GlycoLab: Functional Glycobiology

Understanding the specific molecular interactions between proteins and $\beta$1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for $\beta$1,3-1,4-mixed-linked over $\beta$1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to $\beta$1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the $\beta$1,3-linkage are important for recognition, and identified the tetrasaccharide Glc$\beta$1,4Glc$\beta$1,4Glc$\beta$1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of $\beta$1,3-1,4-mixed-linked glucans. This is mediated by a conformation-selection mechanism of the ligand in the binding cleft through CH-$π$ stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a $\beta$1,3-linkage at the reducing end and at specific positions along the $\beta$1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked $\beta$-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. DATABASE: Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

During the course of fungal infection, pathogen recognition by the innate immune system is critical to initiate efficient protective immune responses. The primary event that triggers immune responses is the binding of Pattern Recognition Receptors (PRRs), which are expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). However, the precise spatial distribution of the ligands that induce immune responses within the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate recognition domains (CRDs) 4-7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and of the $\beta$-1,3 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall of fungi grown in vitro in rich and minimal media. We show that epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic relationships between fungi, and that Fc-lectin binding discriminated between mannosides expressed on different cell morphologies of the same fungus. We also demonstrate CTL epitope differentiation during different phases of the growth cycle of Candida albicans and that MR and DC-SIGN labelled outer chain N-mannans whilst dectin-2 labelled core N-mannans displayed deeper in the cell wall. These immune receptor maps of fungal walls of in vitro grown cells therefore reveal remarkable spatial, temporal and chemical diversity, indicating that the triggering of immune recognition events originates from multiple physical origins at the fungal cell surface.

The relevance of microalgae biotechnology for producing high-value compounds with biomedical application, such as polysaccharides, has been increasing. Despite this, the knowledge about the composition and structure of microalgae polysaccharides is still scarce. In this work, water-soluble polysaccharides from Nannochloropsis oculata were extracted, fractionated, structurally analysed, and subsequently tested in terms of immunostimulatory activity. A combination of sugar and methylation analysis with interaction data of carbohydrate-binding proteins using carbohydrate microarrays disclosed the complex structural features of the different polysaccharides. These analyses showed that the water-soluble polysaccharides fractions from N. oculata were rich in ($\beta$1→3, $\beta$1→4)-glucans, ($\alpha$1→3)-, ($\alpha$1→4)-mannans, and anionic sulphated heterorhamnans. The immunostimulatory assay highlighted that these fractions could also stimulate murine B-lymphocytes. Thus, the N. oculata water-soluble polysaccharides show potential to be further explored for immune-mediated biomedical applications.