Structures, Biosynthesis, and Physiological Functions of (1,3;1,4)-β-d-Glucans

(1,3;1,4)-β-d-Glucans, also named as mixed-linkage glucans, are unbranched non-cellulosic polysaccharides containing both (1,3)- and (1,4)-β-linkages. The linkage ratio varies depending upon species origin and has a significant impact on the physicochemical properties of the (1,3;1,4)-β-d-glucans. (1,3;1,4)-β-d-Glucans were thought to be unique in the grasses family (Poaceae); however, evidence has shown that (1,3;1,4)-β-d-glucans are also synthesized in other taxa, including horsetail fern Equisetum, algae, lichens, and fungi, and more recently, bacteria. The enzyme involved in (1,3;1,4)-β-d-glucan biosynthesis has been well studied in grasses and cereal. However, how this enzyme is able to assemble the two different linkages remains a matter of debate. Additionally, the presence of (1,3;1,4)-β-d-glucan across the species evolutionarily distant from Poaceae but absence in some evolutionarily closely related species suggest that the synthesis is either highly conserved or has arisen twice as a result of convergent evolution. Here, we compare the structure of (1,3;1,4)-β-d-glucans present across various taxonomic groups and provide up-to-date information on how (1,3;1,4)-β-d-glucans are synthesized and their functions.


Introduction
β-Glucans have been found to be highly abundant in plants, algae, fungi, and bacteria as one of the fundamental fibers in the cell walls. The polysaccharides are composed of D-glucopyranosyl units (Glcp) as building blocks. Depending on the glycosidic bonds between the glucose monomers, β-glucans can be classified into two sub-groups, cereal and non-cereal β-glucans. For example, the yeast and fungi β-glucans contain mainly 1,3 and branching 1,6 linkages, whereas cereal β-glucans have linear glucan chain composed of 1,3 and 1,4 glycosidic linkages. (1,3;1,4)-β-D-Glucans, or mixed-linkage glucans (MLGs), have been found rich in rice, wheat, cereal grains and oats, serving as important dietary fibers for our daily consumption, and also impact our metabolic activities by decreasing cholesterol and blood glucose [1].

Viridiplantae
(1,3;1,4)-β-D-Glucans have been found in the cell walls of the grasses and cereal family, also known as Poaceae, which consist of commercially important cereals. During the plant growth and development, the amount of (1,3;1,4)-β-D-glucans in the wall is found to increase proportionally to the cell elongation rate, reaching its maximum during the most rapid phase of cell growth, and is completely hydrolyzed when growth ceases. Not only functioning as structural elements, the (1,3;1,4)-β-D-glucans are present in high abundance in the walls of aleurone layer surrounding the barley, rye, and oats starchy endosperms. These (1,3;1,4)-β-D-glucans are hydrolyzed by specific enzymes, the (1,3;1,4)β-D-glucanases, when germination occurs, allowing mobilization of endosperm cell walls and also providing an extra carbon source that facilitates the germination process [25,26].
(1,3;1,4)-β-D-Glucans are found in the walls of red algae and brown algae, and both are evolutionarily more distant to land plants. A sulfated (1,3;1,4)-β-D-glucan has been isolated from the red algae Kappaphycus alvarezii. This sulfated glucan has only 180 residues, with 64% sulfation on the (1,4)-β-linkages only and the remaining (1,4)-β-linkages were sparse and unlikely to be in long sequences after one and another. The sulfated (1,4)β-linkages likely link to the fibrillar cell wall polymers by their incompatible extraction behavior. It is also speculated that the sulfation helps control construction and positioning of cellulose [31].
The presence of (1,3;1,4)-β-D-glucan in the cell walls of Equisetum arvense, Phaeophyceae (brown algae) in phylum of Stramenopiles that is not closely related to any land plants and green algae, the non-conserved structural characteristics across lineages and the absence of (1,3;1,4)-β-D-glucans in some closely related families of grasses and cereal, suggest synthesis could be highly conserved, or a convergence-independent evolution of (1,3;1,4)-β-D-glucan synthase genes.

