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Protein Science (2004), 13:1859-1864. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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The human serpin proteinase inhibitor-9 self-associates at physiological temperatures

Lauren N. Benning, James C. Whisstock, Jiuru Sun, Phillip I. Bird and Stephen P. Bottomley

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia

Reprint requests to: Stephen P. Bottomley, Department of Biochemistry and Molecular Biology, Monash University, P.O. Box 13D, Clayton, Victoria, 3800, Australia; e-mail: steve.bottomley{at}med.monash.edu.au; fax: 61-3-9905-4699.

(RECEIVED March 1, 2004; FINAL REVISION April 8, 2004; ACCEPTED April 8, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
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°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°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.

Keywords: protein misfolding; aggregation; conformational disease; serpin

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04715304.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
Serine protease inhibitors (serpins) are a large protein superfamily of which there are at least 800 members (Silverman et al. 2001). Although termed serine protease inhibitors, members of this family also inhibit cysteine proteases and some are noninhibitory (Stein et al. 1990; Schick et al. 1998). These biological functions play critical roles in a diverse range of processes such as coagulation, inflammation, fibrinolysis, apoptosis, neoplasia, viral pathogenesis, protein folding, and complement activation (Carrell et al. 1987; Potempa et al. 1994; Silverman et al. 2001). Members of the serpin superfamily all display similar tertiary structures comprising nine {beta}-helices which surround three {beta}-sheets and a mobile reactive center loop (RCL).

This native structure is metastable, a characteristic that is absolutely required for inhibitory function (Im et al. 1999; Lee et al. 2000; Cabrita and Bottomley 2004). During pro-tease inhibition, the serpin molecule undergoes a dramatic conformational change to a more stable conformation. This favorable change in free energy is used to trap the protease in an inactive state (Kaslik et al. 1995; Huntington et al. 2000; Tew and Bottomley 2001). However, this conformational change, termed the stressed-to-relaxed transition, can also occur in the absence of protease. The serpin architecture is therefore finely tuned, and small changes in solution condition or mutation can cause an inappropriate conformational change that inactivates the molecule. One such state, which is of increasing medical importance, is the polymer conformation, where the RCL of one molecule inserts into the {beta}-sheet of another. Various forms of polymer architecture have been observed, including loop-s4A, loop-C-sheet, loop-s7A, cleaved loop-s4A, and polymers formed through intramolecular and intermolecular disulfide bonds (Lomas et al. 1995; Chang et al. 1997; Bottomley et al. 1998; Huntington et al. 1999; Dunstone et al. 2000; Zhou et al. 2001; Marszal et al. 2003; Wilczynska et al. 2003). Depending on the conditions, {alpha}1AT (serpinA1) can form A- or C-sheet polymers as well as cleaved polymers, and PAI-1 has been shown to form loop-s7A polymers (Bottomley et al. 1998; Huntington et al. 1999; Dunstone et al. 2000; Zhou et al. 2001). Mutations or environmental conditions that destabilize the native state can result in polymerization (Chow et al. 2004a). One of the most well-characterized examples is Z-{alpha}1AT (342Glu-Lys). This mutant polymerizes within the endoplasmic reticulum of hepatocytes resulting in liver cirrhosis while also inhibiting {alpha}1AT secretion (Lomas et al. 1992). Lack of secretion results in a deficiency of circulating plasma {alpha}1AT, which can lead to emphysema (Lomas et al. 1992). Other mutants that have similar disease-causing effects in vivo include the Mmalton (52Phe-del) and Sii-yama (53Ser-Phe) variants of {alpha}1AT (Sproule et al. 1983; Takabe et al. 1992). A number of studies investigating the process of polymerization have been performed. It was initially proposed by Kang et al. (1997) that the polymerization of the {alpha}1AT may be caused by a kinetic trap during protein folding, resulting in the accumulation of the polymerogenic intermediate. Subsequent studies identified a polymerogenic intermediate on the folding pathway, indicating that polymerization in vivo occurs during protein folding (Kim and Yu 1996). Furthermore, kinetic polymerization studies confirmed the formation of an intermediate prior to polymerization (Dafforn et al. 1999).

