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J. Biol. Chem., Vol. 277, Issue 19, 17367-17373, May 10, 2002

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Mutant Neuroserpin (S49P) That Causes Familial Encephalopathy with Neuroserpin Inclusion Bodies Is a Poor Proteinase Inhibitor and Readily Forms Polymers in Vitro^*

Didier Belorgey^§, Damian C. Crowther^¶, Ravi Mahadeva, and David A. Lomas

From the  Respiratory Medicine Unit and ^¶ Neurology Unit, Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge CB2 2XY, United Kingdom

Received for publication, January 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by intraneuronal inclusions of mutant neuroserpin. We report here the expression, purification, and characterization of wild-type neuroserpin and neuroserpin containing the S49P mutation that causes FENIB. Wild-type neuroserpin formed SDS-stable complexes with tPA with an association rate constant and K_i of 1.2 × 10⁴ M¹ s¹ and 5.8 nM, respectively. In contrast, S49P neuroserpin formed unstable complexes with an association rate constant and K_i of 0.3 × 10⁴ M¹ s¹ and 533.3 nM, respectively. An assessment by circular dichroism showed that S49P neuroserpin had a lower melting temperature than wild-type protein (49.9 and 56.6 °C, respectively) and more readily formed loop-sheet polymers under physiological conditions. Neither the wild-type nor S49P neuroserpin accepted the P7-P2 ₁-anti-trypsin or P14-P3 antithrombin-reactive loop peptides that have been shown to block polymer formation in other members of the serpin superfamily. Taken together, these data demonstrate that S49P neuroserpin is a poor proteinase inhibitor and readily forms loop-sheet polymers. These findings provide strong support for the role of neuroserpin polymerization in the formation of the intraneuronal inclusions that are characteristic of FENIB.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

An increasing number of neurodegenerative disorders are recognized to result from the aggregation of proteins within the tissues of the central nervous system (1-3). In conditions such as Alzheimer's disease, Huntington's disease, and the prion encephalopathies, the protein deposition is associated with a conformational transition within the protein and aberrant -strand linkage. Indeed, these and other disorders have now been grouped together as a new class of conditions, the conformational diseases (4-6). Our understanding of the mechanisms underlying these neurodegenerative diseases has been enhanced by the recent description of a new dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB)¹ (7, 8). FENIB is characterized by the accumulation of neuroserpin as periodic acid-Schiff-positive diastase-resistant inclusions or Collins' bodies within the deep layers of the cerebral cortex. The original FENIB kindred presented with presenile dementia and cognitive deficits that are unlike those of Alzheimer's or Huntington's disease (7-10).

A genetic analysis of two families with FENIB revealed point mutations in helix B of the neuron-specific protein, neuroserpin (8). Neuroserpin is an axonally secreted member of the serine proteinase inhibitor or serpin superfamily (11-15). Members of this family share a similar molecular architecture based on a dominant -sheet A and a mobile inhibitory reactive center loop (16). The reactive center loop presents a peptide substrate to the target proteinase. Following docking, the reactive loop is cleaved, and the proteinase is inhibited by translocation over 70 Å to the lower pole of the inhibitor (16, 17). The most probable target for neuroserpin is tissue plasminogen activator (tPA) (13, 18, 19), and it has been suggested that neuroserpin-tPA interactions play an important role in regulating neuronal and synaptic plasticity and memory (20-27).

The "mousetrap" mechanism is central to the inhibitory function of the serpins but renders them susceptible to mutations that facilitate aberrant conformational change (16). This process is best characterized for the serpin ₁-antitrypsin (28, 29). Point mutations of ₁-antitrypsin perturb the relationship between the reactive center loop and -sheet A. This allows the sequential incorporation of the reactive loop of one molecule into -sheet A of another to form chains of polymers that are retained within hepatocytes (30-32). These polymers tangle to form periodic acid-Schiff-positive, diastase-resistant inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma (33). It was very striking that the mutations that underlie FENIB were in the same shutter domain of the serpin molecule as those that caused polymerization of ₁-antitrypsin with accompanying liver disease (8, 34). Indeed, the examination of protein from the brains of affected individuals demonstrated that the inclusions were composed solely of mutant neuroserpin and that this had formed chains of loop-sheet polymers identical to those of mutant ₁-antitrypsin, which accumulate within the liver (8, 9, 35).

