Plasminogen binding capacity of C. neoformans surface receptors.
(A–B) Representative histograms for JEC21 (A) and B3501A (B) cultured at 25°C for 48 hr in 50 ml YPD. Cells were suspended at 107/ml and labeled for 1 hr at 37°C with 40 µg (solid line), 80 µg (dashed line), or 120 µg (dotted line) plasminogen, followed by exposure to rabbit anti-human plasminogen antiserum and FITC-conjugated secondary antibody. The numbers located above each curve in the histograms indicate plasminogen-labeling concentration (µg) for corresponding populations. Each histogram shows cell number as a function of relative fluorescence obtained for a total of 10,000 events per population. The control population (bold solid line) was treated with primary and secondary antibody in the absence of plasminogen labeling. Greater than 90% of the cells examined stained positive for plasminogen under the growth conditions applied, so gating was not necessary. Each data set is representative of three independent experiments. (C) Plasminogen binding curves for JEC21 (squares) and B3501A (closed triangles) cultured and plasminogen-labeled as in (A–B), averaged from six independent experiments. Data were adjusted for nonspecific binding, which is represented by the baseline. (Kd JEC21 = 900 nM, Kd B3501A = 750 nM). (D) Plasminogen binding curve of JEC21 as detected by Western blot analysis from three independent experiments. Cells were incubated with the indicated concentrations of plasminogen for 1 hr 37°C then examined for surface-bound plasminogen by Western blot analysis. The graph shows the relative signal density detected for the plasminogen concentrations indicated on the abscissa. A representative blot is shown with plasminogen concentrations (µg) concentrations indicated below each band.