There is a subgroup of the serpin superfamily known as the ovserpins that have been reclassified as clade b of the serpin superfamily (Remold-O’Donnell 1993; Silverman et al. 2001). Little work has been done on the polymerization behavior of these proteins, even though the intracellular serpin PAI-2 has been shown to polymerize under physiological conditions. Furthermore, it is the only wild-type serpin that has been shown to do so (Mikus et al. 1993; Mikus and Ny 1996). Clade b serpins have been implicated in a variety of processes including regulation of protein processing, cell motility, cell invasion, angiogenesis, tumorigenesis, inflammation, and protection from autolysis by granule proteinases and apoptosis (Sheng et al. 1994, 1996; Zou et al. 1994; Bird et al. 1998; Annand et al. 1999; Zhang et al. 2000). The intracellular serpin proteinase inhibitor-9 (PI-9) otherwise known as serpin B9, is nucleocytoplasmic and expressed in cytotoxic lymphocytes, other immune cells, immune-privileged cells, and epithelial cells, where it probably protects against misdirected granzyme B during an immune response (Sun et al. 1996; Bird et al. 1998; Bladergroen et al. 2001; Buzza et al. 2001).

Here we study the aggregation/polymerization behavior of PI-9. Our data show that PI-9 in vitro aggregates under physiological conditions in a manner that is distinct from all previously studied serpins.


   Results
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 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Thermal stability of PI-9 and {alpha}1-AT
In producing PI-9 for studies in our laboratory we observed that it is particularly prone to aggregation at low or ambient temperatures during purification and storage. This is in marked contrast to the behavior of plasma serpins such as {alpha}1AT, which do not readily polymerize at physiological temperatures or below. Accordingly, we compared the self-association properties of PI-9 with {alpha}1-AT at various temperatures. The proteins were incubated for 10 min at temperatures ranging from 30°C to 80°C and the loss of monomer was assessed using nondenaturing-PAGE (Fig. 1Go). Monomeric PI-9 disappeared between 40°C and 50°C and no protein was detectable in the running gel, indicating that PI-9 had either aggregated or polymerized into long chain polymers that were unable to enter the gel. By contrast, {alpha}1-AT did not begin to polymerize until temperatures in excess of 60°C had been reached.



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  		Figure 1. Induction of self-association. Ten-microliter samples of PI-9 and {alpha}1-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).

 
To investigate this behavior further, we determined the thermal melting temperature (TM) of both PI-9 and {alpha}1-AT. Both proteins were heated at a rate of 1°C/min, and the changes in secondary structure were measured using far-UV CD at 230 nm (Fig. 2Go). {alpha}1-AT shows its characteristic un-folding curve with a transition centered around 61°C, followed by the formation of polymeric material with substantial secondary structure (Fig. 2Go; Dafforn et al. 2004). During the first transition, which is concentration independent (Table 1Go), there is a loss of approximately 15% of the secondary structure.



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  		Figure 2. Thermal melts using far-UC CD. PI-9 and {alpha}1-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.

 

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  		Table 1. Midpoints of thermal unfolding
 
In contrast, PI-9 displays two distinct transitions. The first transition occurs at approximately 52°C, which is 10°C lower than that of {alpha}1-AT. This transition also results in the loss of approximately 15% of the far-UV signal. Further investigation using a range of PI-9 concentrations revealed that this transition is concentration independent (Table 1Go). Following this transition there is an almost total loss of signal as the protein precipitates out of solution in a concentration dependent process (Table 1Go). Taken together, these data indicate that PI-9 is less resistant to heat than {alpha}1-AT. Indeed, the midpoint of 52°C is similar to the disease-causing Z variant of {alpha}1-AT (Dafforn et al. 1999). In addition, it is clear that both proteins form an intermediate species, with a similar loss of secondary structure, which has the propensity to aggregate.

Aggregation properties of PI-9
To investigate the facile aggregation of PI-9, we measured the change in light scatter over time at 37°C (Fig. 3Go). {alpha}1-AT showed no change in light scatter over 4 h, consistent with previous data (Mahadeva et al. 2002). By contrast we observed rapid aggregation of PI-9. This reaction was concentration dependent and completed by 50 min at a concentration of 0.5 mg/mL. Ordered protein aggregation often occurs by a nucleation-dependent mechanism, this is frequently revealed by the presence of a lag phase during which nuclei of misfolded protein form (Chow et al. 2004a). Despite repeated attempts under numerous solution conditions, we were unable to detect any lag phase during the aggregation reaction.



