A TIR domain variant of MyD88 adapter-like (Mal)/TIRAP results in loss of MyD88 binding and reduced TLR2/TLR4 signaling

Abstract

The adapter protein MyD88 adapter-like (Mal), encoded by TIR-domain containing adapter protein (Tirap) (MIM 606252), is the most polymorphic of the five adapter proteins involved in Toll-like receptor signaling, harboring eight non-synonymous single nucleotide polymorphisms in its coding region. We screened reported mutations of Mal for activity in reporter assays to test the hypothesis that variants of Mal existed with altered signaling potential. A TIR domain variant, Mal D96N (rs8177400), was found to be inactive. In reconstituted cell lines, Mal D96N acted as a hypomorphic mutation, with impaired cytokine production and NF-kappaB activation upon lipopolysaccharide or PAM2CSK4 stimulation. Moreover, co-immunoprecipitation studies revealed that Mal D96N is unable to interact with MyD88, a prerequisite for downstream signaling to occur. Computer modeling data suggested that residue 96 resides in the MyD88 binding site, further supporting these findings. Genotyping of Mal D96N in three different cohorts suggested that it is a rare mutation. We, thus, describe a rare variant in Mal that exerts its effect via its inability to bind MyD88.

FIGURE 1.

Human Mal carrying the D96N mutation is unable to activate either NF-κB or IRF5. HEK293T cells were transfected with different variants of Mal and either NF-κB luciferase (A) or IRF5 and ISRE-luciferase reporters (B). 48 h post-transfection, lysates were analyzed for luciferase activity. Renilla-luciferase activity was used to normalize for transfection efficiency. Results are reported as the mean of triplicate determinations ±S.D. Each graph is representative of three (A) or two (B) independent experiments. RLU, relative luciferase units.

FIGURE 2.

D96N has impaired cytokine production in response to TLR2 or TLR4 ligands. A, functional restoration of Mal-deficient immortalized macrophage cell line. Immortalized Mal knock-out (ko) cells were transduced with a retrovirus carrying FLAG-tagged Mal. Single cell clones were selected and tested for their responsiveness to LPS and PAM₂CSK₄. B, the same strategy was used to generate cell lines expressing the different variants of Mal. For each cell line clones were chosen for further study based on similar levels of expression of Mal or the Mal variant. IB, immunoblot. C, immortalized Mal-deficient macrophage cell lines expressing either WT Mal or one of the variants were stimulated with LPS, PAM₂CSK₄, and poly I:C overnight. The supernatants were then analyzed for TNFα levels. Poly I:C, a TLR3/TRIF ligand, was used as a control for Mal-independent signaling. Results are reported as the mean of triplicate determinations ±S.D. Each graph is representative of three independent experiments. D, immortalized WT and Mal-deficient (Mal ko)- and Mal-deficient macrophages (Mal ko + D96N)-expressing D96N were stimulated for 2 h with LPS (100 ng/ml). Total RNA was extracted. Levels of mRNA for IFN-β were determined by quantitative real-time-PCR and normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) level of expression. Results are reported as the mean of duplicate determinations ±S.D. This graph is representative of one of two independent experiments. Bl, blank.

FIGURE 3.

Mal D96N fails to degrade IκB-α effectively. NF-κB activation in the reconstituted cell lines was examined via IκB-α degradation assay. The cells were stimulated with LPS (left panel) or PAM₂CSK₄ (right panel) for the indicated time points, and the whole cell lysates loaded on a denaturing gel, transferred to nitrocellulose membrane, and blotted with IκB-α antibody. Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) act as an internal loading control. WT, wild type; ko, knock out; WB, Western blot.

FIGURE 4.

D96N fails to interact with MyD88. HEK293T cells were plated to half confluence in six-well plates. After attachment, cells were transiently transfected with either TLR2-YFP (A), TLR4-CFP (B), or MyD88-CFP (C) and FLAG-tagged Mal WT or Mal D96N. Two days after transfection, supernatants were removed, and cells were lysed. Anti-GFP polyclonal antibody was used for immunoprecipitation (IP). Immunoprecipitates were loaded on a denaturing gel, transferred to nitrocellulose membrane, and visualized with a monoclonal anti-FLAG-horseradish peroxidase antibody. The immunoprecipitation of TLR2-YFP, TLR4-CFP, and MyD88-CFP was confirmed by immunoblotting the membrane with anti-GFP antibody. The same antibodies were used to check the level of expression of the proteins in whole cell lysates. IB, immunoblot.

FIGURE 5.

The D96N mutation results in a loss of negative charge on the surface of the Mal-TIR domain in a region predicted to be involved in MyD88, but not TLR interactions. A, comparison of the electrostatic surfaces of wild-type (left) and Asn-96 (right) Mal-TIR models, indicating a loss of negative charge (red) in the region of residue 96 (circled). B, model of the TLR4-TIR homodimer (cyan)/Mal TIR domain (magenta) complex (20), indicating the location of Mal-D96 (red, space fill), its proximity to the predicted MyD88 interaction surface (pattern 108–122, yellow), and its distance from the BB-loop region (green) that is vital for the interaction of Mal with TLR4 (cyan).