Abstract
We have isolated a full-length cDNA clone encoding a human alpha1, 2-mannosidase that catalyzes the first mannose trimming step in the processing of mammalian Asn-linked 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 approximately 2.8 kilobase pairs that was ubiquitously expressed in human tissues. Expression of an epitope-tagged full-length 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 alpha1,2-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).
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF145732
Identification of Putative C. elegans Open Reading Frames and Human Expressed Sequence Tag (EST) Encoding Homologs of Processing Mannosidases—We anticipated that the mammalian endoplasmic reticulum (ER) mannosidase I sequence would be homologous to the mammalian Golgi processing ␣-mannosidases IA [42] and IB [48] and the yeast ER mannosidase I [49] based on two lines of evidence
In vitro assay data have indicated that the mammalian ER mannosidase I was inhibited by dMNJ and kifunensine but not swainsonine or 1,4-dideoxy-1,4-imino-D-mannitol, and the enzyme required Ca2ϩ for catalytic activity [12]
Summary
Man8C from Man9GlcNAc2 with the predominant product being Man8A [42]. The endomannosidase can produce. In contrast to ER ␣-mannosidase I, a second ER-resident ␣-mannosidase activity, termed ER mannosidase II, has been identified in mammalian cells and has been shown to cleave Man9GlcNAc2 to a distinct Man8GlcNAc2 isomer (Man8C; Fig. 1) and potentially smaller structures [12, 13]. This enzyme was sensitive to inhibition by dMNJ and 1,4-dideoxy-1,4-imino-Dmannitol and to partial inhibition by swainsonine but not kifunensine or EDTA. These data indicate that the cDNA encodes human ER mannosidase I and will provide the basis for further studies on the localization of the enzyme and the role of the enzyme in glycoprotein maturation and catabolism
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