Neuroserpin Portland (Ser52Arg) is trapped as an inactive intermediate that rapidly forms polymers Implications for the epilepsy seen in the dementia FENIB Didier Belorgey 1 , Lynda K. Sharp 1 , Damian C. Crowther 1 , Maki Onda 2 , Jan Johansson 3 and David A. Lomas 1 1 Cambridge Institute for Medical Research, Department of Medicine, University of Cambridge, UK; 2 Department of Environmental Sciences, Faculty of Science, Osaka Women’s University, Sakai, Japan; 3 Department of Molecular Biosciences, Swedish University of Agricultural Sciences, Uppsala, Sweden The dementia f amilial e ncephalopathy with n euroserpin inclusion bodies (FENIB) is caused by point mutations in the neuroserpin gene. We have shown a correlatio n between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relationship with t he age of onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential i nteraction between the reactive centre loop of one neuroserpin molecule with b-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms l oop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The P ortland mutant h as a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, w ith those o f t he CD analysis, are in keeping with the r eactive centre l oop of neuroserpin Portland being partially inserted into b-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclu- sions and t he severity of dementia in FENI B associated with neuroserpin P ortland. Moreover the inactivity of the m utant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in indi- viduals with more severe forms of the disease. Keywords: c onformational d iseases; neuroserpin; polymer- ization; serpin; serpinopathies. The autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) results from point mutations in the neuroserpin gene and is c haracterized by inclusions of neuroserpin within c ortical a nd subcortical neurons [1–3]. Neuroserpin is a member of the serine proteinase inhibitor or serpin s uperfamily. It inhibits the enzyme tissue plasminogen activator (tPA) and may be important in regulating neuronal plasticity and memory [4–7]. We have recently expressed, purified, and character- ized wild-type neuroserpin and neuroserpin with the Ser49Pro mutation, w hich was identified in the fi rst r eported family with F ENIB [6]. T he mutation reduced the inhibitory activity of neuroserpin by  1 00-fold and increased the formation of polymeric protein under physiological condi- tions. Neuroserpin polymers result from the sequential insertion of t he reactive centre loop of one molecule into b-sheet A of another [1,6]. The resulting s pecies is inactive as a proteinase inhibitor and accumulates in the endoplasmic reticulum in cell models of disease [8] and in vivo [2]. Three other mutants of neuroserpin are now recognized to cause FENIB: Ser52Arg, His338Arg and Gly3 92Glu [3]. This condition is unusual among neurodegenerative dis- orders in that there is a striking correlation between the number of inclusions within the cerebral cortex and an inverse relationship with the age of onset of disease [3]. F or example, individuals with the Ser52Arg and Gly392Glu neuroserpin mutation h ave 3 and 9.5 times more inclusions within the cerebral cortex than individu als with the Ser49Pro mutant. This c orresponds t o an age of onset of symptoms in individuals with Ser49Pro, Ser52Arg and Gly392Glu neuroserpin o f 48, 24, and 13 years, respectively. There is also a change in phenotype, with the S er49Pro mutation causing predominantly dementia whereas the Ser52Arg, His338Arg and Gly392Glu mutants cause both dementia and severe progressive epilepsy. In addit ion to the striking genotype–phenotype correlation, FENIB is also unusual in that the mutant neuroserpin forms ordered polymers within the endoplasmic reticulum [1,8]. This contrasts with other conditions such as Parkinson’s and Huntington’s disease in which the mutant proteins form disordered aggregates within the cytoplasm [9]. We have expressed and characterized the Ser52Arg variant of neuroserpin (neuroserpin Portland) to determine if the rate of polymer formation can explain t he correlation between the mutation, the number of intraneuronal inclusions, and Correspondence to D. Belorgey, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 2XY, UK. Fax: +44 1223 336827, Tel.: +44 1223 336825, E-mail: db301@cam.ac.uk Abbreviations: FENIB, familial encephalopathy with neuroserpin inclusion bodies; tPA, tissue plasminogen activator. (Received 20 March 2004, revised 2 8 May 2004, accepted 28 June 200 4) Eur. J. Biochem. 