Overexpression of the Golgi-localized enzyme alpha-mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl-N-acetylchitobiose to tetramannosyl-N-acetylchitobiose in the N-glycan-processing pathway.
Golgi alpha-mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn(1)M(5)Gn(2) to Gn(1)M(3)Gn(2) during biosynthesis of N-glycans. Previously, we isolated a cDNA encoding a protein homologous to alpha-mannosidase II and designated it alpha-mannosidase IIx. Here, we show by immunocytochemistry that alpha-mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with alpha-mannosidase II, alpha-mannosidase IIx colocalizes with alpha-mannosidase II in COS cells. A protein A fusion of the catalytic domain of alpha-mannosidase IIx hydrolyzes a synthetic substrate, 4-umbelliferyl-alpha-D-mannoside, and this activity is inhibited by swainsonine. [(3)H]glucosamine-labeled Chinese hamster ovary cells overexpressing alpha-mannosidase IIx show a reduction of M(6)Gn(2) and an accumulation of M(4)Gn(2). Structural analysis identified M(4)Gn(2) to be Man alpha 1-->6(Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc. The results suggest that alpha-mannosidase IIx hydrolyzes two peripheral Man alpha 1-->6 and Man alpha 1-->3 residues from [(Man alpha 1-->6)(Man alpha 1-->3)Man alpha 1-->6](Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc, during N-glycan processing.
Oh-Eda
M
M
Glycobiology Program, The Burnham Institute, La Jolla, CA 92037, USA.
Nakagawa
H
H
Akama
T O
TO
Lowitz
K
K
Misago
M
M
Moremen
K W
KW
Fukuda
M N
MN
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D017026:Swainsonine / Q000494:pharmacology; D014162:Transfection
D004187:Disaccharides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C032438:chitobiose; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase; D017026:Swainsonine