Endoplasmic reticulum (ER) class I ␣1,2-mannosidase (also known as ER ␣-mannosidase I) is a critical enzyme in the maturation of N-linked oligosaccharides and ERassociated degradation. Trimming of a single mannose residue acts as a signal to target misfolded glycoproteins for degradation by the proteasome. Crystal structures of the catalytic domain of human ER class I ␣1,2-mannosidase have been determined both in the presence and absence of the potent inhibitors kifunensine and 1-deoxymannojirimycin. Both inhibitors bind to the protein at the bottom of the active-site cavity, with the essential calcium ion coordinating the O-2 and O-3 hydroxyls and stabilizing the six-membered rings of both inhibitors in a 1 C 4 conformation. This is the first direct evidence of the role of the calcium ion. The lack of major conformational changes upon inhibitor binding and structural comparisons with the yeast ␣1,2-mannosidase enzyme-product complex suggest that this class of inverting enzymes has a novel catalytic mechanism. The structures also provide insight into the specificity of this class of enzymes and provide a blueprint for the future design of novel inhibitors that prevent degradation of misfolded proteins in genetic diseases. Endoplasmic reticulum (ER)1 class I ␣1,2-mannosidase (also known as ER ␣-mannosidase I and Man 9 GlcNAc 2 -specific processing ␣-mannosidase, EC 3.2.1.113) is a key enzyme in the maturation of N-linked oligosaccharides in mammalian cells (for reviews, see Refs.

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In the endoplasmic reticulum (ER), misfolded proteins are retrotranslocated to the cytosol and degraded by the proteasome in a process known as ER-associated degradation (ERAD). Early in this pathway, a proposed lumenal ER lectin, EDEM, recognizes misfolded glycoproteins in the ER, disengages the nascent molecules from the folding pathway, and facilitates their targeting for disposal. In humans there are a total of three EDEM homologs. The amino acid sequences of these proteins are different from other lectins but are closely related to the Class I mannosidases (family 47 glycosidases). In this study, we characterize one of the EDEM homologs from Homo sapiens, which we have termed EDEM2 (C20orf31). Using recombinantly generated EDEM2, no alpha-1,2 mannosidase activity was observed. In HEK293 cells, recombinant EDEM2 is localized to the ER where it can associate with misfolded alpha1-antitrypsin. Overexpression of EDEM2 accelerates the degradation of misfolded alpha1-antitrypsin, indicating that the protein is involved in ERAD.

We have isolated a full-length cDNA clone encoding a human ␣1,2-mannosidase that catalyzes the first mannose trimming step in the processing of mammalian Asnlinked oligosaccharides. This enzyme has been proposed to regulate the timing of quality control glycoprotein degradation in the endoplasmic reticulum (ER) of eukaryotic cells. Human expressed sequence tag clones were identified by sequence similarity to mammalian and yeast oligosaccharide-processing mannosidases, and the full-length coding region of the putative mannosidase homolog was isolated by a combination of 5-rapid amplification of cDNA ends and direct polymerase chain reaction from human placental cDNA. The open reading frame predicted a 663-amino acid type II transmembrane polypeptide with a short cytoplasmic tail (47 amino acids), a single transmembrane domain (22 amino acids), and a large COOH-terminal catalytic domain (594 amino acids). Northern blots detected a transcript of ϳ2.8 kilobase pairs that was ubiquitously expressed in human tissues. Expression of an epitope-tagged fulllength form of the human mannosidase homolog in normal rat kidney cells resulted in an ER pattern of localization. When a recombinant protein, consisting of protein A fused to the COOH-terminal luminal domain of the human mannosidase homolog, was expressed in COS cells, the fusion protein was found to cleave only a single ␣1,2-mannose residue from Man 9 GlcNAc 2 to produce a unique Man 8 GlcNAc 2 isomer (Man8B). The mannose cleavage reaction required divalent cations as indicated by inhibition with EDTA or EGTA and reversal of the inhibition by the addition of Ca 2؉ . The enzyme was also sensitive to inhibition by deoxymannojirimycin and kifunensine, but not swainsonine. The results on the localization, substrate specificity, and inhibitor profiles indicate that the cDNA reported here encodes an enzyme previously designated ER mannosidase I. Enzyme reactions using a combination of human ER mannosidase I and recombinant Golgi mannosidase IA indicated that that these two enzymes are complementary in their cleavage of Man 9 GlcNAc 2 oligosaccharides to Man 5 GlcNAc 2 .The maturation of Asn-linked oligosaccharides in mammalian cells is initiated in the endoplasmic reticulum (ER) 1 through the cleavage of three glucose residues and as many as two mannose residues soon after the Glc 3 Man 9 GlcNAc 2 oligosaccharide is transferred to the nascent polypeptide chain (1, 2). For glycoproteins that are destined for secretion or transport to other intracellular compartments, additional mannose trimming occurs in the Golgi complex through the action of members of a multigene family of mannosidases that remove the remaining ␣1,2-mannose residues to yield a Man 5 GlcNAc 2 structure (2). Further maturation by the action of GlcNAc transferase I, Golgi mannosidase II, the collection of branching GlcNAc transferases, and additional glycosyltransferases results in the array of complex oligosaccharides that are found on cellular and secreted glycoproteins (1).The initial stages of mannose tr...

Three subfamilies of mammalian Class 1 processing ␣1,2-mannosidases (family 47 glycosidases) play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally unfolded proteins by ER-associated degradation. In an effort to define the structural basis for substrate recognition among Class 1 mannosidases, we have crystallized murine Golgi mannosidase IA (space group P2 1 2 1 2 1 ), and the structure was solved to 1.5-Å resolution by molecular replacement. The enzyme assumes an (␣␣) 7 barrel structure with a Ca 2؉ ion coordinated at the base of the barrel similar to other Class 1 mannosidases. Critical residues within the barrel structure that coordinate the Ca 2؉ ion or presumably bind and catalyze the hydrolysis of the glycone are also highly conserved. A Man 6 GlcNAc 2 oligosaccharide attached to Asn 515 in the murine enzyme was found to extend into the active site of an adjoining protein unit in the crystal lattice in a presumed enzyme-product complex. In contrast to an analogous complex previously isolated for Saccharomyces cerevisiae ER mannosidase I, the oligosaccharide in the active site of the murine Golgi enzyme assumes a different conformation to present an alternate oligosaccharide branch into the active site pocket. A comparison of the observed protein-carbohydrate interactions for the murine Golgi enzyme with the binding cleft topologies of the other family 47 glycosidases provides a framework for understanding the structural basis for substrate recognition among this class of enzymes.