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.
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).
Gonzalez
D S
DS
Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Karaveg
K
K
Vandersall-Nairn
A S
AS
Lal
A
A
Moremen
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D008358:Mannose / Q000378:metabolism*; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis; D016366:Open Reading Frames / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment
D018076:DNA, Complementary; D009844:Oligosaccharides; D012333:RNA, Messenger; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D008358:Mannose