Identification and structural characterization of a mannose‐6‐phosphate containing oligomannosidic N‐glycan from human erythropoietin secreted by recombinant BHK‐21 cells

[摘要]

A sialidase resistant mono-charged N-glycan was isolated from glycosylation site I (Asn-24) of recombinant human eruthropoietin expressed from baby hamster kidney (BHK-21) cells and constituted approximately 2–4% of the oligosaccharide material at this glycosylation site. Mass spectrometry and both 1- and 2-dimensional NMR techniques revealed a high mannose type structure (Man₆) with a phospho-diesterbridged N-acetylglucosamine as follows:

[学科分类] 生物化学/生物物理

[关键词] Recombinant human erythropoietin;Baby hamster kidney cells (BHK-21);Phosporylated oligomannosidic glycan;Mannose-6-phosphate;1- and 2-D NMR spectroscopy;Lysosomal recognition signal;BHK;baby hamster kidney;CHO;Chinese hamster ovary;COSY;correlation spectroscopy;EPO;erythropoietin;GC;gas chromatography;GlcNAc;N-acetylglucosamine;Hex;hexose;HexNAc;N-acetylhexosamine;HPAE-PAD;high-pH anion-exchange chromatography with pulsed amperometric detection;HPLC;high performance liquid chromatography;MALDI/TOF MS;matrix-assisted laser desorption/ionization time-of-flight mass spectrometry;Man;mannose;NMR;nuclear magnetic resonance;NOESY;nuclear Overhauser enhancement and exchange spectroscopy;PNGase F;polypeptidase: N 4-(N-acetyl-glucosaminyl)asp aragine-amidase F;TOCSY;total correlation spectroscopy