These results, with those of the CD analysis, are in keeping with the reactive centre loop of neuroserpin Portland being partially inserted into b-sheet A to adopt a conformation similar
Trang 1Neuroserpin Portland (Ser52Arg) is trapped as an inactive
intermediate that rapidly forms polymers
Implications for the epilepsy seen in the dementia FENIB
Didier Belorgey1, Lynda K Sharp1, Damian C Crowther1, Maki Onda2, Jan Johansson3and David A Lomas1
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;3Department of Molecular Biosciences, Swedish
University of Agricultural Sciences, Uppsala, Sweden
The dementia familial encephalopathy with neuroserpin
inclusion bodies (FENIB) is caused by point mutations in
the neuroserpin gene We have shown a correlation between
the predicted effect of the mutation and the number of
intracerebral inclusions, and an inverse relationship with the
age of onset of disease Our previous work has shown that
the intraneuronal inclusions in FENIB result from the
sequential interaction 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 loop-sheet polymers but
at a faster rate, in keeping with the more severe clinical
phenotype The Portland mutant has 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, with those of the CD analysis, are in keeping with the reactive centre loop 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 the severity of dementia in FENIB associated with neuroserpin Portland Moreover the inactivity of the mutant 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: conformational diseases; 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 characterized
by inclusions of neuroserpin within cortical and subcortical
neurons [1–3] Neuroserpin is a member of the serine
proteinase inhibitor or serpin superfamily 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, which was identified in the first reported
family with FENIB [6] The mutation reduced the inhibitory
activity of neuroserpin by 100-fold and increased the
formation of polymeric protein under physiological
condi-tions Neuroserpin polymers result from the sequential
insertion of the reactive centre loop of one molecule into
b-sheet A of another [1,6] The resulting species 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 Gly392Glu [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] For example, individuals with the Ser52Arg and Gly392Glu neuroserpin mutation have 3 and 9.5 times more inclusions within the cerebral cortex than individuals with the Ser49Pro mutant This corresponds to an age of onset of symptoms in individuals with Ser49Pro, Ser52Arg and Gly392Glu neuroserpin of 48, 24, and 13 years, respectively There is also a change in phenotype, with the Ser49Pro mutation causing predominantly dementia whereas the Ser52Arg, His338Arg and Gly392Glu mutants cause both dementia and severe progressive epilepsy In addition 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 the 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 28 May 2004,
accepted 28 June 2004)
Eur J Biochem 271, 3360–3367 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04270.x
Trang 2the clinical phenotype Our data show that the Ser52Arg
mutation favours the rapid formation of polymers as the
protein is locked as an inactive folding intermediate These
polymers explain the increased number of inclusions in
individuals with Ser52Arg compared with those with the
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 Calbiochem (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 from 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 pQE81L expression vector [6] by
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 proteins were assessed by SDS, nondenaturresult-ing and
transverse urea gradient PAGE, and activity was assessed
against tPA [6]
Complex formation assays
Wild-type and Ser52Arg neuroserpin were incubated in
various ratios with tPA at 25C as described previously [6]
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 [10] 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
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], using the
progress-curve method [11,12] Rate constants of inhibition were measured at 25C in inhibition buffer [50 mMHepes,
150 mMNaCl, 0.01% (w/v) dodecyl maltoside, pH 7.4] by adding tPA (20 nM) to a mixture of wild-type (from 200 nM
to 1000 nM) or Ser52Arg (6600 nM) neuroserpin and the substrate S-2288 (1 mM) 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 mM sodium phosphate buffer,
pH 7.4 Polymers were formed by heating wild-type or mutant neuroserpin at 0.5 mgÆmL)1 and 45C for 24 h Changes in the 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 signal at 216 nm in a 150-lL cuvette between 25C and 95 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 Tm[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)1in NaCl/Pi, pH 7.4, at 37C
or 45C Aliquots were taken over time, and 2 lg protein was loaded on a 7.5% (w/v) nondenaturing gel To avoid evaporation during the experiment, the different samples were covered with mineral oil The proteins were visualized
by staining with GelCode Blue Stain Reagent (Pierce, Tattenhall, Cheshire, UK) or by silver staining