Thus, it seems likely that the accumulation of neuroserpin polymers within the brain causes neuronal loss and hence dementia. However, there is evidence that neuroserpin is neuroprotective (36), and that a shift in the balance between plasminogen and plasmin balance can also cause neuronal cell death (37). Moreover, it is impossible to predict whether the shutter domain mutants retain inhibitory activity. We report here the expression, purification, and characterization of wild-type neuroserpin and neuroserpin containing the S49P mutation that underlies FENIB. We show that S49P neuroserpin is not effective as a proteinase inhibitor and rapidly forms polymers under physiological conditions.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Hitchin, UK), and oligonucleotides were synthesized by Invitrogen (Paisley, UK). The expression vector pQE31 was from Qiagen (Crawley, UK). Ampicillin, kanamycin, and isopropyl--D-thiogalactopyranoside were from Melford Laboratories Ltd. (Ipswich, UK). HiTrap chelating HP and HiTrap Q-Sepharose were from Amersham Biosciences. The tPA substrate S-2288 (H-D-Ile-Pro-Arg-para-nitroanilide) was from Chromogenix (Quadratech, Epsom, UK). 1,5-Dansyl-Glu-Gly-Arg-chloromethylketone and tPA were from Calbiochem. Unless otherwise stated, all experiments were carried out in a solution referred to as the buffer (50 mM Tris-HCl, 50 mM KCl, pH 7.4). Synthetic peptides corresponding to the P7-P2² sequence of the reactive center loop of ₁-antitrypsin (FLEAIG) and P14-P3 sequence of antithrombin (SEAAASTAVVIA) were synthesized and purified by Genosys Biotechnologies Inc. (Cambridge, UK) and MWB (Cambridge, UK), respectively. They were dissolved in 50 mM Tris-HCl, pH 7.4, and water, respectively.

Construction and Mutagenesis of Expression Plasmids-- The pUAST vector containing the cDNA for human neuroserpin was kindly provided by Dr. Clare Green (Department of Genetics, University of Cambridge, UK). Neuroserpin was amplified by the polymerase chain reaction and inserted into the expression vector pQE31 by the restriction sites BamHI and KpnI. The S49P mutation was introduced by a two-step polymerase chain reaction. The sequence of the wild-type and mutant neuroserpin was confirmed by DNA sequencing of the entire neuroserpin gene. Recombinant proteins were expressed with a six-histidine tag at the N terminus.

Expression and Purification of Recombinant Proteins-- Escherichia coli were transformed with the plasmid containing wild-type and mutant neuroserpin, and the proteins expressed as described previously for ₁-antitrypsin (38). The cells were harvested by centrifugation, resuspended in buffer A (50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, pH 7.8), and disrupted by sonication. The cell pellet was washed three times with buffer A containing 0.05% v/v Triton X-100 and once again with buffer A alone prior to solubilization in 10 mM Tris-HCl, 100 mM NaH₂PO₄, 6 M guanidinium-HCl, pH 8.0. The protein was then refolded drop by drop overnight at 4 °C in 20 mM Na₂HPO₄, 100 mM NaCl, pH 7.8. This solution was loaded onto a HiTrap chelating column precharged with 0.1 M NiSO₄. The bound protein was eluted as a single peak with an imidazole gradient (0-0.3 M). The fractions were collected, dialyzed against buffer B (20 mM Tris-HCl, 20 mM NaCl, pH 7.4), and loaded onto a HiTrap Q-Sepharose column. The bound protein was eluted with a NaCl gradient (0-1 M), dialyzed against 50 mM Tris-HCl, 50 mM KCl, pH 7.4, concentrated, and then stored at 70 °C. Purified neuroserpin migrated as a single band on SDS-PAGE and >90% was in a monomeric form when assessed by nondenaturing and transverse urea gradient PAGE (39).

Complex Formation Assays-- Wild-type neuroserpin (NS) and mutant neuroserpin (NS(S49P)) were incubated with varying amounts of tPA at 25 °C. Samples were taken at different time intervals, and the reaction was stopped by the addition of 1 mM 1,5-dansyl-Glu-Gly-Arg-chloromethylketone (final concentration) to inhibit any free tPA (40). The samples were then mixed with SDS-PAGE loading buffer, snap-frozen in liquid nitrogen, and stored until the completion of the experiment. They were then thawed and boiled for 3 min. Proteins were resolved on a 10% w/v SDS gel and visualized by staining with Coomassie Blue. The density of the complex band was determined by densitometry scanning with the data being analyzed by a semilog plot against time using the software Quantity One (Bio-Rad).

