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.

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Alterations in the glycosylation of human IgG have been shown to occur in rheumatoid arthritis (RA). However, the precise nature and location of these changes have not been fully established. Therefore we carried out a detailed analysis of the oligosaccharides chemically released from intact human serum IgG and fragments of the molecule. Serum samples were from three healthy ('normal') individuals, and from four patients with RA. Site-specific glycolsylation of the glycoprotein was shown to occur, which extended to sites even within the Fab fragment. These were differences in galactosylation, sialylation and the presence of a bisecting N-acetylglucosomide. Disease related alterations were also shown to be site-specific. In particular, an increase in the proportion of agalactosylated oligosaccharides occurred on the Fc fragment in RA (P=0.057), but, in contrast to previous reports there was an increase on the light chain in the proportion of fully galactosylated, bisected and core fucosylated oligosaccharides (from 13% of total in normal to between 18 and 35% in RA, P=0.057)). There was also an Fab-specific increase in oligosaccharides bearing a bisecting N-acetylglucosamine and a core fucose (P=0.075) The site-specific glycosylation changes described in this paper reveal the complexity of the regulatory mechanism, perhaps reflecting the many levels at which regulation can occur.

Assignment of most of the proton NMR resonances of bovine pancreatic RNase B has been achieved using standard NMR techniques and by comparison with the published assignments for RNase A. A comparison of the NMR spectra of RNasc B with RNasc A shows that glycosylation of the enzyme has little overall effect on the conffinnz,tion of the protein in solution. Comparisons of hydrogen&uterium solvent exchange rates for the NH protons of RNase A and RNasc B were made using two-dimensional 'H correlation spectroscopy. In the case ofthc glycosylated enzyme the exchange rates decreased for the NH protons of residues g-14. 23-24, 32,34-35, 39110,434l, 4S-49,60,71.75-7G, 80,83-85, 100-101. 107. I I 1 and 122, relative to the unglycosylated RNasc A. These resulu arc consistent with the presence of the oligosaccharidc inducing enhanced global dynamic stability and consequent changes to the unfolding equilibrium of the enrymc. The enhanced stability is observed not only for residues in the vicinity of the glycosylation site, asparagine-34, but also at residues remote from this site, as much as 30 A away.Hydrogen exchange; NMR; RNasc A and B: Effects of glycosylation

Massive release of tumor necrosis factor is responsible for the potentially fatal larisch-Herxheimer reaction that follows antibiotic treatment of relapsing fever due to Borrelia recurrentis. We have undertaken the quantitative purification of the components of B. recurrentis that stimulate human monocytes to produce tumor necrosis factor. We show that the predominant factor inducing tumor necrosis factor is a variable lipoprotein homologous to the variable major protein of B. hermsii. We found antibodies to different forms of variable major protein in two patients with louse-borne relapsing fever. The three purified variable major proteins studied here differ in their ability to induce tumor necrosis factor production, which may partly explain the variable clinical severity of borrelial infection. These results may be of considerable relevance for the pathogenesis of Lyme disease and other forms of human borreliosis.