Extended Data Fig. 4: Extended data for Fig. 3.
From: Epithelial cells remove precancerous cells by cell competition via MHC class I–LILRB3 interaction
a, A computational search using VaPRoS predicted the binding model between MHC class I and LILRB3. The predicted complex of LILRB3 (red) and HLA-G (green) were show in a ribbon diagram and the interaction site in a blue circle, and 8 amino acids (8mer peptides) are further shown and highlighted as blue squares. Amino acid sequences of Human HLA-B and Canis DLA88 (MHC class Ia), which are most similar to the region of HLA-G including the predicted interaction site are shown below HLA-G. Same as MHC class I, amino acid sequence of Canis AltR, which is most similar to the region of LILRB3 including the predicted interaction site is shown below as well. b, Constructs of recombinant proteins and predicted 8mer synthetic peptides. Gray boxes with broken lines show original domain structure of Canis MHC class Ia and LILRB3/AltR. Bold-lined boxes represent structures of recombinant protein or synthetic peptide of Canis MHC class Ia (blue) or AltR (pink). Rec.GST–MHC-α3-Δ8mer–Flag is a truncated mutant of rec.GST–MHC-α3–Flag which lacks the region of the predicted interaction site (amino acid sequence: PISDHEVT). Amino acid sequences of 8mer synthetic peptides are listed as follows: α3-8mer PISDHEVT; AltR-D1-8mer VLYKDGYP; LILRB3-D1-8mer RLHKEGSP; SS, signal sequence region; TM, transmembrane region. c,e, The effects of rec.MHC-α3 and rec.AltR-D1/D2 proteins on apical extrusion. Normal/RasV12-MDCK cells were mixed and cultured with the indicated concentration of rec.GSTDα1/α2–Flag (α1/2) or rec.GST–MHC-α3–Flag (α3) (c), or rec.GST–AltR-D1/D2–HA (D1/D2) or rec.GST–AltR-D3/D4–HA (D3/D4) (e) upon doxycycline treatment for 24 h. The efficiency of apical extrusion is shown in graphs. d, Deletion of the predicted minimal binding site in Canis MHC class Ia cancelled the promotion of apical extrusion by the rec.MHC-α3 treatment. Nomal/RasV12-HaCaT cells were mixed and cultured with 3 μM of rec.GST-α3-Flag (α3) or rec.GST-α3-Δ8mer-Flag protein upon the doxycycline treatment for 16 h. The efficiency of apical extrusion is shown in a graph. f, 8mer synthetic peptides, α3-8mer and D1-8mer, promoted and suppressed apical extrusion, respectively. HaCaT and HaCaT-RasV12 cells were mixed and cultured with α3-8mer, AltR-D1-8mer, or LILRB3-D1-8mer upon the doxycycline treatment for 16 h. The efficiency of apical extrusion is shown in a graph. c-f, Data are presented as mean ± s.d.; n = 3 biologically independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired two-sided Student’s t-test; ns., not significant.
