Secretion and phospholipid binding of the variant 134 compared to wtPS.
(A) Relative transient expression levels of recombinant wtPS and variant 134 in COS-1 cells. The concentrations of PS in cell supernatants were normalized according to reporter control (secreted alkaline phosphatase) values and expressed as a percentage of the wtPS. Each experiment was performed 3 times in duplicate. The error bars represent ± 2 SD. Unpaired t test was used to compare the levels between wild-type and variant 134 (*P = .0007). (B) Pulse-chase analysis of wild-type (WT) and variant 134 PS. Transfected cells were metabolically radiolabeled for 30 minutes and cell lysate (L) and conditioned medium (M) were collected at 0, 2, 4, 8, and 24 hours, immunoprecipitated with a polyclonal anti-PS antibody, and separated by SDS-PAGE. Molecular weight markers were used to calibrate each gel and the arrows identify the PS bands. At 24 hours the amount of PS secreted, as a proportion of the initially labeled material, was 68% ± 1% for wtPS and 36% ± 9% for the 134 variant. (C) Binding of recombinant wtPS and variant 134 to phospholipid. The binding of a range of rPS concentrations (wild-type, ♦; variant 134, ▪) to plates coated with PC/PS/PE (20:30:50) vesicles was detected with a polyclonal anti-PS antibody in the presence of 3 mM CaCl2. We did not detect any nonspecific binding to control wells without immobilized phospholipid or in the presence of EDTA (ethylenediaminetetraacetic acid). A wide range of PS concentrations was tested (0-21.4 nM) showing that variant 134 and wtPS bound to the phospholipid vesicles in a saturable manner with a very similar half-maximal binding.