Unfolding of wild-type and Ser52Arg neuroserpin in urea Neuroserpin at 25 lgÆmL)1was incubated at 20C with various concentrations of urea (from 0 to 9M) in 50 mM sodium phosphate buffer, pH 7.4, and unfolding was monitored by measuring the intrinsic tryptophan fluores-cence by excitation at 295 nm The fluoresfluores-cence spectra were measured with a PerkinElmer LS50B fluorimeter with both the excitation and emission slit widths set to 10 nm The spectrum data 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 length and slit width were 1.0 cm and
2 nm, respectively The fluorescence and CD measurements were performed at the incubation times of 3, 6, 12, and 24 h
to confirm equilibrium in urea, with no difference being observed between the 12 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 2–9M
Trang 3Results 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 This compares with an
average of 5 and 1 mg for wild-type and Ser49Pro
neuroserpin, respectively, when expressed under the same
conditions Wild-type, Ser49Pro and Ser52Arg neuroserpin
migrated as single bands on SDS, nondenaturing, 8Murea
and isoelectrofocusing PAGE
Ser52Arg neuroserpin is inactive as an inhibitor of tPA
Wild-type neuroserpin forms complexes with tPA with a
stoichiometry of inhibition of 1 and an association rate
constant (kass) of 1.2· 104M )1Æs)1[6] At higher ratios of
enzyme to inhibitor (i.e [tPA] [neuroserpin]), there
was cleavage 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 lM Ser52Arg
neuroserpin (Fig 1) To determine if the formation of a
complex was possible between Ser52Arg and tPA, higher
concentrations (in the 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 after 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, as there was always a significant proportion
of Ser52Arg neuroserpin that remained uncleaved even after incubation for 1 h at 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 it is in the wild-type protein
Ser52Arg neuroserpin forms polymers more rapidly than wild-type or Ser49Pro neuroserpin
Polymerization was assessed by incubating wild-type, Ser49Pro and Ser52Arg neuroserpin at 37C or 45 C and separating the resulting mixture by nondenaturing PAGE Ser52Arg neuroserpin readily formed polymers at 0.4 mgÆmL)1 and 37C, which were apparent as a reduction in the intensity of the monomeric band after
6 h of incubation (Fig 3) After 52 h, this mutant had formed higher-order aggregates which were stacked at the
Fig 1 Progress curves for tPA-catalysed hydrolysis of 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 upper curve represents tPA
alone.
Fig 2 Assessment of complex formation between tPA and wild-type or Ser52Arg neuroserpin at 25 °C (A) 10% w/v SDS/PAGE of Ser52Arg neuroserpin and tPA incubated at different ratios and times Lane 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 10; lanes 6–8 correspond to an incubation of 15 min at a Ser52Arg neuroserpin to tPA ratio of 1, 5 and 10; lanes 9–11 correspond to an incubation of 1 h at a Ser52Arg neuroserpin to tPA 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 Ser52Arg neuroserpin incubated with increasing amount
of tPA Lane 1, 2 lg wild-type neuroserpin; lane 2, 2 lg Ser52Arg neuroserpin; lane 3, 2 lg tPA; lanes 4–6 correspond to an incubation
of 5 min at a tPA to wild-type neuroserpin ratio of 1, 5 and 10; lanes 7–9 correspond to an incubation of 1 h at a tPA to wild-type neuro-serpin ratio of 1, 5 and 10; lanes 10–12 correspond to an incubation
of 5 min at a tPA to Ser52Arg neuroserpin ratio of 1, 5 and 10; lanes 13–15 correspond to an incubation time of 1 h at a tPA to Ser52Arg neuroserpin ratio of 1, 5 and 10 N, Intact native neuroserpin;
Cl, neuroserpin cleaved at the reactive centre loop; Cpx, the complex between neuroserpin and tPA.
3362 D Belorgey et al (Eur J Biochem 271) FEBS 2004
Trang 4top of the gel The rate of polymerization was determined
by measuring the reduction in density of the monomeric
band Wild-type neuroserpin had not formed polymers at
a measurable rate after 24 days at 0.4 mgÆmL)1and 37C
compared with a rate of 5.3· 10)6s)1 for Ser49Pro
neuroserpin and 7.9· 10)5s)1 for Ser52Arg neuroserpin
(Table 1 and Fig 3) The same effect was apparent if the
polymerization experiments were conducted at 45C
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 neuroserpin at 0.4 mgÆmL)1and 37 °C Top, wild-type
neuroserpin Lanes 1–8 correspond to 0, 4, 7, 11, 15, 18, 21 and 24 days
of incubation, respectively Middle, 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 incubation, respectively.
Table 1 Rate of polymerization of neuroserpin at 0.4 mgÆmL)1 as measured by densitometry from nondenaturing PAGE The results are the mean of at least three experiments.
Rate (s)1)
37 C
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)1and 45 °C Top, wild-type neuroserpin Lanes 1–8 correspond to 0, 0.5, 1, 2, 3, 4, 5 and 6 h of incubation, respectively Middle, Ser49Pro neuroserpin Lanes 1–8 correspond to 0, 5, 10, 15, 30, 45, 60 and 90 min of incubation, respectively Bottom, Ser52Arg neuroserpin Lanes 1–8 correspond to
0, 3, 7, 12, 18, 30, 45 and 60 min of incubation, respectively.
