Structural and Mechanistic Insight Into N-Glycan Processing by Endo-Α-Mannosidase
Total Page：16
File Type：pdf, Size：1020Kb
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.