BIGH3 (TGFBI) Arg124 mutations influence the amyloid conversion
of related peptides
in vitro
Implications in the BIGH3-linked corneal dystrophies
Clair-Florent Schmitt-Bernard
1,2
, Alain Chavanieu
3
, Gudrun Herrada
3
, Guy Subra
3
, Bernard Arnaud
4
,
Jacques G. Demaille
1
, Bernard Calas
3
and A
´
ngel Argile
´
s
1
1
Institut de Ge
´
ne
´
tique Humaine, CNRS UPR 1142, Montpellier, France;

b-pleated sheet structures in those with enhanced amyloid
yielding. We designed a peptide (BB1) likely to counteract
the role of Val112-Val113 in amyloid fibril formation.
Incubation of Cys124 peptide with BB1 indeed resulted in a
35% inhibition of amyloid fibril formation.
Our results are in keeping with the clinical observations of
Arg124 mutation-linked amyloidosis and show the import-
ance of Val112–Val113, disulfide and hydrogen bonding in
increasing the b-pleated conformation and amyloid forma-
tion. These findings shed new light on the molecular mech-
anisms of TGFBI protein amyloidogenesis and encourage
further research on the use of specifically designed peptides
as putative therapeutic agents for these disabling diseases.
Keywords: amyloidosis; keratoepithelin; lattice corneal dys-
trophy; granular corneal dystrophy; synthetic peptide.
Hereditary corneal dystrophies are a cause of blindness.
These dystrophies are characterized by a progressive
alteration of the particular structure of the cornea resulting
in loss of its transparency. Based upon their clinical
characteristics, hereditary corneal dystrophies form a
distinctive group of corneal diseases. Some of them involve
the corneal stroma where deposits begin to appear
during the first decades of life and severely impair visual
acuity in adulthood. Their therapy is restricted to keratopl-
asty and phototherapeutic keratectomy by Excimer laser.
Unfortunately, the benefits of these therapies remain
transient as recurrence of the deposits is the rule.
Genetic studies have recently confirmed that a group of
hereditary corneal dystrophies have a common molecular
mechanism: the involvement of the BIGH3 (TGFBI,

Abbreviations:BIGH3,beta-inducedgene-human3;big-h3, BIGH3
gene product; TGFBI, transforming growth factor beta-induced gene;
TGFb, transforming growth factor beta; LCD, lattice corneal dys-
trophy; CDB, corneal dystrophy of Bowman’s layer; GCD, granular
corneal dystrophy; ThT, thioflavin T.
(Received 5 March 2002, revised 6 August 2002,
accepted 23 August 2002)
Eur. J. Biochem. 269, 5149–5156 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03205.x
dystrophy, mutation Arg555Gln) and Granular corneal
dystrophy type 1 (GCD1, mutation Arg555Trp). Codon
Arg124 seems to be particularly critical in corneal dystro-
phies as four different phenotypes are associated with four
different mutations of this residue. These mutations are:
Arg124Cys (R124C) [1] in lattice corneal dystrophy type 1
(LCD1) characterized by amyloid deposits, Arg124Ser
(R124S) [12] in a phenotypic variant of granular corneal
dystrophy type 1 (GCD1), Arg124His (R124H) [1] in
granular corneal dystrophy type 2 (GCD2, Avellino
dystrophy) a mixed type of amyloid and granular deposits,
and Arg124Leu (R124L) [13] in corneal dystrophy of
Bowman’s layer type 1 (CDB1, GCD3, Reis-Bu
¨
cklers
corneal dystrophy) a phenotypic variant of GCD1 charac-
terized by superficial granular deposits. The biochemical
mechanisms responsible for the alteration of protein behav-
ior following mutations at codon Arg124 remain unknown.
We have recently described an effective in vitro system to
produce amyloid fibrils from TGFBI protein 110)131
derived peptides [14]. In the present report, we used our