271, 3360–3367 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04270.x the clinical phenotype. Our d ata show that the Ser52Arg mutation favours t he rapid f ormation of polymers as the protein is locked as an inactive folding intermediate. These polymers explain the increased number of inclu sions in individuals with Ser52Arg compared with those with t he Ser49Pro mutation. Moreover the inactivity of the mutant may result in uncontrolled activity of tPA, and so explain the epileptic seizures seen in individuals with more severe forms of the disease. Materials and Methods Materials Oligonucleotides were synthesized by MWG-Biotech AG (Ebersberg, Germany). The expression vectors pQE81L and Ni-nitrilotriacetate agarose were from Qiagen (Crawley, Sussex, UK), HiTrap Q Sepharose was from Amersham Biosciences (Chalfont St Giles, Bucks., UK), and the tPA substrate S-2288 (H- D -Ile-Pro-Arg-p-nitroanilide) was from Chromogenix (Quadratech, Epsom, Surrey, UK). 1,5-Dansyl-Glu-Gly-Arg-chloromethylketone and tissue plasminogen activator (tPA) were from C albiochem (Merck Biosciences, Nottingham, UK). Mineral oil was from either Sigma Chemical Co. (catalogue number M-3516, M-8410, M-5904, M-1180, M -5310) or f rom Fluka ( Buchs, Switzerland; catalogue number 69808). Expression and purification of recombinant proteins The Ser52Arg mutation was introduced into the cDNA of human neuroserpin in the p QE81L expr ession vector [6] b y a two-step PCR. The gene was fully sequenced to ensure that there were no PCR errors. Recombinant wild-type, Ser49Pro and Ser52Arg neuroserpin were expressed with a six-histidine tag at the N-terminus and purified as described previously [6], except that the HiTrap chelating column was replaced by Ni-nitrilotriacetate agarose. The result- ing p roteins w ere a ssessed by SDS, nondenaturing and transverse urea gradient PAGE, and activity was assessed against tPA [6]. Complex formation assays Wild-type and Ser52Arg neuroserpin were incubated i n various ratios with tPA at 2 5 °C as described previously [6]. Samples were t aken at different t ime intervals, and t he reaction was stopped by the addition of 1 m M 1,5-dansyl- Glu-Gly-Arg-chloromethylketone (final concentration) to inhibit any free tPA [10]. The s amples 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 separated by SDS/PAGE [10% ( w/v) gel] and visualized by staining with Coomassie Blue. Determination of the reaction parameters describing tPA inhibition Inhibition rate constants for the inhibition of tPA by wild-type or mutant neuroserpin were determined under pseudo-first-order conditions, i.e. [I] ¼ 10[E] 0 ,usingthe progress-curve method [11,12]. Rate constants o f inhibition were mea sured at 25 °C i n i nhibition buffer [50 m M Hepes, 150 m M NaCl,0.01%(w/v)dodecylmaltoside,pH7.4]by adding tPA ( 20 n M ) to a mixture of wild-type ( from 200 n M to 1000 n M ) or Ser52Arg (6600 n M ) neuroserpin and the substrate S-2288 (1 m M ) and recording the release of product as a function of time. The progress curves were analyzed as described previously [12,13]. CD CD experiments were performed using a Jasco J-810 spectropolarimeter in 100 m M sodium phosphate buffer, pH 7.4. Polymers were formed by heating wild-type or mutant neuroserpin at 0.5 mgÆmL )1 and 45 °C f or 24 h. Changes in t he secondary structure of wild-type or Ser52Arg neuroserpin with time and temperature were measured by monitoring the CD signal at 216 nm for 24 h with protein at a concentration of 0 .5 mgÆmL )1 .When possible, the data were fitted to a single exponential function. Thermal unfolding experiments were performed by monitoring the CD s ignal at 2 16 nm in a 150-lLcuvette between 25 °Cand95 °C using a heating rate of 1 °CÆmin )1 at a concentration of 0.7 mgÆmL )1 . The second derivative of the resulting data was used to calculate the inflection point of the transition and hence the T m [14]. Assessment of the polymerization of wild-type and Ser52Arg neuroserpin Polymerization of wild-type, Ser49Pro and Ser52Arg neu- roserpin was assessed by incubating the