Introduction

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Conformational disorders arise when a protein undergoes a change in topology that leads to self-association, tissue deposition, and disease (1, 2). The serine proteinase inhibitor, or serpin, superfamily includes some of the best-characterized proteins involved in this group of disorders. The most extensively characterized serpin is ₁-antitrypsin (₁-AT), which is composed of 394 amino acids arranged into three -sheets (A, B, and C), nine -helices (A through I), and a mobile inhibitory reactive center loop (3, 4). The severe Z deficiency allele of ₁-AT (³⁴²Glu Lys) is carried by 4% of the Caucasian population and results in the accumulation of ₁-AT at its site of synthesis within the endoplasmic reticulum of the hepatocyte. This accumulation of protein results in neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma (5). The lack of circulating protein predisposes the Z homozygote to early onset panlobular emphysema (6).

The accumulation of Z ₁-AT within hepatocytes results from an ordered polymerization process that is initiated by protein misfolding (7). In the case of the Z variant, the rate of protein folding is much slower than that of the wild-type M ₁-AT (7, 12). This leads to the accumulation of a long-lived intermediate with the propensity to polymerize, especially during transient increases in core body temperature, such as fever (13, 14). The conformation of the polymerogenic intermediate of ₁-AT is unknown but we have predicted that the Z mutation causes an expansion of the dominant A -sheet and partial insertion of the reactive center loop (15). This opening of -sheet A then allows the reactive center loop of another molecule to insert and so to initiate the polymerization process. Z ₁-AT can also polymerize in vivo from the native state inasmuch as polymers have been identified in the lung lavage fluid of Z homozygotes with emphysema (16). Polymerization has also been shown to occur in the circulation with the Siiyama and Mmalton deficiency variants of ₁-AT (17), and this process underlies the deficiency of other members of the serpin superfamily: antithrombin, ₁-antichymotrypsin, C1-inhibitor, and neuroserpin in association with thrombosis (14, 18), emphysema (19), angioedema (20, 21), and dementia (22), respectively.

The most effective inhibitors of serpin polymerization are peptides with homology to the reactive center loop. These can anneal to the A -sheet of the native molecule and so prevent insertion of the loop of a second molecule (23). However, the reactive loop peptides are promiscuous and can also anneal to the A -sheet of other serpins (24). This peptide incorporation inactivates the serpin as a proteinase inhibitor and so is not an ideal therapy to prevent polymerization in vivo. We have therefore explored other strategies to stabilize the serpin and thus ameliorate conformational disease. Trimethylamine N-oxide (TMAO) is an osmolyte found in the cells of elasmobranchs that has been demonstrated to stabilize intracellular proteins against denaturant stress (25, 26). In vitro, TMAO has been shown to protect proteins from thermal denaturation and aggregation (27). More recently, TMAO has been shown to force destabilized proteins to fold to a native-like state (30, 31). We show here that TMAO can block the polymerization of native and the severe Z deficiency variant of ₁-AT. These findings open new therapeutic strategies to ameliorate serpin polymerization in vivo.

	Materials and Methods

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Chemicals

TMAO (Sigma Chemical Co., St. Louis, MO) was prepared analytically by weight and the concentration determined from the refractive index, as previously described (32). 4,4'-di-anilino-1,1'-binaphthyl-5,5'-disulfonate (bis-ANS) was purchased from Molecular Probes (Eugene, OR).

Production of Human ₁-AT

M and Z ₁-AT were prepared from the human plasma of known homozygotes by 50 and 75% ammonium sulfate fractionation, followed by thiol exchange and Q-sepharose chromatography as described previously (13). The purity of the preparation was checked by both sodium dodecyl sulfate- and nondenaturing polyacrylamide gel electrophoresis (PAGE). ₁-AT was stored in 50 mM Tris, 50 mM NaCl, and 0.1% (vol/vol) -mercaptoethanol, pH 7.8, and its concentration was determined by measurement of ultraviolet (UV) absorbance at 280 nm using an extinction coefficient (1 mg/ml) of 0.52.