Chemicals
Trifluoroacetic acid and acetonitrile (HPLC grade) were
purchased from SDS (Peypin, France). All compounds
used for solid-phase peptide synthesis (solvents, resins
and protected amino acids) were from PE Biosystem
(Framingham, USA).
Peptide synthesis
Peptide synthesis was carried out at a 0.2-mmol scale using a
continuous flow apparatus (PE Biosystem, Pioneer,
Framingham, USA) starting from Fmoc-PAL-PEG-PS
resins. The coupling reaction was performed with 0.5
M
of
HATU in presence of 1
M
of DIEA. Protected group
removal and final cleavage were carried out with trifluoro-
acetic acid/H
2
O/EDT/phenol [14] and crude peptides were
purified by reverse-phase HPLC on a C18 semipreparative
column. Electrospray ionization mass spectra were in
complete agreement with the proposed structure.
In vitro
processing of the peptides
The peptides were processed as previously described [14].
Briefly, 0.1 lmolofeachpeptidewasdilutedin250lLof
1/15
M
phosphate-buffered solution (pH 7.4). The solutions

peptide solution was dialyzed against 1/15
M
phosphate-
buffered solution at pH 7.4 and ultracentrifugated at 10
5
g,
for 1 h, at 4 °C. The pellets were resuspended in 50 m
M
glycine-NaOH pH 8.5 containing 100 l
M
ThT in an
assay volume of 500 lL, and processed immediately.
Table 1. Synthetic peptides designed for the study.
Peptide H
2
N-sequence-CONH
2
R110)131 LGVVGSTTTQLYTDRTEKLRPE
R114)131 GSTTTQLYTDRTEKLRPE
C110)131 LGVVGSTTTQLYTDCTEKLRPE
C114)131 GSTTTQLYTDCTEKLRPE
C110)131Acm LGVVGSTTTQLYTDCTEKLRPE
Acm
H110)131 LGVVGSTTTQLYTDHTEKLRPE
S110)131 LGVVGSTTTQLYTDSTEKLRPE
big-h3 110)122 LGVVGSTTTQLYT
5150 C F. Schmitt-Bernard et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Fluorescence spectroscopy was performed on a Quanta-
Master System Spectrofluophotometer (Photon Technology
International, Monmouth Junction, NJ, USA) at 25 °Cas

area over the curve was performed with an overlap
method after baseline correction. Anti-parallel b-pleated
structures were quantified at 1625 cm
)1
wave number
and antiparallel b-aggregates were determined at
1685 cm
)1
.
Beta-breaker methods
Two peptides, five amino-acid long, were synthesized as
described above. Their sequences were decided on the
grounds of the results of the amyloid fibril experiments. BB1
was designed to interfere with the Val112–Val113 of the
C110)131 peptide and consisted of H
2
N-LPVVD-CONH
2
.
An unspecific peptide BB2 (H
2
N-LPFFD-CONH
2
)was
used as control for comparison to evaluate the effect of BB1.
1 l
M
of each peptide was incubated with 0.05 l
M
of

the C114–131 peptide and (F) material obtained from dialysis of the
big-h3 110–122 peptide. This morphological analysis showed more
dichroic material in solutions containing big-h3 110–122 and
C110–131 peptides than in those containing H110–131, S110–131,
R110–131, and C114–131 peptides.
Ó FEBS 2002 TGFBI protein Arg124 mutations and amyloid formation (Eur. J. Biochem. 269) 5151
to the baseline, which corresponds to ThT auto-fluores-
cence. ThT fluorescence was the highest for C110)131, and
diminished significantly for each of the following peptides
H110)131, S110)131, and R110)131, respectively (Fig. 2).
No amyloid material was observed with the same
peptides suspended in distilled water without NaCl/P
i
dialysis at 2 and 24 h. A discrete red/green dichroism was
noticeable by day 3 on smears with the C110)131 peptide,
while R110)131, H110)131, and S110)131 remained
negative. Spontaneous amyloid formation was only ob-
served with prolonged incubation periods. ThT fluorescence
showed increasing emission spectra until day 15 (Fig. 3). By
this time point, ThT emission spectra were significantly
higher for C110)131, and H110)131, compared to that of
S110)131 and R110)131. Therefore, the results of both
spontaneous and dialysis-based precipitations showed the
importance of the residue 124 in determining its tendency to
precipitate into amyloid fibrils.
Analysis of the secondary structure of both R110)131
(Fig. 4A) and C110)131 (Fig. 4B) in H
2
O revealed a switch
in the IR spectrograms from an antiparallel b-pleated sheet