Physiological Function of (1,3;1,4)-β-D-Glucan in Viridiplantae
The (1,3;1,4)-β-D-glucans in plants provide structural roles, such as strength, flexibility and elasticity. They are also important in the transport by providing porosity during active growth, and the exchange of water, nutrients and other small molecules, such as phytohormones between adjacent cells [48]. In Poaceae, (1,3;1,4)-β-D-glucan is associated with the elongating cells differentiating from the meristematic cells, and is largely absent in the meristematic cells and the mature tissues where growth has ceased [49][50][51]. Additionally, in the Poaceae (1,3;1,4)-β-D-glucan, around 30% are β-1,3-glycosidic linkages, and these primarily consist of DP3s and DP4s, with only 10% of the oligosaccharides being larger than DP4s. With no consecutive (1,3)-β-bonds existing, (1,3;1,4)-β-D-glucan is linear with "kinks", caused by the single (1,3)-β-linkage inserted within β-1,4 oligosaccharides, which makes it more flexible and soluble [52]. One could see it as a cis-double bond in lipids, which are harder to stack due to it being folded. It is possible that the irregularity of the (1,3;1,4)-β-D-glucan "kinks" makes the (1,3;1,4)-β-D-glucans unable to lay neatly on each other.  [44]. In addition, the CslF6 knockout mutant rice had reduced in height by 1/3 and seed production after pollination, also known as seed set, was halved compared to the wild type [44]. The reduction of seed set was most likely the result of deformed male reproductive tissues. The CslF6 is suggested to play an important role in the growth of the rice plants, but it is uncertain if this is due to a lack of (1,3;1,4)-β-D-glucans or some other possible mechanism that the CslF6 might take part in.
Thus far, very little is known about the function of (1,3;1,4)-β-D-glucans in algae. A recent study suggests that (1,3;1,4)-β-D-glucans in brown algae appeared to bond tightly to the cell walls. Their insolubility in water with consistent conformation suggests the structural functions [9].
Besides the lichens, a non-lichen fungus, Aspergillus fumigatus, was found to have (1,3;1,4)-β-D-glucans in an alkali-insoluble fraction from its cell wall, which is suggested to be related to fungal cell wall rigidity [55]. The (1,3;1,4)-β-D-glucan was also detected in the cell wall of Neurospora crassa with monoclonal antibody [56]. A high percentage of (1,4)-linked glucose was reported in the glycosyl composition and linkage analysis of fungal cell wall from a filamentous fungus N. crassa [57]. It is highly likely that the occurrence of (1,3;1,4)-β-D-glucans is not restricted in the A. fumigatus and N. crassa. How the particular variation in the structures of (1,3;1,4)-β-D-glucans found may be related to their function is largely unknown. Other β-glucans, for example, the (1,3;1,6)-β-D-glucan, is a common β-glucan found in the cell walls of various fungal species, and has shown to possess immunomodulatory activities in reducing SARS-CoV-2-induced cytokine storm in the infected patients [58]. Further β-glucan structural survey of taxa in other lichen and fungal families would be interesting to provide evidence in understanding structurefunction relationships between β-glucan structures and their functions, and their potential implications for pharmaceutical research and development.