protein at concen- trations of 0.1 or 0.4 mgÆmL )1 in NaCl/P i ,pH7.4,at37°C or 45 °C. Aliquots were taken over time, and 2 lgprotein was loaded on a 7.5% (w/v) nondenaturing gel. To avoid evaporation during the experiment, the different samples were covered w ith mineral oil. The proteins were visualized by staining with GelCode Ò Blue Stain R eagent (Pierce, Tattenhall, Cheshire, UK) or by silver staining. Unfolding of wild-type and Ser52Arg neuroserpin in urea Neuroserpin at 25 lgÆmL )1 was incubated at 20 °Cwith various concentrations of urea (from 0 to 9 M )in50m M sodium phosphate buffer, pH 7.4, and unfolding was monitored by measuring the i ntrinsic tryptophan fluores- cence by excitation at 295 nm. The fluorescence spectra were measured with a P erkinElmer LS50B fluorimeter with both the excitation and emission slit widths set to 10 nm. The spectrum d ata were obtained as the average of five traces, and the wavelength at the emission maximum was determined by PerkinElmer FL WinLab software. The unfolding of wild-type neuroserpin was also monitored by CD ellipticity at 222 nm with a Jasco J-810 spectropola- rimeter. The path l ength and slit width were 1.0 cm and 2 nm, respectively. The fluorescence and CD measurements were performed a t the incubation times of 3 , 6, 12, and 2 4 h to confirm equilibrium in urea, with no difference being observed between the 1 2 h and 24 h data. The transition midpoint of unfolding was determined by fitting of the triplicate experimental data to a theoretical sigmoidal equation at a urea concentration of 2–9 M . Ó FEBS 2004 Biochemical properties of a neuroserpin mutant (Eur. J. Biochem. 271) 3361 Results and Discussion The expression of Ser52Arg neuroserpin resulted in a poor yield, with only 0.1–0.5 mg pure monomeric protein being obtained from 3 L culture medium. T his c ompares with a n average of 5 and 1 mg for wild-type and Ser49Pro neuroserpin, respectively, when expressed un der the same conditions. Wild-type, Ser49Pro and Ser52Arg neuroserpin migrated as single bands on SDS, nondenaturing, 8 M urea and isoelectrofocusing PAGE. Ser52Arg neuroserpin is inactive as an inhibitor of tPA Wild-type neuroserp in forms complexes with t PA with a stoichiometry of inhibition of 1 and an association rate constant (k ass )of1.2· 10 4 M )1 Æs )1 [6]. At higher ratios of enzyme to inhibitor (i.e. [tPA]  [neuroserpin]), there was c leavage of the reactive centre loop [15] and loss of the 4-kDa C-terminal fragment. In contrast, it was not possible to determine a rate of inhibition of tPA by Ser52Arg neuroserpin. Indeed there was no inhibition of tPA even at concentrations as high as 6.6 l M Ser52Arg neuroserpin (Fig. 1). To determine if the formation of a complex was possible b etween Ser52Arg and tPA, higher concentrations (in t he micromolar range) of both species were used to favour complex formation. On SDS/PAGE, there is only a transient band corresponding to the complex between Ser52Arg neuroserpin and tPA (Fig. 2A). Incubation of Ser52Arg neuroserpin with an excess of tPA resulted in the formation of a transient complex, which represented only a small fraction of the total amount of Ser52Arg neuroserpin, consistent with the lack of inhibition observed previously (Fig. 2B). The addition of tPA to wild-type neuroserpin resulted in complete cleavage of the inhibitor a fter a 1 -h incubation at an enzyme to inhibitor ratio of 1 : 1 (Fig. 2B). The reactive loop of Ser52Arg neuroserpin was more resistant to cleavage, a s there was always a significant proportion of Ser52Arg neuroserpin that remained uncleaved even after i ncubation for 1 h a t a tPA to Ser52Arg neuroser- pin ratio of 10 : 1. These data show that the reactive centre loop is not as readily accessible in Ser52Arg neuroserpin as i t is in the wild-type p rotein. Ser52Arg neuroserpin forms polymers more rapidly than wild-type or Ser49Pro neuroserpin Polymerization was assessed by incubating wild-type, Ser49Pro and Ser52Arg neuroserpin at 37 °Cor45°C and separating the resulting mixture by nondenaturing PAGE. Ser52Arg neuroserpin readily formed polymers at 0.4 mgÆmL )1 and 37 °C, which were apparent as a reduction in the intensity of the monomeric band after 6 h of incubation (Fig. 3). After 5 2 h, this mutant had formed higher-order aggregates which were s