The Saccharomyces cerevisiae Processing α1,2-Mannosidase Is an Inverting Glycosidase

137

110

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...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Gonzalez

Karaveg

Nairn

et al. 1999

137

110

“…Relevant fractions were pooled, dried, and used as substrate for digestion with the lysosomal ␣-mannosidase. Man 9 -GlcNAc was isolated from soybean agglutinin as described previously (43,44). Natural Substrate Digestions and Chromatography-Enzyme reactions with oligosaccharide substrates were carried out in 100 mM sodium acetate (pH 4.5) in a volume of 220 l. At individual time points, aliquots were removed and adjusted to 70% acetonitrile and 50-l aliquots were run on a Hypersil APS-2 NH 2 high performance liquid chromatography column (250 ϫ 4.6 mm, Keystone Scientific, Bellefonte, PA) with an initial buffer of 70% acetonitrile, 30% 100 mM sodium phosphate (pH 4.0).…”

Section: Methodsmentioning

confidence: 99%

“…An enzyme activity with these characteristics has been identified and purified from several species sources including Dictyostelium discoideum (5), and a variety of mammalian tissues (6 -11). Metabolic radiolabeling studies have indicated that the human enzyme is initially synthesized as a polypeptide of ϳ110 kDa that is subsequently processed into two subunits of [40][41][42][43][44][45][46]. Proteolytic processing of the precursor appears to be variable in different mammalian species, with reported subunit sizes for the enzyme ranging from 20 to 70 kDa.…”

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confidence: 99%

Cloning, Expression, Purification, and Characterization of the Human Broad Specificity Lysosomal Acid α-Mannosidase

Liao

Lal

Moremen

1996

We have cloned and expressed two cDNAs encoding the human lysosomal ␣-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA. This enzyme is required for the degradation of N-linked carbohydrates during glycoprotein catabolism in eucaryotic cells. The shorter of the two cDNAs (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encode an enzyme that could cleave high mannose oligosaccharides, oligosaccharides isolated from ␣-mannosidosis fibroblasts, and p-nitrophenyl-␣-D-mannopyranoside substrates. In addition, the Pichia-expressed enzyme was inhibited by swainsonine, and had a pH optimum, K m , and V max characteristic of the enzyme purified previously from human liver. The second, larger RT-PCR product (3.6 kb) was found to contain an insertion and a deletion relative to the 3-kb spleen amplimer and encoded a truncated coding region, indicating that it resulted from alternate transcript splicing. No ␣-mannosidase activity could be detected in Pichia transformants containing this coding region, indicating that it did not encode a functional enzyme. Antiserum raised to the recombinant product of the 3-kb ␣-mannosidase cDNA immunoprecipitated lysosomal ␣-mannosidase activity from human fibroblast extracts. Northern blots identified a 3-kb RNA transcript in all human tissues tested, including ␣-mannosidosis fibroblasts, while minor transcripts of 3.6 kb were also present in several adult tissues. Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19. Sequence comparisons were made to previously published human cDNA sequences encoding a putative lysosomal ␣-mannosidase (Nebes, V. L., and Schmidt, M. C. (1994) Biochem. Biophys. Res. Commun. 200, 239 -245) and several differences were found relative to the functional lysosomal ␣-mannosidase encoded by the 3-kb spleen cDNA.The lysosomal catabolism of N-glycans on mammalian glycoproteins occurs through the sequential exoglycosidase digestion of oligosaccharides from the non-reducing terminus down to the carbohydrate-peptide core region (1). Included among the hydrolytic activities responsible for this oligosaccharide degradation is a broad specificity exo-␣-mannosidase activity (EC 3.2.1.24) that catalyzes the hydrolysis of ␣1,2-, ␣1,3-, and ␣1,6-mannoside linkages present in complex, hybrid, and high mannose Asn-linked glycans (2). This enzyme is distinguished from other ␣-mannosidase activities by a combination of a low pH optimum (pH 4.5), a broad natural substrate specificity, activity toward the synthetic substrate p-nitrophenyl-␣-mannoside, and sensitivity to inhibition by swainsonine (3, 4). An enzyme activity with these characteristics has been identified and purified from several species sources including Dictyostelium discoideum (5), and a variety of mammalian tissues (6 -11). Metabolic radiolabeling studies have indicated that the human enzyme is initially synthesized as a polypeptide of ϳ110 kDa that is sub...

“…Both groups of enzymes have similar overall threedimensional structures consisting of a novel (␣␣) 7 fold (8 -11). They are calcium-dependent inverting glycosidases (12,13) inhibited by both 1-deoxymannojirimycin and kifunensine.…”

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confidence: 99%

Respiratory Distress and Neonatal Lethality in Mice Lacking Golgi α1,2-Mannosidase IB Involved in N-Glycan Maturation

Tremblay

Kovács

Daniels

et al. 2007

There are three mammalian Golgi ␣1,2-mannosidases, encoded by different genes, that form Man 5 GlcNAc 2 from Man 8-9 GlcNAc 2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi ␣1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on ␣1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The ␣1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.