Search

In this study, we explored structural differences of five commercial samples of yeast β-glucan. Samples were assayed for their β-glucan content and the yeast storage carbohydrate, glycogen. The β-glucan content ranged from 74% to 86%, the glycogen content varied from 0 to 20%. The linkage pattern of each sample was measured by the partially methylated alditol acetate method. This method showed that the samples varied from 1.9% to 9.2% branching. The side chain length distribution for each sample was analyzed by an alkaline degradation assay followed by ion chromatography. The side length distributions of the samples were shown to be similar. The samples were also analyzed by FT-IR and 1HNMR spectroscopy but it was difficult to derive quantitative differences in the samples by these methods. Our findings confirm that each proprietary source of yeast β-glucan has a unique purity profile, branching, and linkage patterns that determine the chemical structure and composition.

Scleroglucan is a β-(1,3)-glucan which is highly branched at the 6-position with a single glucose residue. Acid hydrolysis of a high molecular weight scleroglucan gave a medium molecular weight, freely soluble material. Linkage analysis by the partially methylated alditol acetate method showed that the solubilized material had 30% branching. When the material was subjected to partial Smith degradations, the percent branching was reduced accordingly to 12% or 17%. After the percent branching was reduced, the average molecular weight of the samples increased considerably, indicating the assembly of higher ordered aggregate structures. An aggregate number distribution analysis was applied to confirm the higher aggregated structures. These aggregated structures gave the material significantly enhanced activity in an in vitro oxidative burst assay compared to the highly branched material. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 665–674, 2015.

An enzymatic method to measure β-glucan content (GEM assay) is applicable in a variety of matrices. The method is composed of swelling the sample with KOH and initial digestion with a lyticase, which is followed by treatment with a mixture of exo-1,3-β-d-glucanase and β-glucosidase that converts the β-glucan to glucose. The glucose generated by the enzymatic hydrolysis is measured by another enzymatic method. The method is shown to be accurate and precise. The method is selective and applicable to both highly branched and unbranched β-1,3-glucans.

Scleroglucan is a β-(1,3)-glucan which is highly branched at the 6-position with a single glucose residue. Acid hydrolysis of a high molecular weight scleroglucan gave a medium molecular weight, freely soluble material. Linkage analysis by the partially methylated alditol acetate method showed that the solubilized material had 30% branching. When the material was subjected to partial Smith degradations, the percent branching was reduced accordingly to 12% or 17%. After the percent branching was reduced, the average molecular weight of the samples increased considerably, indicating the assembly of higher ordered aggregate structures. An aggregate number distribution analysis was applied to confirm the higher aggregated structures. These aggregated structures gave the material significantly enhanced activity in an in vitro oxidative burst assay compared to the highly branched material.

The conformation of polysaccharide PGG-Glucan, isolated from yeast cell walls, in aqueous solution was investigated by small angle x-ray scattering (SAXS) and multidetector gel permeation chromatography coupled with postcolumn delivery (GPC/PCD) techniques in comparison with scleroglucan. It was shown that both polysaccharides exhibit a rigid rod-like conformation in aqueous solution by SAXS experiments. The mass per unit length (M/L) and radius (R) of rod cross section of PGG-Glucan were measured to be 6300 daltons/nm and 1.89 nm, while those of scleroglucan are 2300 and 0.83, respectively. Utilizing a GPC/light scattering technique, the average aggregation number of PGG-Glucan is 9, while that of scleroglucan is around 3. From the comparison of the M/L and R of the respective rod cross sections as well as their aggregation number data, it is concluded that PGG-Glucan is composed of triple helices, which tend to aggregate as triplets in solution, whereas scleroglucan is composed of a single triple helix. The aggregation number distribution of PGG-Glucan was found to range from 1 to about 25 determined by GPC/PCD. From the observation of a Debye–Scherrer ring type of peak in the macroscopic scattering cross section of PGG-Glucan by SAXS, the existence of a small amount of ordered clusters of PGG-Glucan can be deduced. The “lattice parameter” of these ordered fasces-like clusters is consistent with the radius of the individual triple-helical rods forming a microfibrillar superstructure. These results indicate that higher aggregated forms of PGG-Glucan containing up to 8 triple helices behave as ordered fasces-like clusters. We conclude that PGG-Glucan is triple-helix aggregates formed by rigid rods stacking together side by side. We propose a molecular structural model for PGG-Glucan conformations. © 1999 John Wiley & Sons, Inc. Biopoly 50: 569–578, 1999

The immunomodulatory properties of yeast β-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate β-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble β-glucans. Using a well-characterized, pharmaceutical grade, soluble yeast β-glucan, this study evaluated and characterized the binding of soluble β-glucan to human neutrophils and monocytes. The results demonstrated that soluble β-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble β-glucan in these cells. Binding of soluble β-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble β-glucan was demonstrated by detection of iC3b, the complement opsonin on β-glucan-bound cells, as well as by the direct binding of iC3b to β-glucan in the absence of cells. Binding of β-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding.

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects β-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate β-glucan polymers, Dectin-1 signalling is only activated by particulate β-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.

Interest in the use of derivatives of 3, 5-hexadienoic (deconjugated sorbic) acid as precursors to various cycloaddition substrates prompts us to report a convenient preparation of the ethyl ester (2d). This methos avoids the somewhat cumbersome and costly use of lithium diisorpropylamide reured in the reported preparations of these esters which involve the formation and kinetic protonation of the trienolate anion from the sorbic ester.1.