Thermal Deactivation

The thermal stability of ₁-AT in the absence and presence of TMAO was assessed using two assays. Gel analysis of the polymerization reaction was accomplished by first incubating each protein sample in 50 mM Tris and 50 mM NaCl, pH 7.8, at 60°C. Samples were then removed at various time intervals and placed directly into ice-cold nondenaturing loading buffer for subsequent analysis. Nondenaturing PAGE was performed as previously described (33). Heat stability was also followed by measuring the kinetics of inactivation at 60°C. ₁-AT was incubated at 60°C in 50 mM Tris, 50 mM NaCl, and 10 mM CaCl₂, pH 7.8; aliquots were removed at various time intervals; and the amount of remaining ₁-AT activity was determined as previously described (11).

Determination of the Inhibitory Parameters

The association rate constant (k_ass) for the interaction of ₁-AT with bovine -chymotrypsin was determined by progress curve kinetic experiments (34). The assays were performed at 37°C in reaction buffer containing 50 mM Tris and 50 mM NaCl, pH 7.8, and differing concentrations of TMAO. The reaction was started by the addition of 0.1 nM chymotrypsin and the progress of inhibition was determined by continuously monitoring the appearance of p-nitroaniline at 405 nm. The stoichiometry of inhibition (SI) between chymotrypsin and ₁-AT was determined as described previously (34). The reaction mixture in the assay contained 50 nM chymotrypsin, with the residual enzyme activity being determined after incubation with varying concentrations of ₁-AT for 15 min at 37°C.

Fluorescence Measurements

All intrinsic tryptophan fluorescence measurements were performed in 50 mM Tris and 50 mM NaCl, pH 7.8. The fluorescence measurements were performed on a Perkin-Elmer LS50B spectrofluorimeter; a constant temperature was maintained with a thermostatted cuvette holder and a circulating water bath. For all of the kinetic measurements the solution in the cuvette (1 ml) was constantly stirred and the fluorescence data recorded every 1 s. The excitation and emission slit widths were 5 and 4 nm, respectively. The excitation wavelength (_ex) was 390 nm and the emission wavelength (_em) was 480 nm. The effect of TMAO on ₁-AT in the presence of bis-ANS was determined as detailed previously (13).

Circular Dichroism

Circular dichroism (CD) spectra were measured on a Jasco 820s spectropolarimeter at 25°C. Far UV spectra from 190 to 250 nm were collected with 5 s/point signal averaging. Changes in secondary structure with temperature were measured by monitoring the CD signal at 230 nm and a protein concentration of 0.05 mg/ml in 50 mM Tris and 50 mM NaCl, pH 7.8. The temperature within the cuvette was maintained by a computer-controlled water bath connected to a water jacket integral to the cuvette holder and monitored by a sensor directly located in the holder. Thermal unfolding was performed using a heating rate of 60°C/h.

	Results

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The polymerization of ₁-AT occurs within the hepatocyte as the nascent polypeptide chain folds to its tertiary structure (7, 35). Polymerization can also occur from the folded state, inasmuch as polymers of Siiyama and Mmalton ₁-AT have been detected in the circulation in vivo (36, 37) and polymers of Z ₁-AT have been identified in the lungs of Z ₁-AT homozygotes with emphysema (16). In this study we examined the effect of TMAO upon polymer formation by both of these pathways, i.e., the effect on the refolding of denatured protein and the effect on polymer formation from correctly folded, but unstable, protein.

