Expression and functional studies on MCP mutants. (A) Flow cytometry analysis of MCP (CD46) expression in PBMCs from 5 mutation carriers (C1X ho: patient F166 099, homozygous for the IVS1 –1G>C mutation; C1Y he: unaffected healthy carrier of family no. 024, heterozygous for the 147G>A mutation, mother of patients F106 and F108; R25X/C1Y: patient F106 24, compound heterozygous for the 218C>T and 147G>A mutations; C65R he: patient S207 199, heterozygous for the 338T>C mutation; 238-242del* he: patient S045 169, heterozygous for the 858-872del 15bp+875C>T mutation) and from healthy controls (Control, n = 6). Percentages of CD46+ cells and median fluorescence intensity (MFI) are presented (control: mean ± SD). PBMCs separated by density gradient centrifugation were incubated with an FITC-conjugated mouse anti–human CD46 monoclonal antibody (mAb) or with FITC-mouse IgG1 (isotype control, empty curve) and analyzed by FACSort. 238-242del* indicates deletion of 238-242 amino acids+D243N+P244S; ho, homozygous; and he, heterozygous. No PBMCs could be obtained from patients carrying the 39–amino acid change + L72Stop, the 62-95del+G96I+Y97I+Y98I+L99Stop, or the F208C or A304V mutations. (B) Western blot of CHO cell lysates probed with a rabbit polyclonal Ab to MCP. Lane 6 shows the phenotype of wild-type MCP as expressed by transfected CHO cells.29 Lanes 1 to 5 are the MCP mutations identified in HUS patients. The precursor form is predominant for the C1Y, C65R, and D243N+P244S mutants, indicating an altered folding with minimal processing to the mature form so that the proteins do not get expressed on the cell surface. Both the C1Y and C65R mutants give a faint signal on Western blot, likely due to degradation of unstable precursor protein. The F208C and the A304V mutations show a normal phenotype on Western blot. Lane 7 is a CHO cell not expressing MCP. (C) C3b (left) and C4b (right) binding activity of MCP derived from lysates of CHO cells. F208C and A304V indicate CHO cells expressing these mutants; MCP pos, CHO cells transfected with wild-type MCP; and MCP neg, CHO cells not expressing MCP. An ELISA format was used for ligand binding in which C3b or C4b were coated onto wells of a microtiter plate. Binding assay was performed using diluted CHO extracts (5 × 106 to 25 × 106 MCP molecules as quantified in ELISA). Data are from 1 representative experiment of 6.
