Variations in oligosaccharide-protein interactions in immunoglobulin G determine the site-specific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides

Biochemistry. 1997 Feb 11;36(6):1370-80. doi: 10.1021/bi9621472.

Abstract

Glycoproteins, such as immunoglobulin G (IgG), consist of an ensemble of glycosylated variants, or glycoforms, which have different oligosaccharides attached to a common peptide. Alterations in the normal glycoform populations of IgG are associated with certain disease states, notably rheumatoid arthritis and its remission during pregnancy. In this paper, we show that two sets of IgG Fc glycoforms have quite different physical properties. The first set has 1,6 arm terminal galactose residues which interact with the protein, resulting in glycan binding to the protein surface, in agreement with the crystal structure. In contrast, the second set of glycoforms which lack galactose does not bind to the protein surface. Recently developed HPLC techniques combined with enzymatic digestion and mass spectrometry have been used to assign the glycan structures on IgG, Fab, and Fc. Comparison of Fab with Fc shows that glycosylation is site-specific. Two major glycan structures are present on Fab (fucosylated digalacto-bianntenary with and without bisect) and three on Fc (fucosylated agalacto-, 1,6 arm monogalacto-, and digalacto-bianntenary). In comparison to Fab, Fc glycans contain (i) lower levels of bisecting GlcNAc, (ii) lower levels of galactose, (iii) higher than expected levels of 1,6 arm galactose relative to 1,3 arm, and (iv) no 1,6 arm sialylation. We interpret these differences to indicate a role for both the protein quaternary structure and specific protein-glycan interactions in determining the glycoform populations. NMR relaxation measurements have been used to probe the mobility of the glycans in the Fc. By comparing two samples with different glycoform populations, we conclude that this mobility is dependent on the primary sequence of the glycan. Glycans carrying a galactose residue on the 1,6 arm have relaxation properties very similar to those of the peptide backbone and thus do not have independent motion. Glycans lacking galactose have relaxation rates 30 times slower than that of the peptide and thus a higher degree of mobility. These agalactosyl glycans do not interact with the protein, resulting in exposure of previously covered regions of the peptide surface and making the glycan more accessible. This implies that at the early stages of glycan processing the Fc glycans are mobile and only partially protected by the protein quaternary structure. Immobilization of the glycans occurs as a consequence of addition of galactose to the 1,6 arm and results in increased protection.

MeSH terms

  • Carbohydrate Sequence
  • Chromatography, High Pressure Liquid
  • Glycosylation
  • Humans
  • Immunoglobulin Fc Fragments / metabolism*
  • Immunoglobulin G / metabolism*
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Oligosaccharides / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Immunoglobulin Fc Fragments
  • Immunoglobulin G
  • Oligosaccharides