tacked at the Fig. 1. Progress curves for tPA-ca talysed hydrolysis o f 1 m M H- D -Ile- Pro-Arg-p-nitroanilide in the presence of wild-type neuroserpin (lower curve) or Ser52Arg neuroserpin (middle curve). [tPA] ¼ 20 n M , [wild- type] ¼ 1 l M , [Ser52Arg] ¼ 6.6 l M . The u pper c urve rep resents tPA alone. Fig. 2. Assessment of complex formation between tPA and wild-type or Ser52Arg neuroserpin at 2 5 °C. (A) 10% w/v S DS/PAGE of Ser52Arg neuroserpin a nd tP A incubated at different ratios and t im es. L ane 1, 2 lg Ser52Arg neuroserpin; lane 2, 2 lg tPA; lanes 3–5 correspond to an incubation of 5 min at a Ser52Arg neuroserpin to tPA ratio of 1, 5 and 1 0; lanes 6–8 correspond to an incubation of 15 min at a Ser52Arg neuroserpin to tPA ratio o f 1, 5 a nd 10; lanes 9–11 correspond to an incubation of 1 h at a Ser52Arg neu roserpin to tP A ratio of 1, 5 and 10; lanes 12–13 correspond to an incubation of 4 h at a Ser52Arg neuroserpin to tPA ratio of 5 and 10. (B) SDS/PAGE (10% gel) of wild-type or Se r52Arg neuroserpin incubated with in creasing amount of tPA. L a ne 1, 2 lg wild-type neuroserpin; lane 2, 2 lg Ser52Arg neuroserpin; lane 3, 2 lgtPA;lanes4–6correspondtoanincubation of 5 min at a tPA to wild-type neuroserpin ratio of 1, 5 and 10; l anes 7–9 correspond to a n i ncubation of 1 h at a t PA t o w ild-type neuro- serpinratioof1,5and10;lanes10–12correspondtoanincubation of 5 min at a tPA t o Ser52Arg neuroserpin ratio of 1, 5 and 10; lanes 13–15 correspond to an i ncubation t ime of 1 h at a tPA to S er52Arg neuroserpin ratio of 1, 5 and 10. N, Intact native neuroserpin; Cl, n euroserpin cleaved at the reac tive centre loop; C px, the complex between neuroserpin a nd tPA. 3362 D. Belorgey et al.(Eur. J. Biochem. 271) Ó FEBS 2004 top of the gel. The rate of polymerization was determined by measuring the reduction in densit y of t he monomeric band. Wild-type neuroserpin had not formed polymers at a measurable rate after 24 days at 0.4 mgÆmL )1 and 37 °C compared with a rate of 5.3 · 10 )6 s )1 for Ser49Pro neuroserpin and 7.9 · 10 )5 s )1 for Ser52Arg neuroserpin (Table 1 and Fig. 3). The same effect was a pparent if the polymerization experiments were conducted a t 4 5 °C. Both mutants formed polymers more rapidly than wild- type neuroserpin but there was no difference between the rates of the two mutants (Fig. 4 and Table 1). The rates of polymerization for wild-type and Ser49Pro neuroserpin are slower than those that we reported previously [6]. The Fig. 3. Polymerization of wild-type neuroserpin, Ser49Pro neuroserpin and Ser52Arg n eurose rpin at 0.4 mgÆmL )1 and 37 °C. Top, wild-type neuroserpin. Lanes 1–8 correspond to 0 , 4, 7, 11, 15, 18, 21 and 24 days of inc ubation, respectively. Midd le, Ser49Pro neuroserpin. Lanes 1–8 correspond to 0, 5, 23, 30, 47, 54, 69 and 168 h of incubation, respectively. Bottom, Ser52Arg neuroserpin. Lanes 1–8 correspond to a 0 , 6, 22, 30, 46, 52, 70, and 78 h of i ncubation, respectively. Table 1. Rate of polymerization of neuroserpin at 0.4 mgÆmL )1 as measured by densitometry from nondenaturing PAGE. The results are the m ean of at least three experiments. Rate (s )1 ) 37 °C Wild-type No rate Ser49Pro 5.3 (± 0.3) · 10 )6 Ser52Arg 7.9 (± 0.4) · 10 )5 45 °C Wild-type 3.3 (± 0.9) · 10 )5 Ser49Pro 2.7 (± 0.8) · 10 )4 Ser52Arg 2.2 (± 0.2) · 10 )4 Fig. 4. Polymerization of wild-type neuroserpin, Ser49Pro neuroserpin and Ser52Arg neuroserpin at 0.4 mgÆmL )1 and 45 °C. Top, wild-type neuroserpin. Lanes 1–8 corre spond to 0, 0.5, 1, 2, 3, 4, 5 and 6 h of incubation, respectively. Mid dle, Ser49Pro n euroserpin. Lanes 1–8 correspond to 0, 5, 10, 15, 30, 45, 60 and 90 m in of incubation, respectively. Bottom, Ser52Arg neuroserpin. Lanes 1–8 correspond to 0, 3, 7, 12 , 18, 30, 45 and 60 min of incubation, r espectively. Ó FEBS 2004 Biochemical properties of a neuroserpin mutant (Eur. J. Biochem. 