Regulation of TLR9 signaling by N-glycosylation of UNC93B1

Abstract

Nucleotide-sensing Toll-like receptors (TLRs), such as TLR3, 7, and 9, reside in the endolysosomal compartments to avoid activation by host DNA or RNA. Proper intracellular localization and signaling by these TLRs depend on the physical interaction with UNC93B1. Specific amino acid residues in UNC93B1 have been identified to affect the functions of endosolysomal TLRs. However, it is largely unknown how UNC93B1 differentially regulates individual TLRs. Protein N-glycosylation controls folding, maturation, stability, localization and protein-interaction of target glycoproteins. In this study, we examined whether the N-glycosylation of UNC93B1 affects its properties and molecular function. Using mutagenesis, we found that the N251 and N272 residues of UNC93B1 become N-glycosylated. We expressed wild type (WT) and N-glycosylation-defective UNC93B1 mutants (N251Q and N272Q) in UNC93B1-deficient cells and found that signaling of TLR9, but not that of other TLRs, were defective in UNC93B1 N272Q-expressing cells. The N272Q mutation did not affect the protein stability, localization, interaction of UNC93B1 and TLRs, nor did it prevent the CpG DNA-binding of TLR9. Nonetheless, upon CpG DNA stimulation, the recruitment of MyD88 to TLR9 was significantly inhibited in UNC93B1 N272Q-expressing cells compared to WT cells. Consequently, poor phosphorylation and degradation of I_B were observed in UNC93B1 N272Q-expressing cells. Combined, our data show that N-glycosylation of UNC93B1 specifically regulates TLR9 signaling by modulating the recruitment of MyD88 to TLR9.