Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing ␣-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I ␣-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded ␣1-antitrypsin variant (null (Hong Kong)) and of TCR␣. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded ␣1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent ␣1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of ␣1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has ␣1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.ER 3 quality control is an elaborate mechanism conserved from yeast to mammals, ensuring that newly synthesized proteins in the ER fold and assemble correctly and that only proteins that acquire their correct conformations are sorted further into the secretory pathway (1-4). During this process, proteins that fail to attain their native conformation due to mutations of the polypeptides or to ER stress conditions adverse for protein folding as well as orphan subunits are degraded in a process known as ER-associated degradation (ERAD) (3, 5-7). The recognition of misfolded proteins for ERAD is still poorly understood, but there is increasing evidence for a role of mannose trimming in the targeting of glycoproteins for ERAD (8, 9). In mammalian cells, overexpression of ER ␣-mannosidase I stimulates ERAD of misfolded glycoproteins (10, 11), whereas the ␣1,2-mannosidase inhibitors kifunensine and 1-deoxymannojirimycin stabilize misfolded glycoproteins (12-16). These observations suggested that Man 8 GlcNAc 2 isomer B, the major product of the ER ␣1,2-mannosidase, is a recognition marker for ERAD of glycoproteins, but this view is being challenged, since there is increasing evidence that trimming to smaller oligosaccharides occurs on ERAD substrates (10,(17)(18)(19). We previously cloned mouse EDEM (ER degradation enhancing ␣-mannosidase-like protein) as a cDNA whose expression is up-regulated by ER stress and showed that EDEM accelerates glycoprotein ERAD (20). EDEM is an integral ER membrane protein that has all the signature motifs of Class I ...

Mannose trimming is not only essential for N-glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins. The crystal structure of the class I α1,2-mannosidase that trims Man 9 GlcNAc 2 to Man 8 GlcNAc 2 isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (αα) 7 -barrel in which an N-glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion. The observed protein-carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases.

Glycoprotein folding and degradation in the endoplasmic reticulum (ER) is mediated by the ER quality control system. Mannose trimming plays an important role by forming specific N-glycans that permit the recognition and sorting of terminally misfolded conformers for ERAD (ER-associated degradation). The EDEM (ER degradation enhancing alpha-mannosidase-like protein) subgroup of proteins belonging to the Class I alpha1,2-mannosidase family (glycosylhydrolase family 47) has been shown to enhance ERAD. We recently reported that overexpression of EDEM3 enhances glycoprotein ERAD with a concomitant increase in mannose-trimming activity in vivo. Herein, we report that overexpression of EDEM1 produces Glc(1)Man(8)GlcNAc(2) isomer C on terminally misfolded null Hong Kong alpha1-antitrypsin (NHK) in vivo. Levels of this isomer increased throughout the chase period and comprised approximately 10% of the [(3)H]mannose-labeled N-glycans on NHK after a 3-h chase. Furthermore, overexpression of EDEM1 E220Q containing a mutation in a conserved catalytic residue essential for alpha1,2-mannosidase activity did not yield detectable levels of Glc(1)Man(8)GlcNAc(2) isomer C. Yet, the same extent of NHK ERAD-enhancement was observed in both EDEM1 and EDEM1 E220Q overexpressing cells. This can be attributed to both wild-type and mutant EDEM1 inhibiting aberrant NHK dimer formation. We further analyzed the N-glycan profile of total cellular glycoproteins from HepG2 cells stably overexpressing EDEM1 and found that the relative amount of Man(7)GlcNAc(2) isomer A, which lacks the terminal B and C branch mannoses, was increased compared to parental HepG2 cells. Based on this observation, we conclude that EDEM1 activity trims mannose from the C branch of N-glycans in vivo.

Kre2p/Mnt1p is a Golgi ␣1,2-mannosyltransferase involved in the biosynthesis of Saccharomyces cerevisiae cell wall glycoproteins. The protein belongs to glycosyltransferase family 15, a member of which has been implicated in virulence of Candida albicans. We present the 2.0 Å crystal structures of the catalytic domain of Kre2p/Mnt1p and its binary and ternary complexes with GDP/Mn 2؉ and GDP/Mn 2؉ /acceptor methyl-␣-mannoside. The protein has a mixed ␣/␤ fold similar to the glycosyltransferase-A (GT-A) fold. Although the GDPmannose donor was used in the crystallization experiments and the GDP moiety is bound tightly to the active site, the mannose is not visible in the electron density. The manganese is coordinated by a modified DXD motif (EPD), with only the first glutamate involved in a direct interaction. The position of the donor mannose was modeled using the binary and ternary complexes of other GT-A enzymes. The C1؆ of the modeled donor mannose is within hydrogen-bonding distance of both the hydroxyl of Tyr 220 and the O2 of the acceptor mannose. The O2 of the acceptor mannose is also within hydrogen bond distance of the hydroxyl of Tyr 220 . The structures, modeling, site-directed mutagenesis, and kinetic analysis suggest two possible catalytic mechanisms. Either a double-displacement mechanism with the hydroxyl of Tyr 220 as the potential nucleophile or alternatively, an S N i-like mechanism with Tyr 220 positioning the substrates for catalysis. The importance of Tyr 220 in both mechanisms is highlighted by a 3000-fold reduction in k cat in the Y220F mutant.

The alpha-1,6-mannosyltransferase (alpha-1,6-ManT) that initiates outer chain synthesis in Saccharomyces cerevisiae was partially purified along with an alpha-1,2-mannosyltransferase (alpha-1,2-ManT) that acts on alpha-methylmannoside. The enzymes were solubilized by extracting a 145,000 g pellet of S.cerevisiae mnn1 mutant with 1% Triton X-100. The extract was then passed through a concanavalin A-Sepharose column and the bound material was eluted with alpha-methylmannoside. After exhaustive dialysis, the fractions containing both mannosyltransferase activities were chromatographed on DEAE-Trisacryl which removed approximately 90% of the alpha-1,2-ManT. The fractions containing alpha-1,6-ManT and residual alpha-1,2-ManT were further purified by sequential chromatography on Sephacryl S-200 and CM-Trisacryl. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of individual fractions eluted from Sephacryl S-200 and from CM-Trisacryl, followed by silver staining of the gels, showed two major bands whose intensity corresponded to the enzyme activities. A protein band of approximately 62 kDa corresponded to the alpha-1,6-ManT and another band of approximately 66 kDa, which was eluted from the Sephacryl S-200 column slightly earlier, corresponded to the alpha-1,2-ManT.