Complete 1H and 13C resonance assignments of a 21-amino acid glycopeptide prepared from human serum transferrin

Abstract

A 21-amino acid glycopeptide (Gp21) was isolated and purified in multi-milligram yields from commercially available human serum transferrin (HSTF) by a combination of tryptic digestion, Con A affinity chromatography, and reverse phase HPLC. The peptide chain of Gp21 contains a single N-glycosylation site to which a diantennary oligosaccharide is attached. The amino acid sequence and the glycan primary structure of Gp21 have been verified by peptide sequencing, electrospray mass spectrometry, and one-dimensional 1H NMR spectroscopy. Different glycoforms were found for the glycan of Gp21 derived from two different batches of commercial HSTF. These glycoforms differ from one another in the number of NeuAc residues (ranging from 0 to 2) and/or the number of Gal residues (ranging from 1 to 2). As for the monogalacto species, in the two-dimensional nuclear Overhauser effect (NOE) spectrum of Gp21, interglycosidic NOEs were observed between Man4 in the α(1 → 3) branch and the terminal GlcNAc β(1 → 2) residue. No interglycosidic NOE was observed between Man4′ in the α(1 → 6) branch and the terminal GlcNAc residue. These observations indicate that the terminal GlcNAc residue in the minor glycoforms of Gp21 is exclusively located in the α(1 → 3) branch of the Gp21 glycan. The occurrence of such a carbohydrate structure in HSTF has not been reported before. The 1H and 13C NMR spectra of Gp21 have been completely assigned by two-dimensional homonuclear and heteronuclear spectroscopy. The close similarity of the 1H and 13C chemical shift values for the Gp21 glycan with the respective values for the peptide-free diantennary oligosaccharide (Wieruszeski et al., Glycoconjugate J., 6 (1989) 183–194) indicates that the 1H and 13C chemical shifts of the diantennary oligosaccharide are not perturbed by the presence of the Gp21 peptide fragment. The complete 1H and 13C resonance assignments and the full characterization of the primary structure of Gp21 will permit us to study the conformation and dynamics of the N-linked diantennary oligosaccharides while covalently attached to a polypeptide fragment.