Extracellular targeting of Neurospora crassa cell wall and secreted glycoproteins by DFG-5
2022
Patel, Pavan K. | Tung, Sook Keng | Porfirio, Sara | Sonon, Roberto | Azadi, Parastoo | Free, Stephen J.
The formation of a cell wall is vital for the survival and growth of a fungal cell. Fungi express members of the GH76 family of α-1,6-mannanases which play an important role in cell wall biogenesis. In this report we characterize the Neurospora crassa DFG-5 α-1,6-mannanase and demonstrate that it binds to the α-1,6-mannose backbone of an N-linked galactomannan found on cell wall glycoproteins. We show that DFG-5 has an enzymatic activity and provide evidence that it processes the α-1,6-mannose backbone of the N-linked galactomannan. Site-directed mutagenesis and complementation experiments show that D116 and D117 are located at the DFG-5 active site. D76 and E130, which are located in a groove on the opposite side of the protein, are also important for enzyme function. Cell wall glycoproteins co-purify with DFG-5 demonstrating a specific association between DFG-5 and cell wall glycoproteins. DFG-5 is able to discriminate between cell wall and secreted glycoproteins, and does not bind to the N-linked galactomannans present on secreted glycoproteins. DFG-5 plays a key role in targeting extracellular glycoproteins to their final destinations. By processing the galactomannans on cell wall proteins, DFG-5 targets them for cell wall incorporation by lichenin transferases. The N-linked galactomannans on secreted proteins are not processed by DFG-5, which targets these proteins for release into the extracellular medium.
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