Physiological Function of (1,3;1,4)-β-D-Glucan in Fungi and Lichens
As one of the components constituting the fungal cell wall, lichenin is believed to contribute to cell wall rigidity in A. fumigatus. However, even as 10% of all the cell wall glucans, (1,3;1,4)-β-D-glucan still seems to be dispensable for the cell growth and fitness of A. fumigatus, as no phenotypic differences were observed compared to the wildtype when (1,3;1,4)-β-D-glucan synthase, Tft1, was deleted or two-fold overexpressed in in vitro growth. Meanwhile, (1,3;1,4)-β-D-glucan is suggested to be relevant in spore formation and enhances β-1,3 glucan synthesis of A. fumigatus, but it would require further studying to confirm if these functions are affected directly by (1,3;1,4)-β-D-glucans [59]. In contrast, in A. nidulans, the deletion of the CelA gene could cause a "balloon" phenotype, which indicates a weakened cell wall. In addition to the changed morphology, the CelA gene-deleted strain showed decreased sensitivity to the drugs against the cell wall, Congo red and dichlobenil. These observations strongly implicate (1,3;1,4)-β-D-glucan in cell wall-related progression of A. nidulans. In another study, an orthologous gene to CelA was found in the pathogenic fungus Drechslera teres, which may be responsible for the production of (1,3;1,4)-β-D-glucans on the cell wall of D. teres, the fungal species known to cause diseases such as net blotch in barleys and have a severe economic impact [62]. Whether (1,3;1,4)-β-D-glucan here plays any role could be up for discussion, as the cell wall of various pathogenic species are often relevant for attachment and pathogenicity. Moreover, in N. crassa, lichenins contribute to 25% of the vegetative cell wall mass and are thought to cross-link glycoproteins to the cell wall [58].
Bacterial cell wall is a complex multilayered structure that protects the bacteria from an unpredictable and often hostile environment. Most bacteria, except mycoplasmas, have a complex cell wall composed of a mixture of polymers made of carbohydrates and amino acids. These are the peptidoglycan polymers that can provide cell rigidity and act as a physical barrier between the cell and its surrounding. In response to the different environment, some species of Gram-positive bacteria and Gram-negative bacteria secrete high concentration of the exopolysaccharides (EXPs) deposited onto the bacterial surface ( Figure 2). Their roles include basic functions such as maintaining structural integrity and preventing desiccation, to more complex activities of facilitating the interaction within bacterial communities. The bacterial EXPs have been found in many pathogenic bacteria and have a direct impact on human health because of their ability to form multicellular conglomerates called biofilms.
Bacterial cell wall is a complex multilayered structure that protects the bacteria from an unpredictable and often hostile environment. Most bacteria, except mycoplasmas, have a complex cell wall composed of a mixture of polymers made of carbohydrates and amino acids. These are the peptidoglycan polymers that can provide cell rigidity and act as a physical barrier between the cell and its surrounding. In response to the different environment, some species of Gram-positive bacteria and Gram-negative bacteria secrete high concentration of the exopolysaccharides (EXPs) deposited onto the bacterial surface (Figure 2). Their roles include basic functions such as maintaining structural integrity and preventing desiccation, to more complex activities of facilitating the interaction within bacterial communities. The bacterial EXPs have been found in many pathogenic bacteria and have a direct impact on human health because of their ability to form multicellular conglomerates called biofilms. A recent study reported that the structure of (1,3;1,4)-β-D-glucan in Gram-negative bacteria S. meliloti is distinctive to the (1,3;1,4)-β-D-glucans of land plants and fungi, for its entire structure is composed of disaccharide repeating units Glcp-β-1,3-Glcp (DP2s) [10]. Methylation analysis showed that the partially methylated alditol acetates (PMAAs) derived from (1,3;1,4)-β-D-glucans of S. meliloti corresponded to (1,3)-and (1,4)-linked glucopyranosyl residues in a 1:1 ratio. Further structural investigation of (1,3;1,4)-β-D-glucans, using two-dimensional Nuclear Magnetic Resonance with Rotating-frame nuclear Overhauser Effect Spectroscopy (2D-NMR ROESY) spectrum, revealed the cross-peaks between H1 of (1,4)-linked Glcp and H3 of (1,3)-linked Glcp, and between H1 of (1,3)linked Glcp and H4 of (1,4)-linked Glcp, corresponded to a -3)-β-D-Glcp-(1,4)-β-D-Glcp(1repetitive polymeric structure. Lichenase digestion only releases DP2, confirming the presence of a novel (1,3;1,4)-β-D-glucan in S. meliloti.

Biosynthesis of (1,3;1,4)-β-D-Glucan in Bacteria
The biosynthesis of bacterial exopolysaccharides is known to share some common characteristics to that in plants. For example, the polysaccharide synthases involved in the bacterial cellulose, alginate and poly-β-D-N-acetylglucosamine production have similar structural motifs, such as the transmembrane-spanning domain, a catalytic D,D,D,QxxRW motif, and a PilZ domain [64]. These motifs are essential for substrate binding and to catalyze the biosynthesis of bacterial exopolysaccharides. Specifically, the PilZ domain is known to involve in the binding to the secondary messenger cyclic diguanylate (c-di-GMP). Two putative genes bgsA and bgsB are proposed to be involved in the biosynthesis of S. meliloti (1,3;1,4)-β-D-glucan. In silico study suggests that the BgsA protein has seventransmembrane spanning domains and a catalytic D,D,D,QxxRW motif, whereas the PilZ domain is absent. Biochemical study of heterologously expressed BgsA C-terminal domain consisting of 139 amino acids suggests that this unknown domain is also involved in the binding of c-di-GMP, but the lack of PilZ suggests that the activation mechanism of BgsA for the production of (1,3;1,4)-β-D-glucans could be different to the biosynthesis of other exopolysaccharides. A recent study showed that putative (1,3;1,4)-β-D-glucan synthase, the bgsA and bgsB operons, have been identified in the genomes of Rhizobium, Agrobacterium and Methylobacterium, all within the order Rhizobiales [10], suggesting that the occurrence of (1,3;1,4)-β-D-glucan in bacteria maybe more frequent than we have previously expected.

Conclusions
(1,3;1,4)-β-D-Glucans are found most commonly in higher plants such as grasses and cereals, and less commonly in the walls of monilophyte Equisetum, some bryophytes, algae, lichens, fungus, a chromalveolate, and two bacterial species. Except for a few taxa, structural variation of (1,3;1,4)-β-D-glucans across lineage is particularly apparent, but little is known about their physicochemical properties and their biosynthesis [10]. This information is needed to understand the evolution of the important cell wall polysaccharides, the (1,3;1,4)-β-D-glucans, as well as for the possibility of greater utilization of the glucans in food and beverage, and biotechnology industries.