the alkaline solvolysis of α-mannosyl fluoride Glu336 is situated on the “α-face” of the sugar occupying the −1 proceeds through a mechanism involving neighboring group subsite, in a position where it might interact with the glycosidic participation in which the 2-OH attacks the anomeric position oxygen, thus fulfilling a role strongly indicative of the catalytic resulting in the formation of the intermediate 1,2-anhydro-β- acid/base in the enzyme mechanism. Consistent with such a mannopyranose. This intermediate is opened by a nucleophile to function, the BtGH99 variant E332A retains approximately 2% ac- afford an α-configured product possessing a retained anomeric tivity, compared to WT, on the activated fluoride substrate but has configuration (27). Although no evidence currently supports the no detectable activity on the natural GlcMan9GlcNAc2 glycan (de- existence of a 1,2-anhydro sugar as an intermediate for glycosi- termined by mass spectrometry) or against Glc3Man7GlcNAc2 dase-catalyzed hydrolysis, it is possible that such a mechanism (determined by HPLC). may occur with endo-α-mannosidase (Fig. 4B). Accordingly, the A enzyme enzyme HO OH Nu HO OH enzyme HO OH Nu HO O HO O HO O O O Nu O HO HO HO HO OR HO HO O H OH HO O ROH HO O HO O O H O O H HO HO H HO Glu336 Glu336 Glu336 O O O

O B O O Glu333 Glu333 Glu333 O H O H O HO HO H O HO O O HO O HO O O O HO O HO HO O HO HO OR HO HO HO O ROH HO O OH O H O H O HO O HO HO O H Glu Glu H HO 336 336 Glu O O 336 O Fig. 4. Putative catalytic mechanisms for GH99 endo-α-mannosidase. (A) Classical two-step double displacement mechanism proceeding via a glycosyl-enzyme intermediate; in the case of the structures reported here, this would require conformational change. (B) Neighboring group participation via a 1,2-anhydro sugar intermediate.

784 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1111482109 Thompson et al. Downloaded by guest on October 1, 2021 kinetically essential and conserved active-center residue Glu333, 1,3-deoxymannonojirimycin, the epimer of Glc-DMJ, is an effec- poised 2.6 Å away from the O2 group of the substrate, would tive inhibitor (20). deprotonate the 2-OH, promoting its nucleophilic attack on C1. Mammalian endo-α-mannosidase has a preference for mono- The anti-arrangement of O2 and the leaving group seen in the glucosylated N-glycans and its activity increases as the number complex with Glc-DMJ is that stereoelectronically required for of mannose residues on the 6′-mannose branch decreases (16). epoxide formation, which requires a linear transition state of The structures of the bacterial orthologs suggest that the majority the attacking nucleophile, the reactive carbon center, and the of substrate specificity is for the −1 and −2 subsites with addi- leaving group. Neighboring group participation by the 2-acetami- tional sugars off the 3-position of the −2 glucose residue likely do group of N-acetyl-β-glycosides is now well established in to project into solvent and not to be involved in significant en- families GH18, 20, 25, 56, 84, and 85 (28). Togetherthe precedent zyme interactions. α-Mannosidases must overcome certain sub- for neighboring group participation in these glycoside hydrolase strate-specific challenges to achieve effective catalysis because families, the lack of an apparent enzymatic nucleophile in the the enhanced anomeric effect in mannose deters substrate distor- GH99 structures, and the established neighboring group partici- tion, and thus an attacking nucleophile suffers destabilizing 1,2- pation mechanism for nonenzymatic hydrolysis of α-mannosyl diaxial interactions with the axial 2-OH group. In retaining GH38 fluoride, provides an enticing precedent to suggest this unortho- (32), and inverting GH47 (33) GH92 (31) α-mannosidases, a dox enzymatic mechanism. divalent metal ion (Zn2þ for GH38, Ca2þ for GH47 and GH92) In order to dissect the leaving-group (“positive” numbers; is found to coordinate the 2- and 3-hydroxyls, possibly assisting in ref. 18) subsites of the GH99 endo-α-mannosidase, additional crys- catalysis through substrate distortion and in delivery of the nu- tallization experiments were conducted with either Glc-DMJ or cleophile (31). By contrast, in the inverting GH125 enzymes Glc-IFG together with α-1,2-mannobiose. Such conditions afford no metal ion is observed in any structures and no substrate dis- complexes with the inhibitors in the −1 and −2 subsites, and with tortion was seen in a pseudo-Michaelis complex with an S-linked the mannobiose disaccharide in the þ1 and þ2 subsites. As with disaccharide spanning the active site (34). It is unclear how this the −2 moiety, the þ2 mannose residue appears solvent accessible, family overcomes the destabilizing diaxial interaction of the at- projecting outside the