Determination of the Reaction Parameters Describing tPA Inhibition-- Inhibition rate constants (k_a and k_d) for the inhibition of tPA by NS or NS(S49P) were determined under pseudo-first order conditions, i.e. [I]  10 [E]₀, using the progress-curve method (41, 42). Rate constants of inhibition were measured at 25 °C in inhibition buffer (50 mM Hepes, 150 mM NaCl, 0.01% w/v dodecyl-maltoside, pH 7.4) by adding tPA (20 nM) to a mixture of NS (from 200 to 1000 nM) or NS(S49P) (2000 nM) and the substrate S-2288 (1 mM) and recording the release of product as a function of time. The progress curves were analyzed according to Equation 1 (42, 43):

(Eq. 1)

where v_z is the initial velocity, v_s is the steady-state velocity at completion of the reaction, and k_obs is the pseudo-first order rate constant for the approach to the steady state. The values for the different variables were obtained by fitting the progress curve to Equation 1 using nonlinear regression analysis and were used to calculate k_a according to Equation 2 assuming that the inhibition conforms to a simple bimolecular reaction (42, 43)

(Eq. 2)

where K_m of S-2288 at 25 °C in the inhibition buffer was 1.2 mM (±0.1) as measured independently. The final kinetic parameters are the mean ± S.D. of at least three experiments.

Circular Dichroism-- Circular dichroism (CD) experiments were performed using a JASCO J-810 spectropolarimeter in buffer (50 mM Tris-HCl, 50 mM KCl, pH 7.4). Changes in the secondary structure of NS or NS(S49P) with time and temperature were measured by monitoring the CD signal at 216 nm for 24 h with protein concentrations between 0.5 and 1 mg/ml. The data were fitted to single or double exponential functions. Thermal unfolding experiments were performed by monitoring the CD signal at 216 or 222 nm between 25 and 95 °C using a heating rate of 1 °C/min at a concentration of 0.5 or 1 mg/ml. Melting points (T_m) were calculated using an expression for a two-state transition as described previously (44, 45). The results are the average of three experiments.

Polymerization of NS and NS(S49P)-- Polymerization of NS and NS(S49P) was assessed by nondenaturing PAGE in buffer (50 mM Tris-HCl, 50 mM KCl, pH 7.4). NS or NS(S49P) were incubated at a concentration ranging from 0.25 to 1 mg/ml at a temperature from 4 to 45 °C. Aliquots were taken overtime, and 2 µg of protein were loaded on a 7.5% w/v nondenaturing gel. The proteins were visualized by staining with Coomassie Blue or by silver staining.

Peptide Insertion-- NS or NS(S49P) were incubated for 24 h at 0.5 mg/ml in the presence of a 100-fold molar excess of a 6-mer peptide corresponding to the P7-P2 region of the reactive center loop of ₁-antitrypsin or a 12-mer peptide corresponding to the P14-P3 region of the reactive center loop of antithrombin. As the polymerization of NS or NS(S49P) was fast compared with ₁-antitrypsin and antithrombin, the experiments were conducted at both 37 and 15 °C. The results were analyzed by loading the sample onto a 7.5% w/v nondenaturing gel with or without 8 M urea (46). The presence of polymers was visualized by silver staining.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibitory Activity of NS and NS(S49P)-- The progress curve for wild-type neuroserpin did not reach a plateau, but the absorbance steadily increased with time. This indicates that the mechanism of inhibition is reversible, or that the enzyme adsorbs to the walls of the spectrophotometer cuvette and thus escapes inhibition (47). However, the value of v_s was reproducible for a fixed inhibitor concentration, and the profile of v_s versus [I] was hyperbolic and fitted well to a classical, fully competitive inhibition mechanism. The progressive increase in absorbance can therefore be attributed to the dissociation of the complex. This allowed calculation of the dissociation and association rate constants from Equation 2. The apparent pseudo-first order rate constant k_obs increased linearly with the NS concentration up to 1 µM (Fig. 1A). Within this concentration range, the mechanism of inhibition of tPA by NS is consistent with a one-step reaction (41). A k_a value of 1.2 × 10⁴ M¹ s¹ and a k_d value of 7 × 10⁵ s¹ were calculated by regression analysis of the data using Equation 1 (Table I).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Inhibition of tPA by NS or NS(S49P) at pH 7.4 and 25 °C. A, the effect of NS on kobs, the pseudo-first order constant of tPA inhibition in the presence of 20 nM tPA. The theoretical curve has been calculated using Equation 2 and the best estimates of ka and kd. B, a typical progress curve for the inhibition of tPA at 20 nM by NS(S49P) at 2 µM.

