Tài liệu Báo cáo Y học: BIGH3 (TGFBI) Arg124 mutations influence the amyloid conversion of related peptides in vitro Implications in the BIGH3-linked corneal dystrophies pptx

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; 2 Antigone Ophtalmologie, Montpellier, France; 3 Centre de Biochimie Structurale, CNRS UMR 5048, Universite ´ Montpellier, Montpellier, France; 4 Service d’Ophtalmologie, CMC Gui de Chauliac, Montpellier, France Amyloid deposits with Arg124 mutated TGFBI protein have been identified in autosomal dominant blinding corneal dystrophies. We assessed in vitro the mechanisms determin- ing TGFBI protein amyloid transformation involving mutations of Arg124. Eight peptides synthesized following the TGFBI protein sequence, centered on codon Arg124 holding the previously reported amyloidogenic mutations and the respective controls were studied. Cys124 and His124 mutated peptide preparations contained significantly higher amounts of amyloid than the native peptide. Blocking the SH group of Cys124 and deleting the first four NH 2 -terminal amino acids including Val112-Val113 resulted in a decrease in amyloid fibril formation while deletion of the nine CONH 2 -terminal residues increased amyloid fibril concen- tration. Fourrier transformed-infrared spectroscopy analysis of the different peptide solutions showed an increase in 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, transforming growth factor b-induced) gene [1]. Specific BIGH3 mutations have been linked to particular forms of the disease in this group of dystrophies. Autosomal dominant BIGH3-linked corneal dystrophies may present amyloid deposits, granular deposits or a mixture of both (granular and amyloid). The BIGH3 gene encodes for a 683 amino-acid protein inducible by TGFb, the TGFBI protein also known as big-h3. It is a prominent constituent of the cornea, skin, and matrix of many connective tissues [2]. It is a secreted protein with an amino-terminal secretory sequence, a carboxy- terminal Arg-Gly-Asp sequence and four homologous domains of 140 amino acids [2]. The TGFBI protein, as other homologous proteins, may interfere in the cell adhesion process. The Arg-Gly-Asp sequence is known to act as a ligand recognition site for integrins. The particular integrins with binding capacities for the corneal TGFBI protein remain to be fully identified. Kim et al. [3] have recently shown that alpha3-beta1 integrins bind to TGFBI. Two major sites for mutation have been recognized in the BIGH3 gene as inducing four distinct hereditary corneal dystrophies. These mutation sites are located at codon Arg124 and codon Arg555 [4]. Other mutations in the BIGH3 gene have been occasionally reported [5–11]. Mutations in Arg555 are responsible for corneal dystrophy of Bowman’s layer type 2 (CDB2, Thiel-Behnke corneal Correspondence to C F. Schmitt-Bernard, IGH CNRS UPR 1142, 141, rue de la Cardonille, F-34396 Montpellier cedex 5, France. Fax: + 33 4 67 42 39 73, Tel.: + 33 4 67 42 09 83, E-mail: cf.schmitt-bernard@antigone-ophtalmologie.fr 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 in vitro system to elucidate the mechanisms involved in amyloidogenesis. We analyzed the in vitro behavior of several peptides holding the TGFBI protein codon 124, whose sequences were selected according to previous genetic reports of corneal dystrophies linked to TGFBI protein Arg124 mutations. Following our results, that are in accordance with the clinical observations of Arg124 muta- tion linked amyloidosis, we designed a short peptide (BB1) with predicted capacity to inhibit amyloid fibril formation and tested its inhibitory effect on amyloid fibril formation from codon 124 mutated peptides in vitro. EXPERIMENTAL PROCEDURES Synthesis and purification of peptides Eight peptides derived from the TGFBI protein were synthesized (Table 1): four 22 amino-acid long peptides comprising codon 124 in its native (R110)131) and mutated forms (C110)131, H110)131, S110)131); two 18 amino- acid long peptides (R114)131, C114)131) missing amino acids 110–113 (Leu-Gly-Val-Val); one 13 amino-acid long peptide (big-h3 110)122) excluding codons 123 and 124 at the CONH 2 -terminal end; and one peptide homologous to C110)131 in which the SH group was blocked by using Fmoc-L-cystein acetamidomethyl (Acm) to prevent disul- fide bonding (C110)131Acm). The mutated peptides were synthesized following previous reports demonstrating the presence of C, H, and S in the corresponding clinical forms of corneal dystrophies (LCD1, GCD2 and GCD1, respectively). 