Observation of a Partially Opened Triple-helix Conformation in 1→3-β-Glucan by Fluorescence Resonance Energy Transfer Spectroscopy

Abstract

This study used fluorescence resonance energy transfer (FRET) spectroscopy as an indirect method to investigate the effect of NaOH treatment on the conformation of a triple-helix (133)-␤-D-glucan and then evaluated the effect of conformation on biological activity. Previous studies have suggested that treatment of the triple-helix glucans with NaOH produces single-helix conformers. FRET spectra of the triple-helix glucan, laminarin, doubly labeled with 1-aminopyrene as donor probe and fluorescein-5-isothiocyanate as acceptor probe attached at the reducing end, showed that a partially opened triplehelix conformer was formed on treatment with NaOH. Increasing degrees of strand opening was associated with increasing concentrations of NaOH. Based on these observations we propose that a partially opened triplehelix rather than a single helix, is formed by treating the triple-helix glucans with NaOH. After neutralizing the NaOH, changes in FRET indicated that the partially opened conformer gradually reverts to the triple-helix over 8 days. Laminarian was stabilized at different degrees of partial opening and its biological activity examined using the Limulus amebocyte lysate assay and nitric oxide production by alveolar macrophage. Both Limulus amebocyte lysate activity and nitric oxide production were related to the degree of opening of the triple-helix. Partially open conformers were more biologically active than the intact triple-helix.(133)-␤-D-Glucans have been shown to systemically enhance the immune system, resulting in antitumor, antibacterial, and wound healing activities (1). Differences in these activities are thought to be related to three factors: molecular conformation, the degree of branching, and the molecular weight (M r ) (2). Studies have shown that high M r (100,000 -200,000 g/mol) glucans with a degree of branching of 0.20 -0.33 are most active (2), however, data describing the effects of molecular conformation on biological activity are less clear. This is in part due to the lack of adequate methodology to characterize the tertiary structure of glucans.Most spectroscopic techniques only provide data about the secondary structure of glucans. Methods such as solid state 13 C NMR spectroscopy (3) or multi-angle laser-light scattering (4) have had some success in relating the tertiary structure of glucans to their conformation based biological activity, however, their use is limited by availability, cost, and efficacy for evaluating glucan structure in solution. A direct observation technique, such as x-ray crystallography would assist in understanding glucan structure and its biological relevance; however, it is difficult to crystallize glucans and only a limited number of these studies have been done (5-7).Fluorescence resonance energy transfer (FRET) 1 is a phenomenon of non-radioactive energy transfer over relatively long distances that has been used to characterize the spatial relationship of donor-and acceptor-labeled molecules in biological systems (8). If a system contains two fluorophores an...

Solution structure of the silkworm βGRP/GNBP3 N-terminal domain reveals the mechanism for β-1,3-glucan-specific recognition

“…In aqueous solution, ␤-1,3-glucan forms a stable triple-helical structure and is known to be converted into a random coil or a single helix by chemical or physical treatments (18)(19)(20). Previous studies have shown that NaOH treatment alters the structure of laminaran (soluble ␤-1,3-glucan) from a triple-helical structure into a partially opened triple-helical structure, and it was indicated that even after neutralization, the partially opened structure was maintained for up to 20 days (21,22). To distinguish whether ␤GRP N102 binds a triple-helical structure or an opened conformer of ␤-1,3-glucan, NaOH treated laminaran and untreated laminaran were prepared and the affinity to ␤GRP N102 was studied.…”

Characterization of β-Glucan Recognition Site on C-Type Lectin, Dectin 1

“…The conformation is formed by hydrogen bonding of C-2 hydroxyl groups of main-chain glucosyl residues. Alkaline treatment of triple-helix (133)-␤-D-glucans results in a conformational change in the random coil structure, and subsequent neutralization results in a single-helix conformation or a partially opened triple-helix conformation (62). Therefore, the significant differences between the triple-and single-helix or partially opened conformations include the accessibility to C-2 hydroxyl groups of the main-chain strand.…”

Antifungal Properties of Cationic Phenylene Ethynylenes and Their Impact on β-Glucan Exposure

“…6), the lack of interaction with the glucan likely allows the molecule to quickly penetrate the cell wall and to access and damage the cell membrane. Abundant lateral noncovalent interactions between ␤-glucan and PPE-DABCO may promote PE-␤-glucan complexation, analogous to the role of interpolymeric hydrogen bonding in stabilizing lateral interactions of individual ␤-glucan polymers in aqueous solution (59,60). PPE-DABCO is far larger than its oligomeric counterpart and has numerous sites where weak interactions with ␤-glucan polymers may form; furthermore, extensive valency of laterally aggregated ␤-glucan would make this interaction very strong.…”