Fibrinogen has chaperone-like activity

Abstract

Partially or completely unfolded polypeptides are highly prone to aggregation due to nonspecific interactions between their exposed hydrophobic surfaces. Extracellular proteins are continuously subjected to stresses conditions, but the existence of extracellular chaperones remains largely unexplored. The results presented here demonstrate that one of the most abundant extracellular proteins, fibrinogen has chaperone-like activity. Fibrinogen can specifically bind to nonnative form of citrate synthase and inhibit its thermal aggregation and inactivation in an ATP-independent manner. Interestingly, fibrinogen maintains thermal-denatured luciferase in a refolding competent state allowing luciferase to be refolded in cooperation with rabbit reticulocyte lysate. Fibrinogen also inhibits fibril formation of yeast prion protein Sup35 (NM). Furthermore, fibrinogen rescues thermal-induced protein aggregation in the plasma of fibrinogen-deficient mice. Our studies demonstrate the chaperone-like activity of fibrinogen, which not only provides new insights into the extracellular chaperone protein system, but also suggests potential diagnostic and therapeutic approaches to fibrinogen-related pathological conditions.

Fig. 1

Chaperone activity of FG tested with citrate synthase (CS). (A) Thermal-induced CS aggregation. CS alone (○) or with 1.2 µM BSA (Δ), 0.3 µM FG (□), 0.3 µM clusterin (◊), 0.9 µM FG (■), 0.9 µM clusterin (♦). (B) The influence of ATP on thermal-induced CS aggregation. CS alone (○) or with 1.2 µM BSA (●), IgG (Δ), lysozyme (▲), transferrin (□), 0.9 µM FG (■), 0.9 µM FG plus 2 mM Mg ATP (◊), 0.9 µM FG plus 2 mM ATPγS (♦) at 43 °C. (C) Thermal-induced inactivation of CS at 43 °C. CS alone (○) or with 0.15 µM FG (Δ), 0.3 µM clusterin (□), 0.6 µM clusterin (■), 0.15 µM HSP90 (◊), 1.2 µM BSA (●). (D) Co-immunoprecipitation (IP) of FG and thermal-denatured CS. IP with antibody against FG; IB with antibody against CS.

Fig. 2

Heat-denatured firefly luciferase and CS bound to FG is competent for refolding. (A) Heated mixture of luciferase and FG added into rabbit reticulocyte lysate (RRL) (Δ), heated mixture of luciferase and BSA added into RRL (□), heated luciferase alone added into RRL (○). Error bars represent the standard error from duplicated measurements. (B) Heated mixture of luciferase and FG added into FG solutions, heated mixture of luciferase and FG added into RRL, heated mixture of luciferase and BSA, IgG, transferrin, lysozyme added into RRL. The height of the column represents the highest activity recovered. (C) Reactivation of CS. CS alone (○) or with 0.9 µM FG (●).

Fig. 3

Inhibitory effect of FG on the process of fibril formation of yeast prion protein Sup35 (NM). (A) Fibril formation of Sup35 (NM) without FG (○) and with 0.5 µM (Δ), 2 µM (□), and 5 µM (◊) FG. (B) AFM profile of Sup35 (NM) without or with 5 µM FG after 24 h incubation. (C) AFM profile of Sup35 (NM) without or with 5 µM FG after 96 h incubation. In (B) and (C), left, without FG; right, with FG.

Fig. 4

SDS–PAGE of precipitated proteins from plasma of FG^−/− mice and wild type mice. Lane 1, precipitation in plasma from FG^−/− mice; lane 2, precipitation in plasma from wild type mice.