The incubation of mutant NS(S49P) with tPA also resulted in a progress curve that did not reach a plateau (Fig. 1B). The progress curve clearly shows a pre-steady-state release of product followed by a steady state, but in addition there is an increase in the rate of substrate hydrolysis. The first derivative of the data indeed shows an increase in the rate of hydrolysis after 30 min (data not shown). We were not able to follow the inhibition of tPA by NS(S49P) at the lowest concentration used for its inhibition by NS, and therefore, the rate constants k_d and k_a were only determined at a NS(S49P) concentration of 2 µM. Because of the increase in the rate of hydrolysis of the substrate after 30 min, the first 30 min of the progress curve were used to determined the kinetic constants. We calculated values of 0.3 × 10⁴ M¹ s¹ and 1.6 × 10³ s¹ for k_a and k_d, respectively (Table I).

Stability of Complexes-- The stability of the neuroserpin-tPA complex was also assessed by SDS-PAGE. NS forms an SDS-stable complex (Fig. 2A) as has been reported previously (18). After formation of the complex and inhibition of free tPA by 1,5-dansyl-Glu-Gly-Arg-chloromethylketone, there is the appearance of reactive loop-cleaved NS over time. This demonstrates dissociation of the covalent complex. The small amount of cleaved NS at the earliest time point in Fig. 2A, lane 2, shows that the substrate pathway has only a minor effect on the turnover of wild-type NS. Densitometric analysis of the SDS-PAGE gel allowed the calculation of a k_d for the NS·tPA complex of 1 × 10⁴ s¹, a value comparable with that determined by direct measurement (Fig. 2B).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   10% w/v SDS-PAGE to assess the complex formed between tPA and NS or NS(S49P). A, dissociation of the NS·tPA complex at a 2:1 molar ratio at 25 °C. Lane 1, NS; lanes 2-14 correspond to 1-, 5-, 15-, 30-, 60-, 90-, 120-, 150-, 180-, 240-, 300-, 360-, and 480-min time points; lane 15, tPA. Lanes 1-14 contain 2 µg of neuroserpin, and lanes 2-15 contain 2 µg of tPA. The native reactive center loop cleaved and complex (cpx) bands are shown. B, the fractional loss (ln(fxn)) of density of the complex bands on the SDS gel was plotted against time to calculate a kd of 1 × 104 s1. C, the formation of the NS(S49P)·tPA complex over a range of molar ratios. Lane 1, molecular mass markers; lanes 2-8 correspond to NS(S49P):tPA ratios of 1, 2, 3, 4, 6, 8, and 12, respectively; lane 9, NS(S49P); lane 10, tPA. Lane 9 contains 2 µg of NS, and lane 10 contains 2 µg of tPA. NS(S49P) and tPA were incubated for 3 min at 25 °C prior to the addition of 1,5-dansyl-Glu-Gly-Arg-chloromethylketone and then incubated for an additional minute. Loading buffer was then added, and the samples were heated and assessed by 10% w/v SDS-PAGE.

The complex formed between NS(S49P) and tPA more readily favors the substrate pathway. The analysis of the gel is complicated by the fact that the K_i describing the interaction between NS(S49P) and tPA is high (~0.5 µM) as determined kinetically. Even at a 12-fold molar excess of inhibitor, the dissociation process is not negligible (t_1/2 dissociation = ~7 min). Moreover, the complex that forms at a high ratio of NS(S49P) to tPA rapidly dissociates to give the reactive loop-cleaved protein, even if the remaining tPA activity is inhibited by 1,5-dansyl-Glu-Gly-Arg-chloromethylketone (Fig. 2C).

