Structural study of the N-linked oligosaccharides of hepatocyte growth factor by two-dimensional sugar mapping

The structures of the N-linked oligosaccharides on recombinant human hepatocyte growth factor (rh-HGF) expressed by Chinese hamster ovary (CHO) cells were studied by two-dimensional sugar mapping. The oligosaccharides released from the glycopeptides by peptide: N-glycosidase F (PNGase F) treatment were tagged with 2-aminopyridine at the reducing ends. The alpha-chain was linked by biantennary, triantennary, and tetraantennary oligosaccharides, but the dominant oligosaccharides linking the beta-chain were biantennary (> 85%). There was no significant difference in oligosaccharide structures between the two glycosylation sites on each chain, that is, Asn263 and Asn371 on the alpha-chain, and Asn535 and Asn622 on the beta-chain. The linkage of sialic acid to the non-reducing terminal galactose was identified as NeuAc alpha(2-3) by 1H-NMR spectrometry. The structures of the N-linked oligosaccharides from rat HGF were also studied. Triantennary oligosaccharides were obtained from the alpha-chain and a biantennary oligosaccharide was obtained from the beta-chain. This result indicates that the alpha-chain is also linked by higher branched oligosaccharides than the beta-chain in rat HGF.