Characterization of the oligosaccharide structures on a recombinant tissue-type plasminogen activator analog synthesized in chinese hamster ovary cells

The asparagine-linked oligosaccharides on a recombinant tissue-type plasminogen activator analog, lacking the epidermal growth factor-like and kringle 1 domains (FK2P), synthesized by Chinese hamster ovary cells, have been characterized. In vivo [3H]mannose labeled glycopeptides were extracted from SDS-PAGE gels of antibody precipitated FKZP and the major oligosaccharide structures were characterized by serial lectin affinity chromatography, size-exclusion chromatography in combination with exoglycosidase digestions, ion exchange chromatography, HPLC and methylation analysis. 44% of the oligosaccharides on the molecule are triantennary (35% 2,4-branched, 9% 2,6-branched), 34% are biantennary and 20% are tetraantennary complex type, all of which are core-fucosylated. Approximately 1% of the structures are high mannose type, in the form of Man6GlcNAc2, Man7GlcNAc2 and Man8GlcNac2. The remaining 1% of the structures appear to be hybrid type. The complex structures contain varying numbers of sialic acid residues, ranging from non- to fully sialylated. No N-acetylgalactosamine was detected in acid hydrolysates of FK2P, consistent with a lack of serine/threonine-linked oligosaccharides. Sequence analysis of tryptic peptides prepared from purified FK2P revealed that only one of the two consensus sites for N-linked glycosylation on the molecule is utilized.