Circular Dichroism-- The melting points for NS and NS(S49P) were measured by monitoring the change in CD signal at 216 and 222 nm (data not shown) while increasing the temperature at 1 °C/min. Fig. 3, A and B, show typical melting curves for NS and NS(S49P) at 216 nm where the change in signal was the most pronounced. In both cases, there was a decrease in signal, but the amplitude was very different. The change in NS was large enough to readily calculate a T_m of 56.6 (±0.3) °C. The change in signal was far less at the same concentration for NS(S49P) with a calculated T_m of 49.9 (±1.2) °C. This difference in amplitude was not the result of a gross conformational transition within NS(S49P), because it had a typical serpin-unfolding transition on transverse urea gradient PAGE (Fig. 3C). These gels also showed that NS was more stable than NS(S49P) with an unfolding transition at ~6 M urea compared with 1 M urea for the mutant protein. NS and NS(S49P) were also incubated at 0.25 mg/ml between 4 and 90 °C for 15 min and then assessed by nondenaturing PAGE. Polymers appeared between 50 and 60 °C for NS and between 40 and 50 °C for NS(S49P), in good agreement with the T_m calculated from the CD spectra (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   CD signal at 216 nm for NS at 1 mg/ml (A) or NS(S49P) at 1 mg/ml (B) at pH 7.4 while increasing the sample temperature at 1 °C/min. C, 7.5% w/v transverse urea gradient PAGE of NS (left) and NS(S49P) (right). The left and right of the gel represent 0 and 8 M urea, respectively.

Polymerization of Neuroserpin-- Differences were observed in the far-UV CD spectra between NS and NS(S49P). Fig. 4A shows that in their native form, the two inhibitors adopt a different configuration. The spectra for NS is very similar to the one observed for native ₁-antitrypsin (48). In comparison, the CD profile for NS(S49P) was significantly more negative with an important increase in magnitude of the signal at 216 nm and a less significant decrease at 222 nm. Spectra were also taken after polymerization at 45 °C for 24 h. In both cases, the spectra were very similar to the one obtained for native NS(S49P) and were consistent with the formation of loop-sheet polymers as observed for ₁-antitrypsin (48). This decrease in the magnitude of the signal at 216 nm was followed over a period of 24 h at different temperatures and used to calculate rates of polymerization (Table II). As noted previously, the amplitude of the change was larger for NS than for NS(S49P) (Fig. 4, B and C). The rate of polymerization of NS was independent of protein concentration over the range of 0.25-1 mg/ml but showed a progressive increase at higher temperatures with a maximum value of 1.32 × 10⁴ s¹ at 45 °C. In comparison, the rate of polymerization at different temperatures was constant for NS(S49P) with a value that was almost 13-fold higher than that obtained for NS at 37 °C.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Far-UV CD spectra of native and polymeric NS and NS(S49P). A, the far-UV CD spectra of native NS (crossed line), NS polymers (solid line), native NS(S49P) (dashed line), and NS(S49P) polymers (dotted line). Polymers were formed by incubating NS or NS(S49P) at 0.5 mg/ml at 45 °C for 24 h and were confirmed by nondenaturing PAGE. The results are the mean ± S.D. of three experiments. B, the change in the CD signal at 216 nm over time for NS at 0.7 mg/ml at 37 °C (upper curve), 41 °C (middle curve), and 45 °C (lower curve). C, shows the change in the CD signal at 216 nm over time for NS(S49P) at 37 °C and 0.5 mg/ml.

The effect of temperature on the polymerization of NS and NS(S49P) was also assessed by nondenaturing PAGE (Fig. 5A). Both NS and NS(S49P) readily formed polymers at 37 °C and 0.25 mg/ml. However, polymers were already visible for NS(S49P) after 2 h of incubation, whereas the ladder of polymers was only clearly visible for NS after 4 h of incubation. After 24 h, both NS and NS(S49P) formed high order aggregates that were stacked at the top of the gel. Similar results were obtained over a concentration range of 0.25 to 1 mg/ml. Polymer formation was also assessed over a range of temperatures. Fig. 5B demonstrates that NS and NS(S49P) formed polymers at temperatures as low as 4 °C. However, whereas NS formed dimers, NS(S49P) formed higher order polymers.

View larger version (63K): [in this window] [in a new window]   Fig. 5.   Polymerization of NS and NS(S49P). A, NS or NS(S49P) were incubated at 37 °C, and 0.25 mg/ml and aliquots taken over time were analyzed by 7.5% w/v nondenaturing PAGE. Lanes 1-5 correspond to a 0-, 2-, 4-, 6-, and 8-h incubation time for NS (lane 1) or NS(S49P) (lane 2). B, the formation of NS or NS(S49P) polymers when monomeric protein was incubated for 24 h at 0.5 mg/ml over a range of temperatures. Lanes 1-5 correspond to 0, 4, 10, 16, and 22 °C incubation temperatures for NS (lane 1) or NS(S49P) (lane 2).

