Identification, Expression, and Characterization of a cDNA Encoding Human Endoplasmic Reticulum Mannosidase I, the Enzyme That Catalyzes the First Mannose Trimming Step in Mammalian Asn-linked Oligosaccharide Biosynthesis

Abstract

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...

A novel function of VCP (valosin-containing protein; p97) in the control of N-glycosylation of proteins in the endoplasmic reticulum

“…However, both upon proteasome inhibition and VCP depletion the glycosylated species of αTCR differed from the glycosylated species present in control cells, since it had an increased proportion of a faster migrating form. The fact that the faster migrating form is absent after DMJ treatment suggests that it is formed through the action of α-mannosidase I, an ER-resident enzyme which catalyzes the removal of mannose residues from Man 9 GlcNAc 2 prior to the transfer of N-glycosylated proteins from ER to cis-Golgi compartments [51]. Moreover, the faster migrating band can be formed from the slower migrating band by the action of jack bean mannosidase, an enzyme usually producing a mix of truncated forms of high-mannose oligosaccharides.…”

Structural Basis for Catalysis and Inhibition ofN-Glycan Processing Class I α1,2-Mannosidases

“…Expression and Purification-The cloning and characterization of the soluble catalytic domain of human ER class I ␣1,2-mannosidase as a protein A fusion (pPROTA-ERManI) were described previously (13). The portion of the cDNA encoding the catalytic domain (amino acids 172-689) was excised from the pPROTA expression vector by digestion with EcoRI and ligated into the EcoRI site of the Pichia expression vector pPICZ␣A (Invitrogen, La Jolla, CA).…”

Elucidation of the molecular logic by which misfolded α1-antitrypsin is preferentially selected for degradation

“…The ability of ␣-1,2-mannosidase inhibition to arrest aberrant glycoprotein turnover indicates that glycoprotein ERAD (GERAD) is initiated through an early glycan-based proteasome-targeting event (4). The intracellular location of the glycan modification, its sensitivity to kifunensine (12,13), and the results of yeast genetics (14) have implicated ER mannosidase I (ERManI) as the most likely cellular candidate responsible for generating the glycan-based component of a putative GERAD signal (4). ERManI generates the glycan-based signal determinant through the slow removal of a single mannose unit from asparaginelinked Man 9 GlcNAc 2 (Fig.…”