Fig. 3: Mutant CADM1 affects protein structure leading to changes in intercellular distance.
a, CADM1 variants have shorter α-CTF. Western blot of H295R cell lysates transfected with WT or mutant CADM1 (G379D or V380D) in a pCX4bsr vector using a custom-made anti-CADM1 C-terminal antibody. Shown are the protein bands for glycosylated full-length CADM1 (~100 kDa) and α-CTF CADM1 (15–20 kDa). Complete immunoblot for CADM1 is shown in Supplementary Fig. 2b. Total protein was estimated by immunoblotting β-actin. The experiment was repeated twice independently with similar results. Full blots are provided as Source Data Fig. 3. b, ADAM10/γ-secretase-mediated cleavage of CADM1. Schematic representation of the CADM1 protein. Ectodomain shedding due to cleavage by proteases can result in the formation of intracellular CTF and secreted NTF. Cleavage by the protease ADAM10 after the O-glycosylation site leads to the formation of α-CTF (blue scissors), whereas cleavage before the O-glycosylation site leads to β-CTF. The CTF can undergo further cleavage by γ-secretase (purple scissors) to release an intracellular domain. Glycosylation sites (N- and O-) are shown in brown. The 4.1- and PDZ-binding motifs are shown in orange and blue, respectively. Ig, Immunoglobulin domain. Schematic adapted from refs. 19,35. c, Change in angle of the TM helix in mutant CADM1 can result in shorter α-CTF. Schematic diagram showing that the fixed distance that ADAM10 cleaves CADM1 from the cell membrane (dashed blue line with scissors) and an increase in the angle of the TM helix in mutant CADM1 could result in a shorter length of cleaved α-CTF. d, Predicted changes in TM helix in mutant CADM1 compared to WT. Effect of the CADM1 variants on angle and length of the TM helix in the cell membrane lipid bilayer as predicted by protein modeling data. The 3D structures of the TM domain (residues, A375–L395) of WT, G379D mutant and V380D mutant CADM1 were analyzed by using QUARK program (https://zhanggroup.org/QUARK/). e, Predicted structural consequences of mutant CADM1 on intercellular distance. Schematic representation of change in angle resulting in an increase in intercellular distance in mutant cells.
