Structures of the sugar chains of rabbit immunoglobin G: occurrence of asparagine-linked sugar chains in Fab fragment

Abstract

The asparagine-linked sugar chains of rabbit immunoglobulin G (IgG) and its Fc and Fab fragments were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Rabbit IgG was shown to contain 2.3 mol of asparagine-linked sugar chains per molecule distributed in both the Fc and Fab fragments. The sugar chains were of the biantennary complex type containing four cores: Man alpha 1----6(Man alpha 1----3)(+/- GlcNAc beta 1----4)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)-GlcNAc. A total of 16 distinct neutral oligosaccharide structures was found after sialidase treatment. The galactose residue in the monogalactosylated oligosaccharides was present on either the alpha 1----3 or alpha 1----6 side of the trimannosyl core. The Fab fragments contained neutral, monosialylated, and disialylated oligosaccharides, whereas the Fc fragment contained only neutral and monosialylated structures. The oligosaccharides isolated from the Fab fragments also contained more galactose and bisecting N-acetylglucosamine residues than those from the Fc fragments.

Mannose receptor interacts with Fc receptors and is critical for the development of crescentic glomerulonephritis in mice

“…Importantly, because altered IgG glycosylation patterns have previously been associated with anti- or proinflammatory effects of Igs in this model (19), we confirmed that no differences in IgG glycosylation were present in the MRdeficient animals compared with their WT counterparts. In addition, we have shown, for the first time to our knowledge, that the Fab fragment of sheep nephrotoxic globulin is capable of binding MR. Glycosylation of the Fab Ig region has long been known, with increased expression of N-acetylglucosamine groups, and is thought to be critical in influencing antibody immunogenicity, efficacy, and clearance (36)(37)(38). Taken together with the colocalization of the 2 receptors, these findings lead us to conclude that FcR and MR could interact through the binding of nephrotoxic globulin.…”

The Emerging Importance of IgG Fab Glycosylation in Immunity

“…Margni and Binaghi (32) investigated the properties of IgG Abs that are retained on a ConA affinity resin (5-15%) and concluded that their Fab arms contain high-mannose glycans. However, ConA also retains complextype biantennary N-linked glycans without bisecting GlcNAc; most likely, the retained IgG Abs contain both types of Fab glycans (33,34). Interestingly, levels of ConA-binding Abs are increased during the second trimester of pregnancy.…”

Antibody variable region glycosylation: biochemical and clinical effects

“…W h e n the structure o f the sugar chains present on r a b b i t I g G was determined, b o t h F a b and F c fragments contained b i a n t e n n a r y oligosaccharides [54]. While the F a b -a s s o c i a t e d c a r b o h y d r a t e contained neutral, m o n o -a n d disialylated oligosaccharides, the F c contained only neutral and monosialylated sugars.…”

“…Observations that some IgG myeloma protein contained odd numbers of sugar moieties led to the speculation that IgG may be asymmetrically glycosylated [54]. It was found that both sheep and rabbit non-precipitating anti-DNP (dinitrophenol) antibodies were unable to precipitate or fix complement with DNP-BSA but were able to do so with DNP-GABA-BSA, while precipitating antibodies would fix complement with both antigens [32].…”

“…In the IgG-associated oligosaccharide, heterogeneity appears to occur in the latter steps of processing, with variability in the degree of sialylation, fucosylation, and the addition of a bisecting N-acetylglucosamine [41]. Species differences observed to date in IgGassociated oligosaccharide structure appear to be largely confined to linkage specificities of sialyltransferases and the presence of the bisecting N-acetylglucosamine [13,44,54]. The presence of carbohydrate affects a number of biological properties of IgG, and abnormal IgG oligosaccharides are reported to have clinical implications [43].…”