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

Abstract

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-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 Man5-7GlcNAc2 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 alpha2, 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 alpha-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 Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.

Biosynthesis, Glycosylation, and Enzymatic Processingin Vivo of Human Tripeptidyl-peptidase I

“…1C). Thus, the observed difference in mass was caused by dissimilar oligosaccharide structures on secreted and intracellular proenzyme of hTPP I, a finding that was reported for other overexpressed mature acid hydrolases (24,25) or their proforms (26). CHO cells also secreted very small amounts of mature enzyme, which could be visualized only after prolonged exposure of autoradiograms and immunoblots following immunoprecipitation (see below).…”

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

“…In addition to the sialylated multiantennary N-glycans, mass spectrometry indicated the presence, in fractions DEAE-2/3a and 2/3b, of three unusual acidic oligomannose structures with molecular masses consistent with fucosylated and non-fucosylated oligomannose 5 structures and an oligomannose 6 structure, each with an additional mass of 80, indicating the presence of either a phosphate or a sulphate residue. Phosphorylated oligomannose structures have been described to occur in recombinant human a-galactosidase A when the enzyme had been produced by overexpression in CHO cells (Matsuura et al, 1998). A high-mannose-type structure (oligomannose 6) with a phospodiester-bridged N-acetylglucosamine has been described to occur on a rEPO expressed in baby hamster kidney cells (Nimtz et al, 1995).…”

“…Although the occurrence of N-acetyllactosamine extensions in rEPO oligosaccharide structures are well documented, N-glycans with incomplete outer chains terminating in N-acetylglucosamine residues had not previously been described in rEPO. However, these structures have been reported to occur in other glycoproteins synthesized in CHO cells, for example by Rudd et al (1999) and Matsuura et al (1998). This rEPO preparation (SpB) may have been enriched with these oligosaccharides through the use of ion-exchange chromatography during its purification to preferentially isolate the more basic isoforms of rEPO.…”

A protein chimera strategy supports production of a model “difficult‐to‐express” recombinant target

“…Alternatively, high mannose/hybrids forms may be released early as a result of cell lysis. The detection of high mannose/hybrid glycoforms has been reported for other secreted recombinant proteins in mammalian cells 55, 56, 57, 58.…”