The human serpin proteinase inhibitor-9 self-associates at physiological temperatures

Abstract

The metastable serpin architecture is perturbed by extremes of temperature, pH, or changes in primary sequence resulting in the formation of inactive, polymeric conformations. Polymerization of a number of human serpins in vivo leads to diseases such as emphysema, thrombosis, and dementia, and in these cases mutations are present within the gene encoding the aggregating protein. Here we show that aggregation of the human serpin, proteinase inhibitor-9 (PI-9), occurs under physiological conditions, and forms aggregates that are morphologically distinct from previously characterized serpin polymers. Incubation of monomeric PI-9 at 37 degrees C leads to the rapid formation of aggregated PI-9. Using a variety of spectroscopic methods we analyzed the nature of the structures formed after incubation at 37 degrees C. Electron microscopy showed that PI-9 forms ordered circular and elongated-type aggregates, which also bind the fluorescent dye Thioflavin T. Our data show that in vitro wild-type PI-9 forms aggregates at physiological temperatures. The biological implications of PI-9 aggregates at physiological temperatures are discussed.

Figure 1.

Induction of self-association. Ten-microliter samples of PI-9 and α₁-AT at a concentration of 0.2 mg/mL were heated at the temperatures shown for 10 min. Aliquots were then removed and snap frozen and analyzed by 10% nondenaturing-PAGE as previously described (Bottomley and Chang 1997).

Figure 2.

Thermal melts using far-UC CD. PI-9 and α₁-AT (both at a concentration of 0.075 mg/mL) were heated at a rate of 1°C/min and the changes in secondary structure monitored at 230 nm using far-UV CD (Dafforn et al. 2004). (Inset) Changes in PI-9 over the temperature range 45°–60°C.

Figure 3.

Light scatter. Samples of PI-9 and α₁-AT were incubated at 37°C with stirring and the change in turbidity measured at 400 nm.

Figure 4.

Electron microscopy. PI-9 aggregates formed from heating PI-9 at 37°C (A) and α₁-AT polymers formed by heating recombinant α₁-AT at 60°C (B) were adsorbed to a carbon-coated grid and negatively stained with 1% uranyl acetate as previously described (Chow et al. 2004b). The bar represents a scale of 100 nm.

Figure 5.

Thioflavin T binding. The increase in Thioflavin T intensity was measured at an emission wavelength of 480 nm; the data represent the average of three independent experiments. Polymeric PI-9 was formed by incubation of the protein at 40°C and polymeric α₁-AT was formed by incubation at 60°C; both proteins were at a concentration of 4.5 μM and Thioflavin T was at a final concentration of 22.5 μM.