Stabilization of Native ₁-AT by TMAO

TMAO had no effect on the fluorescence and CD spectra of M ₁-AT at concentrations of up to 3 M (data not shown). In addition, the SI and the k_ass for the interaction of M ₁-AT with chymotrypsin were unaffected by increasing concentrations of TMAO (Table 1). These data indicate that the osmolyte has no effect on the structure and activity of the protein as resolved by these techniques. Heating of M ₁-AT has been shown to produce polymers through the same mechanism as those that result from the Z mutation in vivo (7, 11, 16). M ₁-AT was incubated at 60°C and aliquots were removed at various time points, placed on ice, and analyzed using native PAGE (Figure 1A). This rapid quenching on ice inhibited further polymerization (data not shown). Polymers of ₁-AT formed rapidly at 60°C with little monomeric ₁-AT remaining after 30 min of incubation (Figure 1A). TMAO inhibited this polymerization process with monomeric ₁-AT being present after at least 120 min incubation at 60°C (Figure 1B). Longer incubations of ₁-AT in the presence of 2 M TMAO produced polymers of similar length to those without TMAO (data not shown), suggesting that the polymers were still formed in a similar manner but that their rate of formation was markedly reduced.

View larger version (61K): [in this window] [in a new window]   Figure 1.   Nondenaturing PAGE (10% wt/vol) of the polymerization of M 1-AT. M 1-AT (10 µM) was incubated at 60°C in the absence (A) and presence (B) of 2 M TMAO for the times indicated. Aliquots were removed and rapidly added to ice-cold nondenaturing loading buffer before electrophoresis.

The rate of thermal inactivation at increasing TMAO concentrations was then assessed with both M and Z ₁-AT by titration against chymotrypsin (Table 1 and Figure 2). These experiments were performed by adding ₁-AT to buffer containing TMAO already equilibrated at 60°C. Aliquots were removed and placed on ice until the end of the incubation. TMAO dramatically increased the stability of Z (Figure 2) and M ₁-AT (Table 1) in a concentration- dependent manner. Z ₁-AT lost activity, with a half-life of 1.8 min when heated at 60°C in the absence of TMAO. However, the half-life was increased by over 100-fold, to almost 200 min, when incubated at 60°C in the presence of 3 M TMAO. The effect on M ₁-AT was even more dramatic, with the half-life being extended from 4 min under control conditions to 550 min in 3 M TMAO.

View larger version (15K): [in this window] [in a new window]   Figure 2.   Heat inactivation of Z 1-AT. Z 1-AT (0.05 mg/ml) was incubated at 60°C with increasing concentrations of TMAO (squares, 0 M; triangles, 1 M; inverted triangles, 2 M; and diamonds, 3 M). Aliquots were taken at various times and placed on ice to prevent further polymerization. The aliquots were subsequently assayed for inhibitory activity against chymotrypsin. The result is representative of five independent experiments.

Previous work has shown that the binding of bis-ANS to ₁-AT during heat-induced serpin polymerization occurred in two distinct phases (8, 10). An initial rapid conformational change was accompanied by a large increase in bis-ANS fluorescence, followed by a slow decrease in bis-ANS fluorescence that represented polymer formation. The polymerization of ₁-AT was then repeated with bis-ANS in the presence and absence of TMAO (Figure 3, Table 1). TMAO reduced the initial rate and amplitude of fluorescence change in both M and Z ₁-AT (Table 1). In the absence of TMAO an initial rate of 0.34 min¹ was observed for M ₁-AT. However, in the presence of 3 M TMAO the initial rate decreased by approximately 560-fold to 6 × 10⁴ min¹. The rate of the fluorescence change was reduced by 120-fold for Z ₁-AT with 3 M TMAO. These data indicate that TMAO exerts much of its action by inhibiting the initial structural changes that occur during ₁-AT polymerization.

View larger version (13K): [in this window] [in a new window]   Figure 3.   Measurement of Z 1-AT polymerization followed by bis-ANS fluorescence. 1-AT was added to buffer containing increasing concentrations of TMAO and bis-ANS prewarmed to 60°C. The fluorescence emission (ex = 390 nm, em = 480 nm) of the bis-ANS was then constantly monitored. The result is representative of five independent experiments.

