Processing of asparagine-linked oligosaccharides in insect cells: evidence for α-mannosidase II

Abstract

The occurrence of α-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 α-mannosidase essentially exhibits the same substrate specificity as mammalian and plant Golgi α-mannosidase II. The α-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 α1,6-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 α-mannosidase II from mung bean seedlings andXenopus 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