Influence of Core Fucosylation on the Flexibility of a Biantennary N-Linked Oligosaccharide

Abstract

Fluorescence energy transfer was used to study the conformation of each antenna of a complex biantennary oligosaccharide. A core fucosylated biantennary oligosaccharide was converted to a glycosylamine which allowed coupling of a naphthyl donor fluorophore directly to the reducing-end GlcNAc 1. After generating an aldehyde at C-6 of residue 6 or 6' using galactose oxidase, a dansyl ethylenediamine acceptor fluorophore was coupled to either antenna of the oligosaccharide resulting in two donor-acceptor pairs. [Formula: see text] The fluorescence properties of the naphthyl group allowed determination of the end-to-end donor-acceptor distance and antenna flexibility of each isomer by steady-state and time-resolved fluorescence energy transfer at temperatures ranging from 0 to 40 degrees C. Extended (20.6 A) and folded (11.4 A) donor-acceptor distance populations were identified for the isomer containing dansyl attached to Gal 6', whereas only a single extended population (19.7 A) was determined when dansyl was attached to Gal 6. The presence of Fuc 1' had a dramatic effect on the conformation of the 6' antenna. Temperature modulation failed to alter the ratio of extended/folded populations when fucose was present. However, following the removal of fucose, the ratio of the extended/folded populations for 6' exhibited a temperature dependent conformational equilibrium allowing calculation of the enthalpy and entropy of unfolding. These results established a unique conformational property for the 6' antenna of a biantennary oligosaccharide that is influenced by core fucosylation. Comparison of the results obtained for the 6 antenna of biantennary with previous fluorescence energy transfer studies on a triantennary glycopeptide also established conformational differences in this antenna which are dependent on oligosaccharide structure.

The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity

“…There are a few possible explanations of why the antibodies with nonfucosylated oligosaccharides give rise to stronger binding to Fc␥RIIIa than those in which the glycoforms are absent. A core Fuc has been shown to influence the conformational flexibility of biantennary oligosaccharides (26,27). The oligosaccharides of IgG appear to be largely sequestered between the CH2 domains and may help to stabilize the CH2 domain (28).…”

?1,6fucosyltransferase is highly and specifically expressed in human ovarian serous adenocarcinomas

“…This suppression could, however, be due to the altered function of ␣5␤1 integrin by concomitant modification of the N-glycans. Addition of an ␣1,6fucose residue to an N-linked oligosaccharide induces an extended conformation in the oligosaccharide (Stubbs et al, 1996). Therefore, it appears that the large conformational change which occurs as a result of ␣1,6fucosylation affects the progression of malignant tumors through modification of the structure and, hence, the functions of these glycoproteins; thus, the elevated expression of ␣1,6FucT may be involved in the potential for malignancy.…”

“…It has been suggested that addition of an ␣1,6fucose residue to the innermost GlcNAc of the N-linked oligosaccharide leads to an unusual extended conformation (Stubbs et al, 1996). Therefore, it is possible that ␣1,6fucosylation leads to modifications of the functions of glycoproteins, such as growth factor receptors, adhesion molecules and extracellular matrices, by inducing conformational changes in the N-glycan structure.…”

Conformational flexibility of N-glycans in solution studied by REMD simulations

“…The currently accepted view is that chemical modification changes binding, by way of either switching the major conformation from one to another or inducing a unique bioactive conformation, rather than by making an additional unique binding site. Past studies, including NMR-based-analysis of model oligosaccharides (Homans et al 1987a), FRET experiments (Stubbs et al 1996), and molecular modeling followed by systematic bioassays with neoglycoproteins carrying synthetic biantennary N-glycans (André et al 2009), indicate a switch-like change in the shape of the glycans upon chemical modification. Figures 3 and 4 show the results of REMD simulations of four N-glycans with and without chemical modification (Nishima et al 2012).…”