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  		Figure 3. Light scatter. Samples of PI-9 and {alpha}1-AT were incubated at 37°C with stirring and the change in turbidity measured at 400 nm.

 
Traditionally, serpins form polymers that have the appearance of "beads on a string"; however the conformation of PI-9 polymers is strikingly different from those observed previously (Lomas et al. 1992; Devlin et al. 2003). We analyzed the morphology of the PI-9 aggregates formed after heating using electron microscopy. We observed ring-like aggregates that were coalesced into longer strandlike aggregates (Fig. 4Go). We probed the architecture of these aggregates with the fluorescent dye Thioflavin T, which binds to fibrils and protofibrillar species (Rogers 1965; Lashuel et al. 1999). The spectra shown clearly indicate that the aggregates bind Thioflavin T, as indicated by a 30-fold increase in emission intensity (Fig. 5Go). In contrast, {alpha}1-AT polymers prepared at 60°C (pH 7.8) only showed a fourfold increase in Thioflavin T emission intensity (Devlin et al. 2003).



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  		Figure 4. Electron microscopy. PI-9 aggregates formed from heating PI-9 at 37°C (A) and {alpha}1-AT polymers formed by heating recombinant {alpha}1-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.

 


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  		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 {alpha}1-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.

 

   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Incubation of all inhibitory serpins, under partially denaturing conditions such as high temperature or pH, causes loop-sheet polymerization (James and Bottomley 1998; Sivasothy et al. 2000; Devlin et al. 2002; Chow et al. 2004a). During this reaction the RCL of one molecule inserts into a {beta}-sheet of another (Huntington et al. 1999; Dunstone et al. 2000; Sivasothy et al. 2000). This mechanism forms the molecular basis for a number of diseases such as emphysema, liver cirrhosis, thrombosis, and dementia (Lomas et al. 1992; Stein and Carrell 1995). In this study we investigated the aggregation properties of PI-9 and demonstrated that wild-type PI-9 aggregates at physiological temperatures.

One striking feature we observed using electron microscopy was the formation of unusual elongated aggregates, instead of the classical "beads on a string" appearance that was previously reported for other serpins (Lomas et al. 1992). The morphology of the aggregates observed with PI-9 has never been previously reported for a serpin. However, very similar ordered aggregates have been observed for other proteins such as the amino terminal domain of HypF (HypF-N) and the SH3 domain of bovine phosphatidyl-inositol-3'-kinase (PI3-SH3; Bucciantini et al. 2002).

Considering the intracellular cytoprotective functions of PI-9, two questions arise: First, why is PI-9 so easily prone to aggregation, and second, is there a cofactor, a chaperone, or a mechanism inside the cell that stabilizes the native conformation? One possibility is that PI-9’s propensity to aggregate is a reflection of its efficiency as a protease inhibitor. Previous work from our laboratory has shown that PI-9 is an extremely efficient inhibitor of granzyme B and therefore it has the ability to undergo rapid conformational change required for trapping of the target protease (Scott et al. 1999).

The only evidence for polymerization of an ov-serpin inside the cell has been provided by Mikus et al. (1993). They showed that PAI-2 polymerization occurs in the secretory pathway of transfected cells expressing higher than physiological levels of PAI-2. Limited evidence for the existence of PAI-2 polymers in placental cell extracts (which contain elevated levels of endogenous PAI-2) was also provided, with the amount of polymerization increasing on extended incubation of the extracts. However, the interpretation of these studies is difficult as PAI-2 can be enzymatically cross-linked into higher order structures by transglutaminase (Ritchie et al. 2000). We suggest that it is unlikely that PI-9 forms aggregates inside the cell, because it is required to be in an active conformation to function as a proteinase inhibitor, although we cannot discount the possibility that the intracellular concentration of PI-9, which is significantly lower than the concentrations used in our experiments, would also play a role. However, we suggest that there is a cellular mechanism that stabilizes the native structure, preventing aggregation inside the cell, or that aggregation-prone intermediates are efficiently recognized and removed by the surveillance quality control machinery.

