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Differences in protein structural regions that impact functional specificity in GT2 family β-glucan synthases.

ABSTRACT: Most cell wall and secreted β-glucans are synthesised by the CAZy Glycosyltransferase 2 family (www.cazy.org), with different members catalysing the formation of (1,4)-β-, (1,3)-β-, or both (1,4)- and (1,3)-β-glucosidic linkages. Given the distinct physicochemical properties of each of the resultant β-glucans (cellulose, curdlan, and mixed linkage glucan, respectively) are crucial to their biological and biotechnological functions, there is a desire to understand the molecular evolution of synthesis and how linkage specificity is determined. With structural studies hamstrung by the instability of these proteins to solubilisation, we have utilised in silico techniques and the crystal structure for a bacterial cellulose synthase to further understand how these enzymes have evolved distinct functions. Sequence and phylogenetic analyses were performed to determine amino acid conservation, both family-wide and within each sub-family. Further structural analysis centred on comparison of a bacterial curdlan synthase homology model with the bacterial cellulose synthase crystal structure, with molecular dynamics simulations performed with their respective β-glucan products bound in the trans-membrane channel. Key residues that differentially interact with the different β-glucan chains and have sub-family-specific conservation were found to reside at the entrance of the trans-membrane channel. The linkage-specific catalytic activity of these enzymes and hence the type of β-glucan chain built is thus likely determined by the different interactions between the proteins and the first few glucose residues in the channel, which in turn dictates the position of the acceptor glucose. The sequence-function relationships for the bacterial β-glucan synthases pave the way for extending this understanding to other kingdoms, such as plants.

SUBMITTER: Oehme DP

PROVIDER: S-EPMC6821405 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Anti-beta-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective beta-glucan epitope(s) and protection mechanisms, we studied two anti-beta-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the beta1,3-beta1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to beta1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to beta1,6- and beta1,4-linked glucose sequences in addition to beta1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-beta-glucan antibodies may affect details of the beta-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize beta-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

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Differences in protein structural regions that impact functional specificity in GT2 family β-glucan synthases.

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