Peptide Insertion-- Neither the 6-mer P7-P2 ₁-antitrypsin peptide nor the 12-mer P14-P3 antithrombin peptide prevented the polymerization of NS or NS(S49P) at 37 °C. The experiments were then repeated at 15 °C in an attempt to reduce the rate of polymerization and hence favor the binary complex between NS and NS(S49P) and the peptide. However, even at 15 °C there was no binary complex between the peptides and NS or NS(S49P) (Fig. 6). We were unable to assess the effects of a 12-mer peptide corresponding to the P14-P3 region of the reactive center loop of neuroserpin because of its poor solubility.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Peptide insertion experiments. 6-mer 1-antitrypsin and 12-mer antithrombin peptides were incubated in 100-fold molar excess with M or Z 1-antitrypsin, NS, or NS(S49P) at 15 °C, and the samples were loaded on a 7.5% w/v nondenaturing gel with 8 M urea. Lane 1, Z 1-antitrypsin; lane 2, Z 1-antitrypsin 24 h; lane 3, Z 1-antitrypsin 24 h + P7-P2 1-antitrypsin 6-mer peptide; lane 4, M 1-antitrypsin; lane 5, M 1-antitrypsin 24 h; lane 6, M 1-antitrypsin 24 h + P14-P3 antithrombin 12-mer peptide; lane 7, NS(S49P); lane 8 is NS(S49P) 24 h; lane 9, NS(S49P) 24 h + P7-P2 1-antitrypsin 6-mer peptide; lane 10 is NS(S49P) 24 h + P14-P3 antithrombin 12-mer peptide; lane 11, NS; lane 12, NS 24 h; lane 13, NS 24 h + P7-P2 1-antitrypsin 6-mer peptide; lane 14, NS 24 h + P14-P3 antithrombin 12-mer peptide. The lower band seen with NS is probably because of a small amount of the latent conformation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The familial dementia, FENIB, is characterized by intraneuronal inclusions of mutant S49P neuroserpin (8). The principal aim of this study was to assess the biochemical differences between NS and NS(S49P), in particular their ability to inhibit tPA, their overall conformation, and the rate at which they formed polymers. Previous work has shown that NS was capable of forming an SDS-stable complex with tPA, but the results also indicated that a proportion of the inhibitor was cleaved (18). It was suggested that this was because of the turnover of NS by tPA in a substrate reaction as has been described for other serpins (16, 18). However, there is evidence that cleaved NS could result from dissociation of the complex. Osterwalder and colleagues (13, 19) have shown the dissociation of the complex between tPA and chicken neuroserpin, which has >80% homology to human neuroserpin and an identical reactive center loop. Our investigations show conclusively that the generation of the reactive center loop-cleaved species of NS was mainly because of the dissociation of the NS·tPA complex.

The progress-curve experiments also demonstrated a dissociation process and allowed us to calculate a k_d of 7 × 10⁵ s¹, close to the one determined directly from the disappearance of the complex on the gel. This value is similar to the value determined for chicken neuroserpin and seems to indicate a general mechanism for the inhibition of neuroserpin in different species. The rate of association k_a determined by our experiments (1.2 × 10⁴ M¹ s¹) was lower than the one determined previously (8 × 10⁴ M¹ s¹) (18). This result may be partly attributed to the use of a recombinant protein produced in E. coli as was previously found for ₁-antitrypsin where the k_a was reduced 2-3-fold compared with the one obtained with ₁-antitrypsin purified from plasma (41). An alternative possibility is that the previous results did not take into account the dissociation effect, thereby overestimating k_a. Nevertheless, NS remains a very good inhibitor of tPA in vitro with an equilibrium dissociation constant K_i in the nanomolar range (5.8 nM). It is not clear whether the dissociation process plays a major role in vivo, in particular in the regulation of tPA activity, because the exact amount of NS in the brain is unknown. This is an important factor, because the yield of NS will determine the in vivo inhibition frequency, and at high concentration, the inhibition by NS could follow a two-step mechanism, leading to an overall faster inhibition of tPA (42).