-terminal sequence of the 124 centered
peptides included in this study was analyzed by comparing
the 110–131 peptides to the 114–131 counterparts. The ThT
emission spectrum from R110)131 was significantly higher
than that of the same peptide without 110Leu-Gly-Val-
Val113 (R114)131) (Fig. 5A). A similar decrease in amyloid
fibril formation was observed when comparing C110)131
to its corresponding peptide lacking the NH
2
-terminal
110Leu-Gly-Val-Val113 (C114)131) (these results are illus-
trated morphologically in Fig. 1, and quantitatively in
Fig. 5B).
On FT-IR spectroscopy, C114)131 appeared to have less
b-sheet component than C110)131 (6% and 23%, respect-
ively) confirming that the NH
2
-terminal sequence of
C110)131 participates in the b-conformation of the peptide
(Figs 4D and 6). These results were observed both in D
2
O
as well as in H
2
O.
Role of the CONH
2
-terminal sequence
The participation of the CONH
2

2
OandH
2
O.
Kinetic studies also showed a higher propensity of big-
h3 110)122 to form amyloid fibrils when compared to the
full-length peptide (big-h3 110)131). Accelerated amyloid
fibril formation was observed in experiments with spon-
taneous amyloid precipitation, without dialysis. None of
the TGFBI protein110)131 peptides analyzed (C110)131,
H110)131, S110)131 and R110)131) displayed any
significant amount of amyloid fibrils by 24 h. By contrast,
big-h3 110)122 peptide displayed a faint red/green biref-
ringence on Congo red smears by 2 h, and by 24 h a
large amount of dichroic material was observed (Fig. 7A).
The amyloid nature of the big-h3 110)122 deposits was
confirmed by electron microscopy showing bundles of
8–10 nm wide filaments (Fig. 7B).
Fig. 4. Secondary structure determination of the peptides by FT-IR spectroscopy in H
2
O. The peptides included are the following: R110)131 (A),
C110)131 (B), big-h3 110)122 (C) as well C110)131 and C114)131 (D). Anti-parallel b-pleated structures were quantified at 1625 cm
)1
wave
number and antiparallel b-aggregates were determined at 1685 cm
)1
wave number. A significant depression in the spectrogram can be observed at
1685 cm
)1
wavenumber by 24 h in R110)131 (A) and C110)131 (B) peptide solutions (arrows) demonstrating their tendency to form b-aggregates.

dogenicity of big-h3 110)122 when compared to its full-
length counterpart was also evident from dialysis-induced
amyloid fibril formation (Figs 2E and 3 illustrate this point
by Congo red stained smears and ThT fluorescence studies,
respectively).
Role of hydrogen bonds
The role of hydrogen bonds in amyloidogenesis was
analyzed by comparing FT-IR spectrograms in H
2
Oand
in D
2
O, known to prevent hydrogen bond formation. The
FT-IR spectrogram of C110)131 in D
2
O displayed signi-
ficantly less b-pleated sheet structures than the same peptide
analyzed in H
2
O (Figs 4B and 6). A similar decrease in
b-pleated sheet structures in D
2
O was observed with
R110)131 and C114)131 peptides (Fig. 6). Comparing
the spectrograms of the different peptides in D
2
O showed
that big-h3 110)122 had the highest percentage of b-sheet
content (34%), while R110)131, C110)131, and C114)131
had 15%, 23% and 6%, respectively.

