CADA inhibits the expression of human CD4 in a signal peptide-dependent way.

<p>(A) Schematic representation of the hCD4 C-terminal deletion constructs used. Numbers represent the corresponding amino acids of the pre-protein. The N-terminal SPs are indicated by a black rectangle. In mutant hCD4-426 the intracellular C-terminal domain has been removed. Construct hCD4-CD8 is composed of the D1 and D2 domains of hCD4 fused to the membrane-bound alpha chain of hCD8. CD4⁻ A2.01 human T-cells stably expressing hCD4WT, hCD4-426, or hCD4-CD8 were treated for 48 h with control medium or CADA (8 µM) and stained with anti-CD4 antibody to determine surface expression levels of WT or mutant CD4 by flow cytometry. Graph shows relative receptor expression levels of CADA-treated cells as compared to non-treated controls. (B) Schematic representation of the human/mouse chimaeric constructs used. Numbers represent the corresponding amino acids of the pre-protein. The N-terminal SPs are indicated by a black rectangle. The human/mouse chimaeric constructs are C-terminally fused to YFP for ease of flow cytometric detection. Construct hCD4-mD1 is composed of hCD4 in which the D1 region is replaced by the corresponding D1 domain of mouse CD4. It still contains the SP of hCD4 and the first seven residues of mature hCD4. Construct hCD4-mD2 is composed of hCD4 in which the D2 is replaced by the corresponding D2 domain of mouse CD4. HEK293T cells were transiently transfected with the expression plasmids and left either untreated or treated with CADA for 24 h. YFP fluorescence was determined and normalized to non-treated controls.