Identification of candidate MYB transcription factors that influence CslF6 expression in barley grain

(1,3;1,4)-β-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located −331 bp to −382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression

Investigating the synthesis and regulation of (1,3;1,4)-β-glucan biosynthesis

Cereals such as rice (Oryza sativa (Os)), barley (Hordeum vulgare (Hv)) and sorghum (Sorghum bicolor (Sb)) provide a considerable portion of our daily energy requirements. Their cell wall constituents, such as (1,3;1,4)-β-glucan, survive relatively intact through much of the upper human digestive system to reach the colon, where they are fermented by a range of commensal microorganisms. The products of this fermentation help reduce blood cholesterol levels and ameliorate diseases including coronary heart disease, type II diabetes and colorectal cancer. Efforts have therefore been directed toward understanding the regulation and mechanism of (1,3;1,4)-β-glucan biosynthesis to enhance the human health potential and industrial utility of cereal grain. Numerous reports suggest that the CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene encodes the synthase responsible for producing the majority of (1,3;1,4)-β-glucan in cereals. These synthase genes contain species-specific polymorphisms that have been shown to influence the amount and structure of (1,3;1,4)-β-glucan produced when they are expressed heterologously in Nicotiana benthamiana and barley grain. Here, a chimeric approach exchanged sections of the barley (Hv) and sorghum (Sb) CSLF6 synthases to identify regions influencing (1,3;1,4)-β-glucan production and structure. Using this approach an 80 amino acid stretch, which contains the conserved TED and QxxRW motifs, was shown to be responsible for much of the difference in (1,3;1,4)-β-glucan production and structure between the barley and sorghum synthases. Of the six amino acid polymorphisms contained within this section, one affected polysaccharide structure whilst another dictated the amount of (1,3;1,4)-β-glucan. Co-expression in N. benthamiana was used to investigate CSLF6 modulation and complex formation. Results from a variety of chimeric, truncated and mutated constructs suggest that a highly variable section of unknown function, termed the class-specific region (CSR), and the NH2-terminal region of CSLF6 are separately able to mediate complex formation and increase (1,3;1,4)-β-glucan production. Expression of a construct missing the CSR indicated that the region was not structurally or functionally required for (1,3;1,4)-β-glucan synthesis in N. benthamiana. A PilZ domain responsible for cofactor binding and cellulose synthase activation in bacteria was also identified at the COOH-terminal end of the NH2-terminal region of CSLF6, and was shown to influence (1,3;1,4)-β-glucan production. Overall, the results presented here have furthered our understanding of the action of the CSLF6 isoform of the (1,3;1,4)-β-glucan synthase enzyme. This brings us closer to having the capacity to precisely control the synthase’s function, and allowing the prospect of manipulating cereal tissues to contain the optimal amount of (1,3;1,4)-β-glucan with a defined structure for specific human health and industrial applications.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2016

(1,3;1,4)-β-Glucan Biosynthesis by the CSLF6 Enzyme: Position and Flexibility of Catalytic Residues Influence Product Fine Structure

<i>Cellulose synthase-like F6</i> (<i>CslF6</i>) genes encode polysaccharide synthases responsible for (1,3;1,4)-β-glucan biosynthesis in cereal grains. However, it is not clear how both (1,3)- and (1,4)-linkages are incorporated into a single polysaccharide chain and how the frequency and arrangement of the two linkage types that define the fine structure of the polysaccharide are controlled. Through transient expression in <i>Nicotiana benthamiana</i> leaves, two CSLF6 orthologs from different cereal species were shown to mediate the synthesis of (1,3;1,4)-β-glucans with very different fine structures. Chimeric cDNA constructs with interchanged sections of the barley and sorghum <i>CslF6</i> genes were developed to identify regions of the synthase enzyme responsible for these differences. A single amino acid residue upstream of the TED motif in the catalytic region was shown to dramatically change the fine structure of the polysaccharide produced. The structural basis of this effect can be rationalized by reference to a homology model of the enzyme and appears to be related to the position and flexibility of the TED motif in the active site of the enzyme. The region and amino acid residue identified provide opportunities to manipulate the solubility of (1,3;1,4)-β-glucan in grains and vegetative tissues of the grasses and, in particular, to enhance the solubility of dietary fibers that are beneficial to human health

