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Human α‐fetoprotein produced from hep G2 cell line: Structure and heterogeneity of the oligosaccharide moiety

1995; Wiley; Volume: 30; Issue: 4 Linguagem: Inglês

10.1002/jms.1190300415

1096-9888

Pasquale Ferranti, Piero Pucci, Gennaro Marino, Immacolata Fiume, B Terrana, Costante Ceccarini, Antonio Malorni,

Enzyme Structure and Function

Abstract The carbohydrate moiety of human α‐fetoprotein, an RMM 67000 glycoprotein produced in a hepatoma cell line (Hep G2), was investigated by the combined use of high‐resolution chromatographic techniques and mass spectrometry. Fast atom bombardment mass spectrometric (FABMS) and reversed‐phase high‐performance liquid chromatographic analysis of α‐fetoprotein obtained from a large‐scale cell culture following tryptic and peptide N ‐glycanase F hydrolysis demonstrated that the protein contains a single glycosylation site at level of asparagine 232. Further, electrospray mass spectrometric measurement of the intact protein molecular mass showed that two main glycoforms are present. The complete definition of the structural heterogeneity of the oligosaccharide moiety was achieved by high‐performance anion‐exchange chromatography with pulsed amperometric detection together with carbohydrate mapping by FABMS of the released oligosaccharides demonstrating (i) that the glycosylation produced by cell culture is of the biantennary complex type typical of human hepatoma α‐fetoprotein and (ii) the presence of two main structures in a ratio of about 2:1 differing in the presence of a fucose residue in the N ‐acetylglucosamine in the non reducing portion of the molecule.