Figure 4.

Plasminogen binds to Aspergillus fumigatus enolase (AfEno1) via lysine residues. (A) AfEno1 binding to plasminogen was assayed by enzyme-linked immunosorbent assay (ELISA). AfEno1 at indicated amounts was added to immobilized plasminogen, and bound AfEno1 was detected with rabbit AfEno1 antiserum. AfEno1 bound to plasminogen dose dependently. AfEno1 did not bind to gelatin. (B) Plasminogen binds to AfEno1. Plasminogen at increasing amounts was added to immobilized AfEno1, and bound plasminogen was detected with goat human plasminogen antiserum. AfEno1 showed no binding to gelatin. (C) AfEno1 recruits plasminogen from serum. Normal human serum (NHS) (10 mM EDTA) at different concentrations was added to immobilize AfEno1, and bound plasminogen was detected as above. (D) The binding affinity of plasminogen to AfEno1 was evaluated by biolayer interferometry. Plasminogen at different concentrations (312, 625, 1,250, and 2,500 nM) was added to AfEno1 immobilized on Ni-NTA biosensors. For each concentration, the association was evaluated for 250 s. Following removal of the analyte, the complex dissociation was assessed for another 250 s. Plasminogen binds to AfEno1 with a K_D = 530 nmol/L. Heat-inactivated (95°C) plasminogen showed no binding to AfEno1 (bottom line). (E) The lysine analog ε-ACA blocks plasminogen binding to AfEno1. ε-ACA, at the indicated concentrations, was incubated with plasminogen for 30 min, and the mixture was added to immobilized AfEno1. The bound plasminogen was detected with goat human plasminogen antiserum. (F) The effect of free amino acids on plasminogen binding to AfEno1 was assayed by ELISA. Lysine, arginine, or glutamic acid at increasing concentrations were added to plasminogen, and the mixture was added to immobilized AfEno1. The bound plasminogen was detected as above. Lysine blocked plasminogen bonding to AfEno1. However, arginine and glutamine acid had no effect on plasminogen binding to AfEno1.