271) 3363 difference was due to the m ineral oil used to o verlay the protein solution. We had previously used mineral oil (Sigma Chemical Co.; M-3516) that was m ore t han 3 years old. Repeating the experiment with newer b atches of oil confirmed the difference between wild-type and Ser49Pro but the rates were 10-fold slower. It was not possible to follow the change in secondary structure of Ser52Arg neuroserpin during polymerization with CD because the signal at 216 nm did not change during the course of the experiment, i.e. after incubation of Ser52Arg neuroserpin at either 37 °Cor45°C for 24 h. Assessment of the conformation of Ser52Arg neuroserpin The most likely cause for the inactivity of Ser52Arg neuroserpin and inaccessibility of the reactive loop is that the mutant had adopted an aberrant conformation. One possibility is that the reactive loop had fully inserted into its own b-sheet A to form a latent conformer [16]. However, this is unlikely as the latent conformer of the serpins is unable to form polymers [17,18]. Other charac- teristics of the latent conformer are a failure to unfold in denaturants and enhanced the rmal stability [17,18]. The conformation adopted by Ser52Arg neuroserpin was therefore assessed by electrophoresis on transverse urea gradient gels. Ser52Arg neuroserpin unfolded with a profile that was similar to wild-type neuroserpin (Fig. 5A), indicating tha t the re w as no gross distort ion of structure. The melting point temperature was determined by mon- itoring the change in CD signal at 216 nm while increasing the temperature at 1 °CÆmin )1 (Fig. 5B). In the case of Ser52Arg neuroserpin, the signal magnitude only allows us to calculate an approximation of the melting temperature (T m ). This gave a T m of  55 °C. This T m was surprising as it is close to the value for wild-type neuroserpin (56.6 °C) and significantly higher than t hat for Ser49Pro neuroserpin (49.9 °C) [6]. Previous studies h ave shown an inverse relationship between rate of polymer formation and T m [19], and thus it was unusual to find that the T m was higher than that of the less severe Ser49Pro neuro- serpin. The overall structure of Ser52Arg neuroserpin was therefore assessed by CD spectroscopy. There were marked differences in the profiles of native wild-type and Ser52Arg neuroserpin (Fig. 5C). The profile for wild- type neuroserpin was comparable to that obtained for other serpins, including a 1 -antitrypsin and a 1 -antichymo- trypsin [19,20]. In comparison, the spectrum of Ser52Arg neuroserpin shows an increase in both b-sheet and a-helical structure content as determined by the large increase in magnitude of the signal at 216 nm and the small increase at 222 nm. This profile is comparable to that obtained for monomeric Ser 49Pro neuroserpin and the polymers of both wild-type and Ser49Pro n euroserpin [6]. Spectra taken after incubation of Ser52Arg neuroser- pin for 24 h at 0.5 mgÆmL )1 and 45 °C (i.e. after the protein was 100% polymers on nondenaturing PAGE) showed a profile that was similar to monomeric Ser52Arg neuroserpin and polymers of wild-type and Ser49Pro neuroserpin. More detailed unfolding experiments were then per- formed to further a ssess the conformation of wild-type and mutant neuroserpin. The proteins were added to increasing concentrations of urea, and the change in fluorescence profile was followed by exciting the protein at 295 nm and measuring the shift in maximum fluorescence Fig. 5. Characterization of the c onformation of Se r52Arg neuroserpin. (A) Transverse u rea gradient PAGE (7.5% gel) of wild-type (left) and Ser52Arg neuroserpin ( right). The leftandrightofthegelrepresent0 and 8 M urea, respectively. (B) CD signal at 216 nm for Ser52Arg neuroserpin at 0.7 mgÆmL )1 with increases in temperature of 1 °CÆmin )1 . The data represent three repeats. (C) Far-UV CD spectra of wild-type neuroserpin (–·–), wild-type neuroserpin polymers (––), native Ser52Arg neuroserpin ( ÆÆÆÆ) and Ser52Arg neuroserpin polymers (- - -). 3364 D. Belorgey et al.(Eur. J. Biochem. 271) Ó FEBS 2004 after a 12 or 24 h incubation time (Fig. 6A). No differences were observed between the two incubation times. The profiles obtained for wild-type neuroserpin, Ser49Pro and Ser52Arg were consistent with the results obtained from both transverse urea gradient gels and assessment of the melting temperature (Fig. 6B). The transition points calculated for wild-type neuroserpin and Ser52Arg were very similar, at 6.4 and 6.3 M urea, respectively. The calculated transition point for S er49Pro was lower, at 5.3 M urea. The CD ellipticity of wild-type neuroserpin was also assessed at 222 nm; the data were identical with those obtained from urea unfolding (Fig. 6). The transition