Trang 5difference was due to the mineral oil used to overlay the
protein solution We had previously used mineral oil
(Sigma Chemical Co.; M-3516) that was more than
3 years old Repeating the experiment with newer batches
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 37C or 45 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 thermal 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 that there was no gross distortion of structure
The melting point temperature was determined by
mon-itoring the change in CD signal at 216 nm while increasing
the temperature at 1CÆmin)1 (Fig 5B) In the case of
Ser52Arg neuroserpin, the signal magnitude only allows us
to calculate an approximation of the melting temperature
(Tm) This gave a Tmof 55 C This Tmwas surprising
as it is close to the value for wild-type neuroserpin
(56.6C) and significantly higher than that for Ser49Pro
neuroserpin (49.9C) [6] Previous studies have shown an
inverse relationship between rate of polymer formation
and Tm[19], and thus it was unusual to find that the Tm
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 a1-antitrypsin and a1
-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 Ser49Pro neuroserpin and
the polymers of both wild-type and Ser49Pro neuroserpin
[6] Spectra taken after incubation of Ser52Arg
neuroser-pin for 24 h at 0.5 mgÆmL)1 and 45C (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 assess 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 conformation of Ser52Arg neuroserpin (A) Transverse urea gradient PAGE (7.5% gel) of wild-type (left) and Ser52Arg neuroserpin (right) The left and right of the gel represent 0 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) FEBS 2004
Trang 6after 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 6.3M urea,
respectively The calculated transition point for Ser49Pro
was lower, at 5.3M 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 CD data for
wild-type neuroserpin was also 6.4M
Correlation of biochemical characteristics with the dementia and epilepsy found in individuals with the neuroserpin Portland (Ser52Arg) mutation
The neuroserpin Portland (Ser52Arg) mutation is associated 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 age 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 complex 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 neuroserpin, which is unusual for a serpin that spontaneously forms polymers
in vitroand in vivo [19]
The polymers of mutant neuroserpin that form in FENIB are analogous to polymers that form with mutants of other members of the serpin superfamily such as a1-antitrypsin [21], antithrombin [22], C1 inhibitor [23,24] and a1 -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 have a common underlying mechanism [26,27]
A mutation in the shutter region of a1-antichymotrypsin (Leu55Pro) resulted in a similar conformer to Ser52Arg neuroserpin in that it was inactive as a proteinase inhibitor, had enhanced thermal stability but still rapidly formed polymers [25] We were able to solve the crystal structure of this d conformer of a1-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-sheet A, which explains the increased stability However, this helix must be readily displaced by the reactive loop of another molecule to form the 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 arginine mutation would cause a significant disruption in this area, thereby forcing b-sheet A into an open 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 also a shutter domain mutation) because Ser49Pro neuroserpin remains partly active as a proteinase inhibitor [6] In keeping with this, the CD 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 the emission maxima of the native wild-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 reactive loop of a second neuroserpin
Fig 6 Unfolding of wild-type, Ser52Arg and Ser49Pro neuroserpin in
urea The proteins were incubated at 25 lgÆmL)1and 20 C for 12 h
with various concentrations of urea in sodium phosphate buffer,
pH 7.4 (A) Intrinsic tryptophan fluorescence spectra of wild-type (––),
Ser52Arg (- - -) and Ser49Pro (ÆÆÆÆ) neuroserpin in the presence of 0 or
9 M urea (B) Unfolding pattern 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 the mean of three repeats.
Trang 7molecule and the formation of a dimer Extension of this
process forms the characteristic loop–b-sheet A polymers
Epilepsy is far more common with 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 growing evidence from animal
models that epilepsy results from an imbalance between tPA
and neuroserpin [29] The inactivity of Ser52Arg
neuro-serpin will contribute to this imbalance in individuals who
carry this mutation and 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 the Medical Research Council
(UK), the Wellcome Trust (UK) and Papworth NHS Trust (UK).
D.C.C is a Wellcome Trust Intermediate Clinical Fellow.
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Fig 7 Pathway of the polymerization of Ser52Arg neuroserpin Left, wild-type a 1 -antitrypsin The position of the shutter domain which controls opening of b-sheet A is shown in blue [30] Middle, proposed structure of Ser52Arg neuroserpin based on the d conformer of a 1 -antichymotrypsin The reactive centre loop (red) is inserted into b-sheet A (green), which explains the inactivity as an inhibitor of tPA and the resistance of the reactive loop to cleavage The lower portion of b-sheet A is filled by unfolding of the F-helix (yellow) Right, the F-helix is displaced by the reactive loop of another molecule of neuroserpin (yellow) to form a dimer which then extends to form chains of polymers.
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