Study of the sugar chains of recombinant human amyloid precursor protein produced by Chinese hamster ovary cells

• Published in: Biochimica et biophysica acta 1999-10, Vol.1472 (1), p.344-358
• Main Authors: Sato, Yuji, Liu, Chen, Wojczyk, Boguslaw S., Kobata, Akira, Spitalnik, Steven L., Endo, Tamao
• Format: Article
• Language: English
• Subjects: Alzheimer’s disease; Amyloid beta-Protein Precursor - chemistry; Amyloid beta-Protein Precursor - genetics; Amyloid beta-Protein Precursor - isolation & purification; Amyloid precursor protein; Animals; Blotting, Western; Carbohydrate Conformation; Carbohydrate Sequence; CHO Cells; Chromatography, Liquid - methods; Cricetinae; Cricetulus; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Sequence Data; N-Glycan; O-Glycan; Polysaccharides - chemistry; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(99)00140-3

The N- and O-glycans of recombinant amyloid precursor protein (APP), purified from Chinese hamster ovary cells transfected with the human 695-amino acid form of APP, were separately released by hydrazinolysis under different conditions. The reducing ends of the released N- and O-glycans were reduced with NaB 3H 4 and derivatized with 2-aminobenzamide (2AB), respectively. After acidic N-glycans were obtained by anion-exchange column chromatography, these were converted to neutral oligosaccharides by sialidase digestion, demonstrating that their acidic nature was entirely due to sialylation. The sialidase-treated N-glycans were then fractionated by lectin column chromatography and their structures were determined by linkage-specific sequential exoglycosidase digestion. These results demonstrated that recombinant APP has bi- and triantennary complex type N-glycans with fucosylated and nonfucosylated trimannosyl cores. In a similar fashion, the 2AB-labeled O-glycans derived from APP were determined to be mono- and disialylated core type 1 structures. Taken together, these results indicate that recombinant APP has sialylated bi- and triantennary N-glycans with fucosylated and nonfucosylated cores and sialylated O-glycans with core type 1 structures.