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 were placed in the dialysis well of a Fast Dialyzer Interbiotech (Interchim S.A., Montluc¸ on, France) mounted with a dialysis membrane of 1000 Dalton cut-off. The sample solutions were dialyzed at 4 °C for 72 h against 1/15 M phosphate-buffered solution (pH 7.4). The dialysate was exchangedevery24hwithafreshbufferedsolution. Spontaneous fibril formation was assessed by the dilution of 0.5 lmol of each peptide in 50 lL of distilled water and studied on day 1, day 3, day 7, and day 15. Congo red stain Pellets obtained by centrifugation of the protein solution at 10 5 g,for1hat4°C were resuspended in 40 lL of distilled water. The samples were spread onto glass microscope slides with gelatin. The smears were air-dried, fixed with 95% ethanol for 5 min and stained with Congo red (Sigma- Aldrich Chemicals, Inc). The presence of amyloid fibrils was confirmed by viewing the typical apple-green birefringence under plane polarized light using a Zeiss microscope (Carl Zeiss, Go ¨ ttingen, Germany). Thioflavine T fluorescence analysis The studies were performed essentially as described in previous reports [16]. This technique has been proved to be adequate in amyloid quantification in other in vitro systems [17]. Thioflavine T (ThT) was purchased from Sigma- Aldrich Chemicals, Inc. (St Quentin Fallavier, France). The 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 described by Naiki et al. [16] with excitation at lambda- ex ¼ 450 nm and emission spectra at lambda-em ¼ 482 nm. Electron transmission microscopy The samples were applied to a formvar carbon-film-coated copper grid and then negatively stained with 1% uranyl acetate for 60 s. The specimens were viewed on a Hitashi H-7000 electron microscope (Hitashi LTD, Tokyo, Japan) with an acceleration voltage of 75 kV. Fourier-Transform Infrared Spectroscopy (FT-IR) The secondary structure of the peptides was studied with FT-IR spectroscopy of the amide I region performed in both H 2 OpH7.4andD 2 Oat25°C immediately after their dilution, and 24 h after their suspension in H 2 O [18,19]. D 2 O was used to prevent intermolecular and intramolecular hydrogen bond formation. Infrared spec- tra were collected on a Perkin-Elmer spectrum-one IR spectroscope (PE Applied Biosystems, Foster City, CA, USA). Absorbance was plotted against the wave number. The spectrograms were Fourier-deconvoluted and the secondary structure was determined by Gaussian curve- fitting. The calculation of each fraction of the total band 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 C110)131 in an assay volume of 50 lL, at 37 °C. Separate experiments were performed and independently processed on day 1, day 3, day 7, and day 14 by Thioflavine T fluorescence analysis in order to determine the amount of amyloid fibril formation. RESULTS Role of amino-acid 124 Congo red stained smears of peptides R110)131, C110)131, H110)131, and S110)131 dialyzed against a phosphate-buffered solution 1/15 M pH 7.4 displayed significant amounts of birefringent material. Transmission electron microscopy showed the fibrillar pattern of this material confirming the amyloid nature. The yield of amyloid was clearly different depending on the considered peptide (Fig. 1). Large quantities of amyloid were found with C110)131 and H110)131 peptides, while amyloid was scarcer with S110)131, and R110)131. The amount of amyloid was quantitatively determined using ThT spectrofluorometry as it can be expressed as the fluorescence emission spectra at lambda-em ¼ 482 nm following excitation at lambda-ex ¼ 450 nm in comparison Fig. 1. Dialysis-based amyloid fibril formation. Left hand side show the Congo red staining and right hand-side panels show polarized light microscopy of the same samples. The slides included correspond to (A) material obtained from dialysis of the C110–131 peptide (B) material obtained from dialysis of the H110–131 peptide (C) material obtained from dialysis of the S110–131 peptide (D) material obtained from dialysis of the R110–131 peptide (E) material obtained from dialysis 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 (1625 cm )1 )tob-aggregation (1685 cm )1 ) when comparing the baseline