Báo cáo khoa học: Neuroserpin Portland (Ser52Arg) is trapped as an inactive intermediate that rapidly forms polymers Implications for the epilepsy seen in the dementia FENIB ppt

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:
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.
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