active site cavity at a distance of approxi- tacking water nucleophile, although it is possible that a favorable mately 7 Å from α-1,3-glucose, consistent with an ability to accom- hydrogen bond between the 2-OH of the sugar and the nucleo- modate the remaining reducing end residues of a complex philic water may assist in nucleophilic attack in a similar way to N-glycan. Phenylalanines at positions 253 and 258 provide an the divalent metals in the GH38, GH47, and GH92 enzymes. A aromatic platform positioning the þ1 mannoside, with Tyr289 notable feature of the GH99 active site observed in both bacterial and Asn298 making hydrogen-bonding interactions with the O3 endo-α-mannosidase orthologs is the absence of a metal ion or and O4 positions of the same sugar. Likewise within the þ2 subsite, indeed of a site for possible metal ion coordination. The en- hydrogen bonding occurs between O3 and O4 and Tyr195, Asp196, zyme-inhibitor complexes with Glc-DMJ and Glc-IFG deter- and Tyr198. Despite the sugar residue occupying the þ1 subsite mined for BxGH99 reveal no obvious substrate distortion in the appearing to be slightly displaced from its likely true position dur- −1 subsite and, despite the presence of an ionic trap in Glc-IFG, ing hydrolytic cleavage of the authentic substrate (O2 of the þ1 no enzymatic nucleophile is apparent. However, a widely con- sugar lies approximately 4 Å from the atom equivalent to the served residue, Glu333, is found in close proximity to O2, and anomeric C1 of the residue in the −1 subsite in both inhibitor com- its mutagenesis results in a near-complete decrease in catalytic plexes), both structures show the carboxylate group of the likely activity. Taken together, these observations suggest that the catalytic acid/base, Glu336, to be in close proximity to the position hydrolysis reaction catalyzed by the GH99 endo-α-mannosidase of the scissile α-1,2-mannosidic bond. family proceeds via both a metal-independent and possibly a uniquely unorthodox mechanism. Discussion Inhibitors that intervene in early steps of N-glycan biosynthesis Endo-α-mannosidase is a resident of the cis/medial compartment have been largely ineffective in suppressing N-linked glycan of the Golgi apparatus and pre-Golgi intermediates, and is a mem- processing. Cells in which glucosidase II has been inhibited or brane-associated protein that is difficult to recombinantly express subjected to genetic knockout retain 40–80% normal N-glycan in soluble form (29). The bacterial orthologs from B. thetaiotaomi- processing function through the intervention of endo-α-manno- cron and B. xylanisolvens are soluble proteins and may have been sidase (8, 35, 36). During a castanospermine (CST)-imposed acquired by horizontal gene transfer because these organisms are α-glucosidase I and II blockade, HepG2 cells produced N-linked common and beneficial components of the human gut (30). It has glycoproteins with the normal glycan structure and resulted been suggested that, under normal conditions, endo-α-mannosi- in concomitant release of free Glc1–3Man oligosaccharides (6). dase acts to deglucosylate folded Glc1Man7–9 glycoproteins that Evidence for the basal action of endo-α-mannosidase in the ab- may reach the Golgi apparatus through being poor substrates sence of glucosidase inhibition or knockout was demonstrated BIOCHEMISTRY for ER α-glucosidase II (7). The biological role of the bacterial through the use of the mannosidase I inhibitor, DMJ, which pre- enzymes is unclear, but may include, as is the case for the exo- vents further processing of the deglucosylated N-linked oligosac- α-mannosidases of family GH92 (31), mannose foraging for basic charides. Man6–8GlcNAc, but not Man9GlcNAc, structures were metabolic inputs. Just as incomplete deglucosylation in the mam- identified, with the Man8GlcNAc structure being the character- malian cell prevents the action of exo-α-mannosidases, a similar istic isomer generated by endo-α-mannosidase cleavage (6). problem with the bacterial exo-α-mannosidases might have led Coadministration of the α-glucosidase I and II inhibitor CSTand to the beneficial acquisition of the enzyme by the bacteria. It is the selective endo-α-mannosidase inhibitor Glc-DMJ resulted in CHEMISTRY notable that whereas the genomes of B. thetaiotaomicron and B. inhibition of biosynthesis of Glc3Man and N-linked glycoproteins xylanisolvens possess many copies of the N-glycan active exo-α- (25). On the other hand, Golgi α-mannosidase II is seen as a mannosidases (24 and 17 GH92 enzymes, respectively; 3 GH125 promising target for therapeutic intervention, as this enzyme acts enzymes each) each genome contains only a single copy of a GH99 after endo-α-mannosidase rejoins the normal pathway and so acts enzyme, which supports its role in