Functional differentiation of glycoside hydrolases family 18 in filamentous Ascomycetes

Sammanfattning: Glycoside hydrolases (GH) family 18 includes enzymes such as chitinases and endo-β–Ν-acetylglucosaminidases (ENGases). They are clustered into three distinct phylogenetic groups A, B and C, and further subdivided into several subgroups. Chitinases are responsible for chitin degradation and are involved in hyphal growth, nutrient acquisition, autolysis and mycoparasitic interactions. ENGases cleave the N,N’-diacetylchiotobiose moiety from high mannose N-linked glycans, and cytosolic ENGases together with peptide:N-glycanases (PNGases) are involved in the endoplasmic reticulum associated degradation process (ERAD) of misfolded glycoproteins. This study investigated the functional role of these enzymes in different aspects of fungal biology using gene deletion and gene expression techniques. Putative GH18 ENGases were enzymatically characterized by heterologous expression in Saccharomyces cerevisiae, and their deglycosylation activity was investigated. The role of ENGases in the generation of free N-glycans was further studied using HPLC techniques. The deletion of the gh18-10 gene, encoding a putative cytosolic ENGase, in Neurospora crassa resulted in a slower growth rate on carbon rich and chitin media, while an increased tolerance to abiotic stress and increased conidiation rate were also observed. In addition, the gh18-10 deletion strain displayed lower extracellular protein secretion and reduced levels of extracellular protease activity. All of these data indicate that improper secretion might result in a more rigid cell wall, affecting the hyphal growth and the tolerance to abiotic stress. The current study proved that the Eng18A and Eng18B ENGases are active deglycosylating enzymes, while the cytosolic Eng18B ENGase in Trichoderma atroviride is the main factor for free N-glycans generation. Furthermore, this enzyme was able to degrade the RTL protein complex, which is an ERAD-specific substrate, implying a potential role in the ERAD pathway. This study also showed that chitinase genes belonging to C-II subgroup and displaying similarities to the Kluyveromyces lactis killer toxin were induced during interspecific interactions. Deletion of certain C-II chitinase genes in Clonostachys rosea and Aspergillus nidulans reduced their growth inhibitory activity of culture filtrates against Botrytis cinerea and Rhizoctonia solani, while reduced conidiation and altered responses to abiotic stress were also observed, indicating that these chitinases might be involved also in cell wall modification and conidiation.

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