midpoint calculated from the C D data for wild-type neuroserpin was also 6.4 M . Correlation of biochemical characteristics with the dementia and epilepsy found in individuals with the neuroserpin Portland (Ser52Arg) mutation The neuroserpin Portland (Ser52Arg) mutation is ass ociated with three times the number of intracellular inclusion bodies (or Collin’s bodies) in neurons compared with dementia associated with the Syracuse mutation (Ser49Pro). The clinical manifestations are more severe, with an age of onset of disease  20 years earlier [3]. This earlier a ge of onset is in keeping with the faster rate of polymerization of Ser52Arg neuroserpin compared with Ser49Pro neuroserpin. It was surprising that Ser52Arg neuroserpin was almost inert when incubated with tPA. There was only transient co mplex formation, and 50% of Ser52Arg neuroserpin remained uncleaved even after incubation with a 10-fold excess of tPA for 1 h. Moreover the melting temperature and unfolding in urea were similar to that of wild-type n eurose rpin, which is unusual for a serpin that s pontaneously forms polymers in vitro and in vivo [19]. The polymers of m utant n euroserpin that form in FENIB are analogo us to polymers that f orm w ith m utants of other members of the serpin superfamily such as a 1 -antitrypsin [21], antithrombin [22], C1 inhibitor [23,24] and a 1 -anti- chymotrypsin [25] in association with cirrhosis, thrombosis, angio-oedema and emphysema, respectively. Indeed we have recently grouped these conditions together as the serpinopa- thies as they h ave a common underlying mechanism [ 26,27]. A m utation in the shutter region of a 1 -antichymotrypsin (Leu55Pro) resulted in a similar conformer to Ser52Arg neuroserpin i n t hat i t w as inactive as a proteinase inhibitor, had enhanced thermal stability but still rapidly formed polymers[25].Wewereabletosolvethecrystalstructureof this d conformer of a 1 -antichymotrypsin and showed that the reactive loop was partially inserted into b-sheet A [25]. The F-helix was unfolded and inserted into the lower part of b-shee t A, which explains the increased stability. However, this helix must be readily displaced by the reactive loop of another molecule t o form t he chains of polymers. This conformation would also explain the data obtained with Ser52Arg neuroserpin. The Ser52Arg mutation is in the shutter domain of the molecule which controls opening of b-sheet A [28]. The a rginine mutation would cause a significant d isruption in this a rea, thereby forcing b-sheet A into an ÔopenÕ or acceptor configuration. This in turn would allow partial insertion of the reactive loop into b-sheet A, with the lower part of b-sheet A being filled by unfolding and insertion of the F-helix (Fig. 7). The reactive loop must be inserted further than in Ser49Pro neuroserpin (which is a lso a shutter domain mutation) because Ser49Pro neuroserpin r emains partly active as a proteinase inhibitor [6]. In k eeping with this, the C D profile of Ser52Arg neuroserpin is similar to that of the polymeric conformation in which b-sheet A is filled with the reactive loop of another neuroserpin molecule. Moreover th e emission maxima of the native w ild-type and mutant proteins (Fig. 6) are also different, in keeping with a different conformation induced by the shutter domain mutants. The F-helix must be readily displaced from the lower portion of b-sheet A during polymeriza- tion of Ser52Arg neuroserpin. This would allow accept- ance of the r eactive loop of a second neuroserpin Fig. 6. Unfolding of wild-type, Ser52Arg and Ser49Pro neuroserpin in urea. The proteins were incubated at 25 lgÆmL )1 and 2 0 °Cfor12h with various concentration s of urea in sodium phosph ate buff er, pH 7.4. (A) I ntrinsic tryptophan flu orescence spectra of wild-type (––), Ser52Arg (- - -) and Ser49Pro ( ÆÆÆÆ) neuroserpin in the presence of 0 or 9 M urea. (B) U nfolding p attern of wild-type (s), Ser52Arg (m)and Ser49Pro (h) neuroserpin assessed by emission maxima of intrinsic tryptophan fluorescence. The unfolding pattern of wild-type neuro- serpin was also assessed by the CD ellipticity at 222 nm and super- imposed ( ·). The data represent th e mean of three repeats. Ó FEBS 2004 Biochemical properties of a neuroserpin mutant (Eur. J. Biochem. 