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

Abstract

Human α-galactosidase A (α-Gal A) is the lysosomal glycohydrolase that cleaves the terminal α-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N-linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man_5–7GlcNAc₂ species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi-, 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N-acetylneuraminic acid and occurred exclusively in α2,3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man₅GlcNAc₂ and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human α-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans-Golgi network, and the predominately Man_5-7GlcNAc₂ cellular glycoforms resulting from carbohydrate trimming in the lysosome.