In conclusion, PI-9 and {alpha}1-AT are structurally homologous proteins. Our data illustrate that although these proteins adopt a similar native structure, their response to temperature-induced conformational change is extremely different. PI-9 forms circular aggregates rapidly at physiological temperatures, whereas {alpha}1-AT forms ordered polymers only at elevated temperatures. The physiological significance of these findings is under investigation.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Expression and purification of recombinant PI-9
N-terminally hexahistidine-tagged PI-9 was expressed in the methylotropic yeast species Pichia pastoris as previously described with the following modifications (Sun et al. 1995): Following growth and expression induction, cells were subjected to centrifugation for 10 min at 2000g. The cells were washed in H2O and subjected to centrifugation under the same conditions. The pellet was resuspended in lysis buffer (20 mM Tris.HCl at pH 8.0, 5% (v/v) glycerol, 5 mM {beta}-mercaptoethanol, 4 mM PMSF) and lysed with glass beads and vortexing. Three milliliters Talon resin (Clonetech) were added to approximately 50 mL of lysate and incubated for 1 h at 4°C with mixing. The mixture was then used to form a column and subsequent steps were carried out at 4°C. The column was washed with 10 column volumes of 20 mM Tris.HCl, 100 mM NaCl, and 5 mM {beta}-mercaptoethanol and 10 column volumes of 20 mM Tris.HCl, 1 M NaCl, and 5 mM {beta}-mercaptoethanol. Bound proteins were eluted with 250 mM imidazole in 20 mM Tris.HCl (pH 8.0), 20 mM NaCl, and 5 mM {beta}-mercaptoethanol. Fractions containing PI-9 were loaded onto a mono-Q column and eluted using a linear gradient from 50 mM–250 mM NaCl in equilibrating buffer. Fractions containing PI-9 were pooled and stored at 4°C and used within 3 d. The preparation was judged to be more than 95% pure by SDS-PAGE and monomeric by native PAGE analysis. PI-9 concentration was determined as previously described (Pace et al. 1995).

Purification of plasma {alpha}1-antitrypsin
Human plasma {alpha}1-AT was purified as previously described (Lomas et al. 1993).

Induction of self-association
Samples of either PI-9 or {alpha}1-AT were heated at various temperatures at a concentration of 0.2 mg/mL. Samples (10 µL) were taken after 10 min and added to ice-cold nondenaturing PAGE loading buffer and stored on ice until the completion of the experiment. All samples were then analyzed using nondenaturing PAGE, as previously described (Bottomley and Chang 1997).

Circular dichroism spectroscopy
Circular dichroism measurements were performed on a Jasco 810 spectropolarimeter using a thermostated, stirred cuvette as previously described (Dafforn et al. 2004). Protein concentrations ranging from 0.01 mg/mL to 0.1 mg/mL were used.

Turbidity
Samples of either PI-9 or {alpha}1-AT, at concentrations of 0.1 mg/mL and 0.5 mg/mL were added to thermostated, stirred cuvettes and monitored for change in absorbance at 400 nm at 37°C on a Perkin-Elmer LS50B spectrophotometer. {alpha}1-AT and PI-9 were measured at the same time. Stirred cuvettes containing only buffer (for antitrypsin, 50 mM KCl, 50 mM Tris, and 0.1% [v/v] {beta}-mercaptoethanol, for PI-9, 50 mM NaCl, 50 mM Tris, and 1 mM {beta}-mercaptoethanol) were used as blanks before each reading was taken throughout all the experiments.

Electron microscopy
Samples for electron microscopy were prepared by heating to induce polymerization at 0.01 mg/mL. The protein was then adsorbed to a carbon-coated grid and negatively stained with 1% (w/v) uranyl acetate (Chow et al. 2004b).

Thioflavin T binding
Readings were recorded at a wavelength of 480 nm with 10 nm slit widths, on a Perkin-Elmer LS50B spectrofluorimeter. A thermostated cuvette at 45°C in a 1-cm pathlength quartz cell was used. The final concentration of Thioflavin T was 22.5 µM and the protein concentration was 4.5 µM.


   Acknowledgments
 
We thank Weiwen Dai for technical support. We also thank the Australian Research Council and the National Health and Medical Research Council (NH&MRC) for their generous support. S.P.B. is an R.D. Wright Fellow of the NH&MRC and Monash University Senior Logan Fellow. J.C.W. is an NH&MRC Senior Research Fellow and Monash University Logan Fellow.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


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 Discussion
 Materials and methods
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