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. 2024 Apr 30;27(6):109860. doi: 10.1016/j.isci.2024.109860

Figure 4.

Figure 4

Mutation of the projected ceramide-binding site on ANXA2 (E142A) inhibited ceramide-mediated mitophagy induction and FimA association

(A) Trimolecular modeling of ANAX2, ceramide, and LC3 in MOE.

(B) Docking of ceramide onto ANXA2. Bimolecular modeling of ANXA2 and ceramide was refined from 100 poses in MOE and CLUSPRO, revealing a preference for ceramide to bind ANXA2 on one face of the enzyme, interacting specifically with Glu142 (E142).

(C) Skeletal model of ceramide interacting with residues of interest, particularly E142, on ANXA2.

(D) Western blotting was performed to detect the protein abundance of stably knocked down ANXA2, and transiently transfected V5-tagged mutant or wildtype ANXA2. Actin was used as the loading control.

(E–G) Uninfected UMSCC1A cells stably expressing shRNA against endogenous ANXA2 were transiently transfected with V5-wiltype ANXA2 or V5-E142A ANXA2 pcDNA3.1+ plasmid and treated with LCL768 (30 μM, 2 h) or vehicle control. Cells were dual labeled with ceramide (green) and V5 (red) antibody for confocal imagery. Similarly, ceramide (green) and LC3 (red), and LC3 (green) and Tom20 (red) confocal images are shown. Images represent three independent experiments. Yellow indicates colocalization. Quantification of colocalization in confocal images was estimated via Pearson’s correlation coefficient and normalized to exogenous wildtype ANXA2 expressing, untreated control cells. Values indicate mean ± SD of n = 3 independent experiments. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(H and I) UMSCC1A cells stably expressing shRNA against endogenous ANXA2 were transiently transfected with either V5-wildtype ANXA2 or V5-E142A ANXA2 pcDNA3.1+ plasmid, infected with WT P. gingivalis for 6 h (100 MOI), and treated with LCL768 (30 μM, 2 h) or vehicle control. Confocal microscopy was conducted on cells dual-labelled with FimA and V5-tag antibody. Quantification of colocalization via Pearson’s correlation coefficient was normalized to exogenous wildtype ANXA2 expressing, untreated, uninfected control cells. Values indicate mean ± SD of n = 3 independent experiments. Ns not significant, ∗∗p < 0.01.