b-pleated sheet configuration and to form amyloid. The
CONH
2
-terminus of the peptides analyzed has an inhibitory
Fig. 6. Secondary structure determination of the peptides by FT-IR
spectroscopy in D
2
O. Spectrograms of solutions containing the same
peptides included in Fig. 4 were analyzed in D
2
O (known to prevent
intra- and intermolecular hydrogen bonds). Note the absence of
inflexion of the curves both at 1625 and 1685 cm
)1
wavenumber, when
compared to the same peptides analyzed in H
2
O, showing that
hycdrogen bonding participates in the b-pleated conformation of the
peptides. The relative areas of the peaks around these wavenumbers
expressed as percentage of the total area over the curve were clearly
diminished for all the peptides (see the text).
Fig. 7. Spontaneous amyloid fibril formation.
(A) Congo red staining and polarized light of
big-h3 110)122 after 24 h of suspension in
distilled water showing a large amount of
amyloid material (· 125). (B) Electron
micrograph of the negatively stained material
from big-h3 110)122 spontaneous precipita-
tion on day 1 displaying bundles of 8–10 nm

formation does facilitate amyloid precipitation, but is not
the only factor responsible for the amyloidogenicity of
C110)131 peptide. The participation of other factors in
amyloidogenesis, unrelated to disulfide bonding, is further
supported by the appearance of amyloid fibrils with
H110)131, and to a lesser extent with the other peptides
studied.
Amyloid deposits are characteristically insoluble. Hydro-
phobicity is an important feature in the formation of
b-pleated insoluble aggregates [20,21]. The NH
2
-terminal
sequence of the peptides included comprises two valine
residues at positions 112 and 113. Our studies with the
peptides lacking the residues 110–113 (excluding the two
valine amino acids), showed a decrease in the formation of
amyloid fibrils, suggesting the importance of the hydropho-
bic NH
2
-end of the peptides. In the same way, removing the
CONH
2
-terminus of the peptides resulted in increased
amyloid fibril formation. This increase may be due to a
direct inhibitory effect of this nine-amino-acid sequence, or
to disequilibrium in the hydrophobic-hydrophilic balance of
the truncated peptide. Based on the importance of the two
Val residues in amyloid fibril formation we designed BB1, to
interfere hydrophobic interactions. The positive results
obtained with BB1 further confirm the importance of

for all the peptides assessed, including the native form, when
the peptide solution was allowed to interact for longer
incubation periods.
As well as the precise factors we demonstrate to
participate in amyloid fibril formation, other factors may
also contribute in TGFBI protein amyloidogenesis. The first
is the putative alteration in the degradation of the TGFBI
protein that would allow amyloid fibril formation. It has
been recently proven that corneas of patients affected by
LCD1, GCD1, and GCD2 have an abnormal pattern of
TGFBI protein content compared with normal corneas,
suggesting the existence of an abnormal degradation of the
protein [22,24]. The second is the putative participation of
local factors on the appearance of the disease. The TGFBI
protein, in addition to the cornea, is abundantly expressed
in the skin. However, the clinical manifestations of BIGH3
related dystrophies are restricted to the cornea and we failed
to find amyloid deposits in the skin in LCD1, even when
carefully checked using electron microscopy [5]. Further, it
has been established that cultured fibroblasts bearing the
R124C mutation do not produce amyloid spontaneously
[22]. Therefore, the involvement of the cornea may be the
consequence of specific constitutive and/or physiological
factors of this tissue that would enhance amyloid formation,
as the presence of the same protein precursor in other
tissues does not result in amyloid deposits. These factors
may include interactions with other constitutive molecules
(glycosaminglycans, [25]), and/or the particular physiology
of the cornea, which acts as a semipermeable membrane
allowing ion and water transfers as in our in vitro system.

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