Functional characterization of a glycosyltransferase from the moss physcomitrella patens involved in the biosynthesis of a novel cell wall arabinoglucan

Mixed-linkage (1,3;1,4)-β-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco (Nicotiana benthamiana) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-β-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana, but (1,4)-β-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots

Hypoxia Controls the Glycome Signature and Galectin-8–Ligand Axis to Promote Protumorigenic Properties of Metastatic Melanoma

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, β1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. In this study, we explored hypoxia-dependent alterations in MM glycome‒associated genes and found that β1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8–ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low β1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed β1,6 N-acetylglucosaminyltransferase 2 in MM cells upregulated TIC marker, NGFR/CD271, whereas loss of MM cell‒intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-N-acetyllactosamines on N-glycans of the TIC marker and prometastatic molecule CD44, among other receptors, and activated prosurvival factor protein kinase B. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-N-acetyllactosamine and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-N-acetyllactosamine and Gal-8 as biomarkers and therapeutic targets of MM

Inhibition of melanoma cell-intrinsic Tim-3 stimulates MAPK-dependent tumorigenesis.

T-cell immunoglobulin and mucin domain 3 (Tim-3) is an immune checkpoint receptor that dampens effector functions and causes terminal exhaustion of cytotoxic T-cells. Tim-3 inhibitors are under investigation in immuno-oncology (IO) trials, because blockade of T-cell-Tim-3 enhances antitumor immunity. Here, we identify an additional role for Tim-3 as a growth-suppressive receptor intrinsic to melanoma cells. Inhibition of melanoma cell-Tim-3 promoted tumor growth in both immunocompetent and immunocompromised mice, while melanoma-specific Tim-3 overexpression attenuated tumorigenesis. Antibody (Ab)-mediated Tim-3 blockade inhibited growth of immunogenic murine melanomas in T-cell-competent hosts, consistent with established antitumor effects of T-cell Tim-3 inhibition. In contrast, Tim-3 Ab administration stimulated tumorigenesis of both highly and lesser immunogenic murine and human melanomas in T-cell-deficient mice, confirming growth-promoting effects of melanoma-Tim-3 antagonism. Melanoma-Tim-3 activation suppressed, while its blockade enhanced, phosphorylation of pro-proliferative downstream mitogen-activated protein kinase (MAPK) signaling mediators. Finally, pharmacologic MAPK inhibition reversed unwanted Tim-3 Ab-mediated tumorigenesis in T-cell-deficient mice and promoted desired antitumor activity of Tim-3 interference in T-cell-competent hosts. These results identify melanoma-Tim-3 blockade as a mechanism that antagonizes T-cell-Tim-3-directed IO therapeutic efficacy. They further reveal MAPK targeting as a combination strategy for circumventing adverse consequences of unintended melanoma-Tim-3 inhibition

Don't sugarcoat it: How glycocalyx composition influences cancer progression.

Mechanical interactions between tumors and the extracellular matrix (ECM) of the surrounding tissues have profound effects on a wide variety of cellular functions. An underappreciated mediator of tumor-ECM interactions is the glycocalyx, the sugar-decorated proteins and lipids that act as a buffer between the tumor and the ECM, which in turn mediates all cell-tissue mechanics. Importantly, tumors have an increase in the density of the glycocalyx, which in turn increases the tension of the cell membrane, alters tissue mechanics, and drives a more cancerous phenotype. In this review, we describe the basic components of the glycocalyx and the glycan moieties implicated in cancer. Next, we examine the important role the glycocalyx plays in driving tension-mediated cancer cell signaling through a self-enforcing feedback loop that expands the glycocalyx and furthers cancer progression. Finally, we discuss current tools used to edit the composition of the glycocalyx and the future challenges in leveraging these tools into a novel tractable approach to treat cancer