N-Glycosylation of Gel1 or Gel2 is vital for cell wall β-glucan synthesis in Aspergillus fumigatus.

Fungal cell wall is a dynamic structure that communicates with and protects the cell from outside stress. In Aspergillus fumigatus, the cell wall β-glucans are mainly elongated by β-1,3-glucanosyltransferases Gels, which consist of seven family members (Gel1-7) utilizing β-1,3-glucan chains as substrates. Previously, we have shown that the mutant deficient of N-glycan processing displays a reduction in the cell wall β-glucans, suggesting that N-glycosylation is required for the proper function of β-1,3-glucanosyltransferase. To verify this hypothesis, in this study, the gene encoding β-1,3-glucanosyltransferase Gel1 or Gel2 was deleted in the Δcwh41 mutant to construct a double-mutant Δgel1Δcwh41 or Δgel2Δcwh41. The growth phenotypes of both double mutants were similar to the single-mutant Δcwh41, suggesting that Gel1 and Gel2 are proteins that are mainly affected by deficient N-glycan processing in Δcwh41. Furthermore, the mutant Δgel1(Gel1-NM) or Δgel2(Gel2-NM), in which all potential N-glycosylation sites on Gel1 or Gel2 were removed by site-directed mutagenesis, showed phenotypes similar to the single-mutant Δgel1 or Δgel2. Biochemical analysis revealed that N-glycosylation was essential for the function of Gel1 or Gel2 and thus required for β-glucan synthesis in A. fumigatus.