In contrast with NS, NS(S49P) showed a decrease in k_a and a dramatic increase in k_d with an overall increase in K_i of almost 100-fold. This high K_i prevented the determination of an exact stoichiometry of inhibition, because even at a concentration of NS(S49P) of 5 µM, the dissociation competed with the association process (t_1/2 ~7 min for the dissociation and t_1/2 ~1 min for the association at equimolar concentration). However, the small amount of NS(S49P)·tPA complex that did form as seen on SDS-PAGE (Fig. 2C), even after a relative short period of time, combined with a measurable k_a by our kinetic experiments indicates that both a substrate pathway and dissociation of the complex are possible. These results clearly indicate a loss of function associated with NS(S49P) and suggest that some of the symptoms of FENIB may be because of a lack of proteinase inhibitor (8).

Our results also provide strong support for the role of polymerization in the formation of the inclusions that are characteristic of FENIB (8). The NS(S49P) mutation that underlies FENIB is in the shutter domain of the protein. This region controls access of the reactive center loop to -sheet A and if perturbed will favor polymer formation (8, 48). Whereas this finding was proven by the examination of ex vivo inclusions, the polymerization of wild-type and mutant NS has never been assessed using purified protein. The polymerization rate of recombinant NS(S49P) at 37 °C was 13-fold faster than NS when assessed spectroscopically (Table II). Polymerization was also apparent at lower temperatures for NS(S49P) with the formation of polymers being seen at temperatures as low as 4 °C (Fig. 5B).

The differences between NS and NS(S49P) were assessed by measuring melting temperature and assessing the overall conformation of the protein using CD. A comparison of the melting curve for NS and NS(S49P) showed a melting point 5 °C lower for NS(S49P) (Fig. 3, A and B). An analysis of the CD spectra showed that polymers of NS and monomeric NS(S49P) have a very similar overall configuration, but that monomeric NS and NS(S49P) show striking differences. This does not result from the NS(S49P) being present in the cleaved form or as denatured protein, because it had an unfolding profile on transverse urea gradient PAGE that is characteristic of a native serpin (Fig. 3C). It is likely that the reactive center loop and -sheet A in NS(S49P) are in a conformation that is intermediate between that of wild-type protein and fully formed polymers. Such a conformation was predicted for Z ₁-antitrypsin (and called M*) and later confirmed by our crystal structure of a shutter domain mutant of another serpin ₁-antichymotrypsin (48, 49). In this conformer -sheet A was widely patent and the reactive loop partly inserted. Thus, it is likely that the configuration of the mutant NS(S49P) is similar to that described for M* ₁-antitrypsin and that this explains the differences seen on CD analysis and the ready formation of polymers in vitro and in vivo.

The polymerization of ₁-antitrypsin and other serpins can be blocked by 11-13-mer peptides that correspond to residues P14-P3 of the reactive center loop of antithrombin (30, 50-53). More recently, we have also demonstrated that the conformation of M* in Z ₁-antitrypsin can be specifically targeted using a 6-mer peptide corresponding to residues P7-P2 of the reactive loop that anneals to the lower part of -sheet A (46). Neither of these peptides was able to anneal to NS or NS(S49P) and prevent polymer formation. This demonstrates that NS has more stringent requirements than other serpins for accepting exogenous reactive center loop peptides.

Taken together, our results strongly support the role of polymerization in the aggregation of mutant neuroserpin in the brain of individuals with FENIB. They also show that any NS(S49P) that does not polymerize will be a poor inhibitor of the target proteinase tPA, which may be important in the pathophysiology of FENIB.

	FOOTNOTES

^* This work was supported by the Medical Research Council (United Kingdom) and the Wellcome Trust (United Kingdom).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 44-1223-336825; Fax: 44-1223-336827; E-mail: db301@cam.ac.uk.

Recipient of Wellcome Trust Advanced Clinical Fellowship.

Published, JBC Papers in Press, March 5, 2002, DOI 10.1074/jbc.M200680200

² By convention, serpin-reactive center loop residues are numbered according to the nomenclature of Schechter and Berger (54) for substrates and proteases. The scissile bond in the serpin is denoted as P1-P1'. Residues that are N terminus to P1 are P2, P3, and so forth, and residues C terminus to P1' are P2', P3', and so forth.

	ABBREVIATIONS

The abbreviations used are: FENIB, familial encephalopathy with neuroserpin inclusion bodies; tPA, tissue plasminogen activator; NS, wild-type neuroserpin; NS(S49P), mutant neuroserpin S49P.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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