A NOVEL C-13 ISOTOPIC LABELING STRATEGY FOR PROBING THE STRUCTURE ANDDYNAMICS OF GLYCAN CHAINS IN-SITU ON GLYCOPROTEINS

A protocol is described for uniform C-13 labelling of terminal galacto se residues of the glycan chains of glycoproteins, using an enzymatic method which does not perturb the protein. The technique is illustrate d by application to the biantennary N-linked glycan chains attached at Asn 297 of immunoglobulin G (IgG). Isotope-edited NMR experiments on this glycoprotein yield data which suggest that the galactose residues on the glycan exist in two discrete environments, with the galactose in one environment having greater mobility than that in the other. The se data are qualitatively consistent with crystallographic data on an Fe fragment, which suggest that one arm of the glycan is in contact wi th the protein, while the other projects into the space between the C( gamma)2 domains. Quantitatively, however, these data cannot be rationa lized with the crystallographic data, which implies subtle differences in oligosaccharide structure and dynamics between the solution and cr ystal states of Fc.