Expression analysis of Duffy and CCR5 mutant constructs.

(A) Schematic diagram of Duffy protein showing the known positions of Fy620 and Fy321 epitopes and the position of the single amino acid polymorphism associated with the Fy^bantigen.4 The sequence of the first cytoplasmic domain and the flanking sequences in the transmembrane (TM) domains 1 and 2 are also depicted. The introduced mutations are listed. To test whether the C residue at position C93, by forming an abnormal disulfide bond with R89C, can be responsible for weak expression, the double mutant R89C/C93A and the single C93A control mutation were constructed. R89 was also mutated to S to introduce a relatively minor sulfur-to-oxygen side-chain substitution; to K, a positively charged residue; to H, which is positively charged only depending on its surrounding microenvironment; to a negatively charged E; and to Q, which is similar in structure to E but is neutral. (B) Flow cytometric analysis of Duffy mutants expressed in 293T cells using murine monoclonal anti-Fy6 (clone K60), anti-Fy3 (clone CRC-512-1), and the human polyclonal reagent anti-Fy^b. Values on the y-axis depict percentage of the median fluorescence intensity relative to wild-type Duffy expression after normalizing for transfection efficiency. As reported previously,11 R89C was expressed weakly as compared with wild-type (WT) Duffy. Substitution of C93 with A resulted in wild-type Duffy expression levels, but with the double mutation R89C/C93A, a low-level expression comparable to that of R89C was seen, indicating that the reduced expression of R89C mutant is not caused by disulfide bond formation with the C93 residue. Only the conservative substitution to K rescued expression to wild-type levels. (C) Schematic diagram of CCR5 with the list of mutations introduced into R60 and the sequence of the first cytoplasmic domain and the flanking TM 1 and TM 2. (D) Flow cytometric analysis of CCR5 constructs expressed in 293T cells using 2D7, which recognizes epitopes on the second extracellular loop of CCR5.22 Values on the y-axis depict percentage of the median fluorescence intensity relative to wild-type CCR5 after normalizing for transfection efficiency. (E) Western blot analysis of 293T cells transfected with wild-type and R60S CCR5 expression vectors using monoclonal antibody 1D4, which recognizes a 9-residue rhodopsin tag15 present in the carboxy-terminal of both constructs. CCR5 protein, indicated by the arrow, is expressed more weakly in R60S CCR5 whole-cell extracts than in wild-type CCR5 and is absent in mock-transfected lysates. (F) Sequence comparison of the predicted first intracellular loop of several members of the chemokine receptor superfamily, highlighting the conservation of the positively charged R60 residue.