The protective effect of TMAO was characterized further by measuring the melting temperature of the protein using CD in the presence and absence of TMAO. M ₁-AT, at 0.05 mg/ml, was heated to 90°C at a rate of 1°C/min. TMAO increased the melting temperature (T_M) in a concentration-dependent manner, with the T_M of M ₁-AT increasing by approximately 10°C in 3 M TMAO (Figure 4A and Table 1). Upon cooling of the samples at the same rate (1°C/min), the CD signal did not return to baseline (Figure 4B). Native PAGE analysis of the samples after heating and cooling showed that the protein had completely polymerized. Thus, the transition observed upon heating native ₁-AT is the generation of the polymeric species.

View larger version (37K): [in this window] [in a new window]   Figure 4.   Thermal denaturation of 1-AT. (A) The TM of M 1-AT in the presence of increasing concentrations of TMAO, as measured by monitoring the change in CD signal at 230 nm while increasing the sample temperature at 60°C/h. The result is representative of five independent experiments. (B) A thermal melt of 1-AT (continuous line) followed by the subsequent cooling from 90°C to 20°C (broken line), at a rate of 60°C/h.

Induction of ₁-AT Polymerization by TMAO upon Renaturation

The studies so far have investigated the effect of TMAO on native ₁-AT. Polymerization can also occur in vivo during protein folding as an off-pathway phenomenon. The effect of TMAO was therefore assessed on the refolding of denatured ₁-AT. M ₁-AT was unfolded in 5 M guanidine hydrochloride (GuHCl) for 30 min before its dilution into refolding buffer in the presence or absence of 1.5 M TMAO. Refolding was allowed to proceed for 4 h. The final concentration of protein was 0.05 mg/ml and the GuHCl was diluted to less than 0.1 M. Samples of the renatured material were then analyzed by native PAGE. A silver-stained gel demonstrated that ₁-AT renatured to a monomeric species in the absence of TMAO (Figure 5, lane c). However, upon refolding in the presence of 1.5 M TMAO, extensive polymerization of the protein was observed (Figure 5, lane b). These results were consistent over a 100-fold protein concentration range (200 nM to 20 µM) and coincided with a loss of > 90% inhibitory activity against chymotrypsin.

View larger version (71K): [in this window] [in a new window]   Figure 5.   Silver-stained nondenaturing PAGE (10% wt/ vol) of refolded 1-AT. 1-AT was unfolded in 5 M GuHCl at room temperature for 60 min. Aliquots of the unfolded material were then diluted into buffer and allowed to refold for 4 h. The final concentration of 1-AT was 0.05 mg/ ml and the final concentration of GuHCl was 0.1 M. Lane a, 1-AT that has not been unfolded and was loaded onto the gel at the same concentration as the refolded samples; lane b, 1-AT refolded into buffer containing 1.5 M TMAO; lane c, 1-AT refolded into buffer containing no TMAO.

The kinetics of refolding of ₁-AT in TMAO were then assessed using bis-ANS. Previous studies have demonstrated that the intermediate present on the equilibrium folding pathway of ₁-AT coincides with a dramatic increase in bis-ANS fluorescence. Denatured protein was added into refolding buffer containing bis-ANS and the change in fluorescence was followed with time. Both of the refolding experiments fitted well to a single exponential decay, indicating the formation of an intermediate within the dead time of the experiment, approximately 10 s (Figure 6). This represents the transition from the intermediate state to the fully folded protein, which occurs with a rate constant of 0.3 min¹. Refolding in the presence of 1.5 M TMAO, which results in polymer formation, occurred with a rate constant of 1.8 min¹. Refolding in TMAO resulted in a final signal intensity that was almost 6-fold higher than that observed in the absence of TMAO. This is consistent with the formation of polymeric protein and our previous equilibrium data (10).

View larger version (17K): [in this window] [in a new window]   Figure 6.   Refolding of 1-AT monitored by bis-ANS fluorescence. 1-AT was unfolded in 5 M GuHCl. An aliquot of this material was added to a constantly stirred cuvette containing refolding buffer with 5.0 µM bis-ANS, and 1.5 M TMAO as appropriate. The final concentration of protein was 1.0 µM and GuHCl was less than 0.1 M. The change in fluorescence emission (ex = 390 nm, em = 480 nm) of the bis-ANS was then constantly monitored. The fluorescence of bis-ANS in the presence of unfolded 1-AT is marked with an arrow.