271) 3365 molecule and the formation of a dimer. Extension of this process forms the characteristic loop–b-sheet A polymers. Epilepsy is far more common w ith Ser52Arg neuroserpin than Ser49Pro neuroserpin. This may be explained by the increased number of inclusions. However, it may also be explained by the lack of inhibitory activity caused by the Ser52Arg mutation. There is g rowing evidence from animal models that epilepsy r esults from an imbalance between tPA and neuroserpin [29]. The inactivity of Ser52Arg neuro- serpin will contribute to this imbalance in individuals who carry this m utation a nd may exacerbate the intrinsic ability of the intracerebral inclusions to cause epilepsy. Acknowledgements We are grateful to Tim Dafforn for help in preparing Fig. 7. We are also grateful to Kerstin Nordling and Ingemar Bjo ¨ rk for helpful comments. This work was supported by t he Med ical Research C ouncil (UK), the Wellcome Trust ( UK) and Papworth NHS Trust (UK). D.C.C. is a Wellcome Trust I nt ermedia te Clinical Fellow. References 1. Davis, R.L., Shrimpton, A.E., Holohan, P.D., Bradshaw, C., Feiglin, D., Sonderegger, P., Kinter, J., Becker, L.M., Lacbawan, F., Krasnewich, D., Muenke, M., Lawrence, D.A., Yerby, M.S., S haw, C M., Gooptu, B., Elliott, P.R., Finch, J.T., Carrell, R.W. & L omas, D .A. (1999) Fa milial dementia caused by polymerisation of mu tant ne uroserpi n. Nature (London) 401, 376–379. 2. Davis, R.L., Holohan, P.D., Shrimpton, A.E., Tatum, A., Dau- cher, J., Collins, G.H., Todd, R., Bradshaw, C., Kent, P., Feiglin, D.,Rosenbaum,A.,Yerby,M.S.,Shaw,C M.,Lacbawan,F.& Lawrence,D.A.(1999)Familialencephalopathy w ith n euroserpin inclusion bodies (FENIB). Am. J. Pathol. 155 , 1901–1913. 3. Davis, R.L., Shrimpton, A .E., Carrell, R.W., Lomas, D.A., Ger- hard, L., Baumann, B., Lawrence, D.A., Yepes, M., Kim, T.S., Ghetti, B., Piccardo , P., T akao, M., L acb awan, L., M ue nke, M., Sifers, R.N., Bradshaw, C.B., Kent, P.F., Coll ins, G.H., Larocca, D. & Holohan, P.D. ( 2002) Association between conformational mutations in n euroserpin and onset and severity of dementia. Lancet 359, 2242–2247. 4. Osterwalder, T., Contartese, J., S toeckli, E.T., Kuhn, T.B. & Sonderegger, P. (1996) Neuroserpin, an axonally secreted serine protease inhibitor. EMBO J. 15, 2944–2953. 5. Hastings, G.A., Coleman, T.A., H audenschild, C.C., Stefansson, S., Smith, E.P., Barthlow, R ., Cherry, S., Sandkvist, M. & Lawrence, D.A. (1997) Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is loc alized primarily in neurones. J. Biol. Chem. 272, 33062–33067. 6.Belorgey,D.,Crowther,D.C., Mahadeva, R. & Lo mas, D .A. (2002) Mutant neuroserpin ( Ser49Pro) that cau ses the fam ilial dementia FENIB is a poor proteinase inhibitor and readily forms polymers in vi tr o. J. Biol. Chem. 277, 1 7367–17373. 7. Barker-Carlson, K ., Lawrence, D.A. & Schwartz , B.S. (2002) Acyl-enzyme complexes between tissue t ype plasminogen ac ti- vator and n euroserpin are short live d in vitro . J. Biol. Chem. 27 7, 46852–46857. 8. Miranda, E., Ro ¨ misch, K. & Lomas, D.A. (2004) Mutants of neuroserpin that cause dementia accumulate as polymers within the endoplasmic reticulum. J. Biol. Chem. 27 9 , 28283–28291. 9. Kopito, R.R. (2000) Aggresomes, inclusion bodies and protein aggregation. Tr ends Cell. Biol . 10, 524–530. 10. Renatus,M.,Engh,R.A.,Stubbs,M.T.,Huber,R.,Fischer,S., Kohnert, U. & Bode , W. (1997) Ly sine 156 p romot es the a no m- alous proenzyme activity o f tPA: X- ray crystal structure of s ingle- chain human tPA. EMBO J. 16, 4797–4805. 11. Belorgey, D. & Bieth, J.G. (1998) Effect of polynucleotides on the inhibition of neutrophil elastase by mucus proteinase inhibitor and alpha 1-proteinase inhibitor. Biochemistry 37, 16416–16422. 12. Bieth, J.G. (1995) Theoretical and practical aspects of proteinase inhibition kinetics. Metho ds Enzymol. 24 8, 59–84. 13. Morrison, J.F. & Walsh, C.T. (1988) The behavior and sig- nificance of slow-binding en zyme inhibitors. Ad v. Enzy mol. Re lat. Areas Mol. Biol. 61, 201–301. 14. Dafforn, T.R., Mahadeva, R., Elliott, P.R., Sivasothy, P. & Lomas, D.A. (1999) A k inetic mechanism f or the p olymerization of a 1 -antitrypsin. J. Biol. Chem. 274, 9548–9555. 15. Briand, C., Kozlov, S.V., Sonderegger, P. & G ru ¨ tter, M.G. (2001) Crystal structure of neuroserpin: a neu ronal se rpin i nvolved i n a conformational disease. FEB S Lett. 25171, 1–5. 