Processing of asparagine-linked oligosaccharides in insect cells: evidence for alpha-mannosidase II.
The occurrence of alpha-D-mannosidase II activity in insect cells was studied using pyridylaminated oligosaccharides as substrates and two-dimensional HPLC and glycosidase digestion for the analysis of products. GlcNAcMan5GlcNAc2 was converted to GlcNAcMan3GlcNAc2 by each of the three cell lines investigated (Bm-N, Sf-21, and Mb-0503). The respective activity was highest in Bm-N cells which were used for further experiments. Man5GlcNAc2 was not degraded by the Bm-N cell homogenate. Thus, this alpha-mannosidase essentially exhibits the same substrate specificity as mammalian and plant Golgi alpha-mannosidase II. The alpha-mannosidase II-like activity from Bm-N cells exhibits a pH optimum of 6.0-6.5, has no requirement for divalent metal ions, and is highly sensitive to swainsonine. The alpha 1,6-linked mannosyl residue is removed first as deduced from the elution time on reversed phase HPLC of the intermediate product. The same branch preference was found with alpha-mannosidase II from mung bean seedlings and Xenopus liver. Upon ultracentrifugation of Bm-N cell homogenate, 72% of the mannosidase acting on the GlcNAcMan5GlcNAc2 substrate was found in the microsomal pellet indicating the enzyme to be membrane-bound.
Altmann
F
F
Institut für Chemie der Universität für Bodenkultur Wien, Vienna.
März
L
L
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D007313:Insecta / Q000201:enzymology*; D008361:Mannosidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D009844:Oligosaccharides; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase; D000118:Acetylglucosaminidase