	Discussion

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Recently, a group of diverse proteins that includes the serpin superfamily, prion, -amyloid, and glutamine-repeat proteins were classified as Conformational Diseases (1, 2). All of these proteins have the ability to undergo unique conformational changes that result in their self-association and tissue deposition, causing diseases such as the encephalopathies and dementias, emphysema, and thromboembolism. The best characterized of these is the severe plasma deficiency of ₁-AT. Z ₁-AT aggregates predominantly through an ordered polymerization process within the hepatocyte (35). This process is initiated by a kinetic trap in the folding pathway that increases the lifetime of a partially folded intermediate (7). The Z ₁-AT that is normally folded and secreted can also polymerize, which indicates that Z ₁-AT polymerization can occur via two distinct pathways: (1) from unfolded protein, and (2) from correctly folded and secreted ₁-AT. It is likely that under stress conditions, such as increased temperature, both of these processes are accelerated, resulting in the accumulation of polymeric material (13, 35). Chemical chaperones have been demonstrated to enhance both the ability of unstable proteins to fold (30, 31, 38) and to increase their conformational stability (39, 40); this dual effect prompted us to examine their role in preventing the polymerization of ₁-AT.

TMAO has a solvophobic effect on the peptide backbone; that is, it drives the folding of proteins by forming unfavorable interactions with the peptide backbone of the unfolded state (32). Thermal induction of the polymerization of Z and M ₁-AT involves partial unfolding of the molecule, specifically expansion of the A -sheet (8, 10). This exposes more of the peptide backbone, which would be unfavorable in the presence of TMAO. Our data demonstrate that TMAO dramatically stabilizes the native form of ₁-AT, thereby slowing the conformational transition underlying the polymerization of both M and Z ₁-AT. Moreover, this substantial stabilization of ₁-AT was achieved without impairment of inhibitory activity. These studies indicate that TMAO, and similar rationales that stabilize the native state, may be useful in protecting circulating ₁-AT against partial unfolding and subsequent inactivation by polymerization.

The TMAO-induced stabilization of the native state of ₁-AT is complicated by our finding that the osmolyte has a deleterious effect on the refolding of denatured protein, actually enhancing polymerization. Like all inhibitory serpins, the native state of ₁-AT is metastable; the thermodynamic minimum state being the reactive loop cleaved, latent, or polymeric structure, in which the A -sheet consists of six rather than five strands (41). Our refolding data is consistent with this because TMAO induced denatured ₁-AT to form the polymeric conformation. These findings explain the recent results of Burrows and colleagues (42), who showed that TMAO did not rescue Z ₁-AT from polymer formation in cell culture models of ₁-AT deficiency.

It has been proposed that ₁-AT folding proceeds through one intermediate that can partition into either the native or polymeric form (8, 10). The presence of only one intermediate in the bis-ANS refolding traces and the speed of the reaction make it unlikely that the native state is formed on the refolding pathway to polymer formation. Therefore, the presence of TMAO must lower the energy barrier dramatically between the intermediate state and the polymeric form in preference to the native state. Indeed, the bis-ANS-monitored refolding kinetics showed that ₁-AT polymer formation at 25°C in the presence of 1.5 M TMAO occurred within 3 min, which is considerably faster than either polymerization at 60°C (which takes over 30 min to complete) or folding to the native state (which takes over 15 min). These data suggest that changes in protein hydration caused by TMAO accelerate the major steps in the polymerization pathway and highlight the importance of hydration effects in controlling protein-protein interactions.

Taken together, our data suggest that TMAO may be useful in stabilizing folded Z ₁-AT that has been secreted into the circulation but is unlikely to be of value in helping Z ₁-AT to fold into a native conformation within the endoplasmic reticulum of the hepatocyte. Moreover, the data highlight the intricacies in designing strategies to prevent the aberrant folding of ₁-AT.