relieving a metabolic bottleneck on all N-linked glycans undergoing processing (25). Swainsonine, in the degradation of glucosylated N-glycans. Alternatively, or a selective inhibitor of Golgi α-mannosidase II, blocks the abnor- additionally, the bacterial endo-α-mannosidase may act on yeast mal formation of complex oligosaccharides, leading to reduced mannans, which contain the epimeric α-mannosyl-1,3-mannose metastasis and tumor growth (37). However, swainsonine also substructure. In this regard, it is interesting that studies of mam- inhibits the closely related lysosomal α-mannosidase, limiting its malian endo-α-mannosidase have indeed shown that α-mannosyl- clinical use (38). Combination therapies of glucosidase I/II inhi-

Thompson et al. PNAS ∣ January 17, 2012 ∣ vol. 109 ∣ no. 3 ∣ 785 Downloaded by guest on October 1, 2021 bitors (e.g., 6-O-butanoyl-CST) and endo-α-mannosidase inhibi- Activity, Kinetics and Stereochemistry. GH99 activity on GlcMan9GlcNAc2 was tors may provide a viable alternative approach. The development studied by MALDI-TOF mass spectrometry of the permethylated products fol- of Glc-IFG as a more potent inhibitor of the bacterial endo-α- lowing overnight incubation at 37 °C (see SI Methods). The ligand affinity of BtGH99 for Glc-DMJ and Glc-IFG was analyzed by ITC using an iTC200 calori- mannosidase serves as a valuable precedent for glucosylation meter (MicroCal). Assays were carried out at 25 °C with Glc-DMJ (6.4 mM) and of inhibitors of exo-α-mannosidases to search for more active in- Glc-IFG (3.0 mM) titrated into the ITC cell containing 460 and 370 μM BtGH99, K hibitors of endo-α-mannosidase, which will be assisted by the respectively. The dissociation constant for each reaction ( d) was then calcu- structural and mechanistic blueprint provided by this work. lated using the Origin 7 software package (MicroCal). Kinetic parameters for the hydrolysis of the synthetic substrate α-glucopyranosyl-1,3-α-mannopyra- Methods nosyl fluoride (Glc-ManF) were determined using a fluoride-selective elec- Gene Cloning, Mutagenesis and Protein Production. The gene encoding trode with NMR analysis used to determine reaction stereochemistry (see BtGH99 was amplified from genomic DNA by PCR and ligated into a modified SI Methods). pET28a vector encoding an N-terminal His6-tag (pETLIC-BtGH99). Site-direc- ted mutagenesis was carried out using the QuikChange mutagenesis kit (Stra- Crystallization, Data Collection, and Structure Solution. The BtGH99 structure was solved using single wavelength anomalous dispersion techniques using tagene). The gene encoding BxGH99 was constructed from synthesized the selenomethionyl protein with data collected at beamline I24 of the Dia- oligonucleotide fragments (Genscipt, Inc.) and also subcloned into pET28a. mond Light Source. Other structures were solved by molecular replacement Protein production and purification was identical for both Bt- and BxGH99 with data collected on beamlines ID23-2 and ID14-1, respectively, of the Eur- enzymes, including all respective variants. Escherichia coli BL21 (DE3) cells opean Synchrotron Radiation Facility, and at beamlines I04-1 and I03 of the harboring the GH99-encoding plasmid were cultured in 0.5 L ZYM-5052 auto- Diamond Light Source. Full details of crystallization, data collection, and −1 induction media (39) supplemented with 50 μgmL kanamycin at 37 °C for structure solution, including programs used, are given in the SI Methods. 8 h, with induction occurring overnight at 16 °C. Cells were harvested and resuspended in 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, and lysed by sonica- ACKNOWLEDGMENTS. G.J.D. thanks the Biotechnology and Biological Sciences tion. Soluble lysate was applied to a NiSO4-charged 5 mL HiTrap chelating Research Council for funding and is a Royal Society/Wolfson Research Merit column (GE Healthcare), preequilibrated in the same buffer. The protein award recipient. T.M.G. is a Sir Henry Wellcome Fellowship recipient. was eluted in an imidazole gradient, dialyzed, concentrated, and further pur- S.J.W. thanks the Australian Research Council and the School of Chemistry, ified on an S75 16∕60 gel filtration column (GE) preequilibrated in 25 mM University of Melbourne, for funding support. T.W. thanks the Netherlands Organisation for Scientific Research for funding support. The York Center of Hepes, pH 8.0, 50 mM NaCl. The BtGH99 selenomethionine derivative was Excellence in Mass Spectrometry was created thanks to a major capital invest- overexpressed in PASM-5052 media (39), otherwise all isolation and purifica- ment through Science City York, supported by Yorkshire Forward with funds tion steps were as described above. from the Northern Way Initiative.

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