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Substrate Recognition and Catalysis by GH47 Α-Mannosidases Involved In Substrate recognition and catalysis by GH47 PNAS PLUS α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway Yong Xianga,b,1, Khanita Karavega,b,1,2, and Kelley W. Moremena,b,3 aComplex Carbohydrate Research Center, University of Georgia, Athens, GA 30602; and bDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602 Edited by Armando Parodi, Fundacion Instituto Leloir, Buenos Aries, Argentina, and approved October 13, 2016 (received for review July 8, 2016) Maturation of Asn-linked oligosaccharides in the eukaryotic secre- cluding ER α1,2-mannosidase I (ERManI), three Golgi α1,2- tory pathway requires the trimming of nascent glycan chains to mannosidases (GMIA, GMIB, and GMIC), which play important remove all glucose and several mannose residues before extension roles in N-glycan trimming in the ER and Golgi complex, and into complex-type structures on the cell surface and secreted three ER degradation-enhancing α-mannosidase-like (EDEM) glycoproteins. Multiple glycoside hydrolase family 47 (GH47) proteins (EDEM1, EDEM2, and EDEM3) that play roles in α-mannosidases, including endoplasmic reticulum (ER) α-mannosi- ERAD (3, 6). In classical models for mammalian N-glycan mat- α dase I (ERManI) and Golgi -mannosidase IA (GMIA), are responsi- uration, Man9GlcNAc2 structures are acted upon first by ERManI ble for cleavage of terminal α1,2-linked mannose residues to tocleaveasingleα1,2-mannose residue (M10) from the glycan B produce uniquely trimmed oligomannose isomers that are neces- branch to generate a Man8GlcNAc2 B isomer (Man8B) (Fig. 1) sary for ER glycoprotein quality control and glycan maturation. (6, 9, 10). This cleavage has been proposed to contribute to the ERManI and GMIA have similar catalytic domain structures, but mannose-timer mechanism for ER quality control and ERAD (1, each enzyme cleaves distinct residues from tribranched oligoman- 3, 5). Following completion of protein folding and release from nose glycan substrates. The structural basis for branch-specific ER chaperones, N-glycoproteins with Man8B isomer glycans are cleavage by ERManI and GMIA was explored by replacing an es- α BIOCHEMISTRY 2+ 3+ transported to the Golgi complex where the remaining three 1,2- sential enzyme-bound Ca ion with a lanthanum (La )ion.This mannosyl residues are cleaved by GMIA, GMIB, or GMIC to ion swap led to enzyme inactivation while retaining high-affinity C – 3+ generate Man5GlcNAc2 (Fig. 1 ) (11 13). In vitro, ERManI and substrate interactions. Cocrystallization of La -bound enzymes the Golgi α-mannosidases exhibit complementary and nonover- with Man GlcNAc substrate analogs revealed enzyme–substrate 9 2 lapping specificities for cleavage of Man GlcNAc substrates. complexes with distinct modes of glycan branch insertion into 9 2 ERManI rapidly cleaves the single M10 (branch B) residue (9, the respective enzyme active-site clefts. Both enzymes had glycan 14) but cleaves the remaining mannose residues on other interactions that extended across the entire glycan structure, but branches with greatly reduced efficiency (Fig. 1D). In contrast, each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme Significance specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential gly- Asn-linked glycosylation of newly synthesized polypeptides can cleavage events. The data also indicate that full steric access to occurs in the endoplasmic reticulum of eukaryotic cells. Glycan glycan substrates determines the efficiency of mannose-trimming structures are trimmed and remodeled as they transit the se- reactions that control the conversion to complex-type structures in cretory pathway, and processing intermediates play various mammalian cells. roles as ligands for folding chaperones and signals for quality control and intracellular transport. Key steps for the genera- Asn-linked glycan processing | α-mannosidase | glycosidase mechanism | tion of these trimmed intermediates are catalyzed by glycoside glycoside hydrolase | bioinorganic chemistry hydrolase family 47 (GH47) α-mannosidases that selectively cleave α1,2-linked mannose residues. Despite the sequence and ammalian Asn-linked glycoproteins are initially synthe- structural similarities among the GH47 enzymes, the molecular sized by cotranslational transfer of a Glc Man GlcNAc basis for residue-specific cleavage remains obscure. The pre- M 3 9 2 – glycan precursor to nascent polypeptide chains on the luminal sent studies reveal enzyme substrate complex structures for α face of the endoplasmic reticulum (ER) (1). During transit two related GH47 -mannosidases and provide insights into