16. Im, H., Woo, M S., Hwang, K.Y. & M H. (2002) Interactions causing the kinetic trap in serpin protein folding. J. Biol. Ch em. 277, 4 6347–46354. Fig. 7. Pathway of the p o lyme rizatio n of Ser52Arg neuroserpin. Left, w ild-type a 1 -antitrypsin. The p osition of th e shutter domain which co ntrols opening of b-sheet A i s shown in blue [30]. Middle, proposed struc ture of Ser52Arg ne uroser pin b ase d on the d conformer of a 1 -antichymotrypsin. Thereactivecentreloop (red) is inserted into b-sheet A (green), whic h explains the inactivity as an inhibitor of tPA and t he resistance of the reactive loop to cleavage. The lo wer portion o f b- sheet A i s fi lled by unfolding of t he F-helix (yellow). Right, the F-helix is displaced by t he re active loop of another m olecule of n euroserpin (yellow) to form a dimer which then extends to form chains of polymers. 3366 D. Belorgey et al.(Eur. J. Biochem. 271) Ó FEBS 2004 17. Lomas, D.A., Elliott, P.R., Chang, W S.W., Wardell, M.R. & Carrell, R.W. (1995) Preparation and characterisation of l atent a 1 -antitrypsin. J. Biol. Chem. 270, 5 282–5288. 18. Wardell, M.R., Chang, W S.W.,Bruce,D.,Skinner,R.,Lesk,A. & C arrell, R.W. ( 1997) Preparative induction a nd characterization of L-antithrombin: a structura l homologue of l atent plasminogen activator inhibitor-1. Biochemistry 36, 13133–13142. 19. Dafforn, T.R., Mahadeva, R., Elliott, P.R., Sivasothy, P. & Lomas, D.A. (1 999) A kinetic description o f the polymerisation of a 1 -antitrypsin. J. Biol. Chem. 274, 9 548–9555. 20. Crowther, D.C., Serpell, L.C., Dafforn, T.R., Gooptu, B. & Lomas, D.A. (2002) Nucleation of a 1 -antichymotrypsin p oly- merisation. Biochemistry 42 , 2355–2363. 21. Lomas, D.A., Evans, D .L., Finch, J.T. & C arrell, R.W. (19 92) The mechanism o f Z a 1 -antitrypsin accumulation in the liver. Natur e (London) 357, 605–607. 22. Bruce,D.,Perry,D.J.,Borg,J Y.,Carrell,R.W.&Wardell,M.R. (1994) Thromboembolic disease due to thermolabile conforma- tional ch ange s o f antithrombin R ouen VI (187Asn fi Asp). J. Clin. Invest. 94, 2265–2274. 23. Aulak, K.S., E ldering, E., Hack, C.E., L ub bers, Y.P.T., Harriso n, R.A., Mast, A., Cicardi, M. & Davis,A.E. III (1993) A hinge region mutation in C1-inhibitor (Ala436 fi Thr) results in non- substrate-like behavior and in polymerization of the molecule. J. Biol. C hem. 268, 18088–18094. 24. Eldering, E., Verpy, E., Roem, D., Meo, T. & Tosi, M. (1995) COOH-terminal substitutions in the serpin C1 inhibitor th at cause loop overinsertion and subsequent multimerization. J. Biol . Chem. 270, 2 579–2587. 25. Gooptu, B., Hazes, B., C hang, W S.W., Dafforn , T.R., Carrell, R.W., R ead, R. & L omas, D.A. ( 2000) Inactive conform ation of the s erpin a 1 -antichymotrypsin indicates two stage in sertion of t he reactive loop; implications for inhibitory function a nd conforma- tional disease. Proc. Natl Acad. Sci. USA 97, 6 7–72. 26. Carrell, R.W. & L omas, D.A. (2002) Alpha 1 -antitrypsin de ficiency: a model for conformational diseases. N. Engl. J . M ed. 34 6, 45–53. 27. Lomas, D.A. & Mahadeva, R. (2002) Alpha-1-antitrypsin poly- merisation and the serpinopathies: pathobiology and prospects for therapy. J. Clin. Invest. 110, 1585–1590. 28. Stein, P.E. & C arrell, R.W. (1995) What do d ysfunctional serpins tell us about m olecular mobility and disease? Nat. Struct. Biol. 2, 96–113. 29. Yepes,M.,Sandkvist,M.,Coleman,T.A.,Moore,E.,Wu,J Y., Mitola,D.,Bugge,T.H.&Lawrence,D.A.(2002)Regulationof seizure spreadin g b y n euroserpin and tissue-type plasm inogen activator i s p lasminoge n indepe ndent. J. Clin. Invest. 10 9, 1571–1578. 30. Elliott, P.R., Pei, X.Y., Dafforn, T.R. & Lomas, D.A. (2000) Topography of a 2.0A ˚ structure of a1-antitrypsin reveals targets for r ational drug design to p revent conformational disease. Protein Sci. 9, 1274–1281. Ó FEBS 2004 Biochemical properties of a neuroserpin mutant (Eur. J. Biochem. 271) 3367 . Neuroserpin Portland (Ser52Arg) is trapped as an inactive intermediate that rapidly forms polymers Implications for the epilepsy seen in the dementia FENIB Didier. the dementia and epilepsy found in individuals with the neuroserpin Portland (Ser52Arg) mutation The neuroserpin Portland (Ser52Arg) mutation is ass ociated with