Human alpha-galactosidase A: characterization of the N-linked oligosaccharides on the intracellular and secreted glycoforms overexpressed by Chinese hamster ovary cells

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolasethat cleaves the terminal alpha-galactosyl moieties of variousglycoconjugates. Overexpression of the enzyme in Chinese hamster ovary(CHO) cells results in high intracellular enzyme accumulation and theselective secretion of active enzyme. Structural analysis of the N -linkedoligosaccharides of the intracellular and secreted glycoforms revealed thatthe secreted enzyme's oligosaccharides were remarkably heterogeneous,having high mannose (63%), complex (30%), and hybrid (5%) structures. Themajor high mannose oligosaccharides were Man5-7GlcNAc2 species.Approximately 40% of the high mannose and 30% of the hybridoligosaccharides had phosphate monoester groups. The complexoligosaccharides were mono-, bi- , 2,4-tri-, 2,6-tri- and tetraantennarywith or without core-region fucose, many of which had incomplete outerchains. Approximately 30% of the complex oligosaccharides were mono- ordisialylated. Sialic acids were mostly N -acetylneuraminic acid andoccurred exclusively in alpha2, 3-linkage. In contrast, the intracellularenzyme had only small amounts of complex chains (7.7%) and hadpredominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 andsmaller species, of which only 3% were phosphorylated. The complexoligosaccharides were fucosylated and had the same antennary structures asthe secreted enzyme. Although most had mature outer chains, none weresialylated. Thus, the overexpression of human alpha-Gal A in CHO cellsresulted in different oligosaccharide structures on the secreted andintracellular glycoforms, the highly heterogeneous secreted formspresumably due to the high level expression and impaired glycosylation inthe trans- Golgi network, and the predominately Man5-7GlcNAc2 cellularglycoforms resulting from carbohydrate trimming in the lysosome.