through the secretory pathway, all three glucose residues and six how these enzymes recognize the same substrates differently of nine mannose residues are trimmed, and the structures are and catalyze the complementary glycan trimming reactions extended further by Golgi glycosyltransferases to generate the necessary for glycan maturation. diverse collection of complex-type glycans found on cell-surface Author contributions: Y.X., K.K., and K.W.M. designed research; Y.X. and K.K. performed and secreted glycoproteins (2). The transiently formed glycan- research; Y.X., K.K., and K.W.M. analyzed data; and Y.X., K.K., and K.W.M. wrote processing intermediates resulting from glucose and mannose the paper. cleavage also play critical roles in the early secretory pathway, The authors declare no conflict of interest. including acting as ligands for ER chaperones, signals for ER This article is a PNAS Direct Submission. quality control and ER-associated degradation (ERAD), and – Data deposition: Crystallography, atomic coordinates, and structure factors reported in targeting signals during intracellular transport (3 6). this paper have been deposited in the Protein Data Bank database (ID codes 5KK7, 5KIJ, Key steps in the generation of the trimmed oligomannose and 5KKB). glycans are catalyzed by a collection of α-mannosidases, termed 1Y.X. and K.K. contributed equally to this work. “class 1” or “Carbohydrate Active Enzymes Database (CAZy) 2Present address: Avitide, Inc., Lebanon, NH 03766. ” α glycoside hydrolase family 47 (GH47) -mannosidases (7, 8), that 3To whom correspondence should be addressed. Email: [email protected]. α selectively cleave 1,2-mannose residues from the Man9GlcNAc2 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. precursor. In mammals, there are seven GH47 members (6), in- 1073/pnas.1611213113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1611213113 PNAS Early Edition | 1of10 Downloaded by guest on September 24, 2021 residue within the catalytic −1 subsite (where bond cleavage A B occurs between the terminal catalytic −1 subsite and the sub- Branch terminal +1 subsites) (Fig. S1B). Previous studies characterized − C B A interactions with glycone-mimicking inhibitors within the 1 Asn Asn Asn subsite (14, 16–19) and a thiodisaccharide substrate analog that M9 M10 M11 Man8A Man8B Man8C bridged the −1 and the adjoining +1 subsites (14, 19) through a 1,2 1,2 1,2 Man8GlcNAc2 isomers highly conserved collection of hydrophobic and H-bonding in- 2+ M6 M7 M8 C In vivo teractions as well as direct coordination with the Ca ion. − + 1,2 In contrast to the highly conserved 1 and 1 subsites, the 1,6 1,3 topologies of the broad clefts (≥+1 subsites) leading to the ac- M4 M5 Fast Fast tive-site cores are distinct among the GH47 family members (10, 1,6 1,3 16–20); these distinct topologies have been proposed to account M3 ER ManI GMIA/GMIB/GMIC for the differences in branch specificities (20). For example, 1,4 D In vitro by ERManI protein–glycan complexes were obtained for both mouse GMIA NAG2 Slow (20) and the yeast ERManI (10), in which the equivalent of ≥+ Fast Man5GlcNAc2 enzymatic products occupied the 1 subsites as 1,4 − NAG1 ligands, but the 1 glycone subsites remained unoccupied. The orientations of the resulting glycans were distinct, and different Man8B (100%) Asn branch termini were docked in the respective active-site clefts. E In vitro by However, these structures did not reveal interactions with the intact Man GlcNAc substrate or the determinants of initial Man9GlcNAc2 GMIA 9 2 Fast glycan branch specificity. A challenge for obtaining structural insights into GH47 Man8A (90%) α-mannosidase glycan cleavage intermediates has been the pro- Legend! gressive digestion of glycan substrates by even partially inacti- Fast Slow Mannose Fast vated mutant enzymes (21). As an alternative approach for enzyme inactivation, we examined the role of the enzyme-bound + N-acetylglucosamine Ca2 ion in catalysis. The role of ion coordination was probed by Man8C (10%) determining the structure of a mutant form of ERManI + (T688A), which reduced the Ca2 ion coordination number and Fig. 1. Diagrams of glycan cleavage specificity for ERManI and GMIA in vivo compromised catalysis while partially enhancing substrate-bind- and in vitro. (A) Schematic diagram of the branched Man9GlcNAc2 Asn- 2+ linked glycan including the residue nomenclature and glycosidic linkages for ing affinity. We further tested replacement of the Ca ion with mannose (M) and N-acetylglucosamine (NAG) residues as indicated by the other ions that could potentially eliminate enzymatic activity but symbol nomenclature shown in the legend.
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