preparations to those at 24 h. Further, comparison of C110)131 to R110)131 showed that the former had approximately 30% more b-sheet conformation than R110)131 both at baseline and by 24 h. Role of the Cys–Cys disulfide bonds The role of disulfide bonding was examined by comparing amyloid formation of C110)131 to that of the same peptide with blocked sulfated radicals of Cys, the C110)131Acm peptide. Blocking the Cys residues resulted in a 50% decrease in ThT signal, suggesting that the increased tendency to form amyloid fibrils observed for C110)131 peptide is, at least in part, due to disulfide bonding (Fig. 5B). Blocking Cys–Cys disulfide bonding resulted in a similar decrease in amyloid fibril formation than trunca- ting the NH 2 -terminals sequence of the C110)131 peptide (peptide C114)131) suggesting both these elements are of equal importance for the formation of amyloid from the C110)131 peptide. Role of the NH 2 -terminal sequence The role of the NH 2 -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 -terminal sequence of the TGFBI protein derived peptides on amyloid precipitation was assessed by comparing the 22 amino-acid-long peptide centered on codon 124 (big-h3 110)131) to its counterpart missing residues 123–131 (big-h3 110)122). The amount of amyloid material obtained with big-h3 110)122 peptide was significantly higher than that obtained with the full length big-h3 110)131 peptide. This was clearly supported by morphological studies (Fig. 1E) and confirmed by quanti- tative analysis (Fig. 2). These differences were observed from dialysis as well as from spontaneous amyloid fibril formation studies, and were confirmed by FT-IR spectros- copy studies (Figs 4C and 6). big-h3 110)122 displayed a Fig. 2. ThT fluorescence profiles of synthetic peptides in solution after dialysis. The excitation wavelength was fixed at lambda-ex ¼ 450 nm. ThT fluorescence was maximal for a lambda-em wavelength of 482 nm and this value was used as the reference for the quantitative meas- urements of the amount of amyloid material contained in each peptide solution. ThT alone was included as for the auto-fluorescence control. big-h3 110)122 and C110)131 produced the most amyloid material. Lesser amounts of amyloid were obtained with H110)131, S110)131, and R110)131 solutions, respectively. Fig. 3. ThT fluorescence kinetic analysis of spontaneous amyloid preci- pitation. big-h3 110)122 appears to have the highest amyloidogenic properties when compared withthe big-h3 110)131 peptides. C110)131 and H110)131 show significant amyloid fibril formation from day 1 to day 15 in contrast to S110)131 and R110)131. Log scale Y axis. 5152 C F. Schmitt-Bernard et al. (Eur. J. Biochem. 269) Ó FEBS 2002 higher beta-content on FT-IR spectrograms than its full- length counterpart, both in D 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. Calculation of the ratio between the total surface area and the surface area at 1625 cm )1 and 1685 cm )1 shows that C110)131 contains approximately 30% more b-structures than R110)131 both at baseline and at 24 h indicating the higher tendency of C110)131 to adopt a b-pleated conformation. (C) big-h3 110)122 (the peptide missing the eight residues of the CONH 2 -terminal end) displayed a strong formation of antiparallel b-sheet conformation at 24 h confirming its high propensity to adopt a b-pleated conformation when compared to the other peptides analyzed (arrow) (profound depression at 1625 cm )1 wavenumber in the solid line plot corresponding to the peptide solution at 24 h). (D) Comparison of C114)131 (missing the hydrophobic Val112–Val113) and C110)131 peptides at 24 h. It can be observed that C114)131 has significantly less b-pleated structures than C110)131 (1625 cm )1 and 1685 cm )1 wavenumber differences arrows). Fig. 5. ThT fluorescence quantitative analysis comparing big-h3 110)131 and big-h3 114)131 peptides after dialysis. R114)131 and C114)131 produce less amyloid than R110)131 (A) and C110)131 (B), respectively, proving the NH 2 -terminal sequence is involved in the amyloid conversion of both the big-h3 110)131 peptides. A similar difference is apparent between C110)131 and C110)131 Acm suggesting that disulfide bonding also accounts for amyloid formation. Ó FEBS 2002 TGFBI protein Arg124 mutations and amyloid formation (Eur. J. Biochem. 269) 5153 ThT analysis confirmed the high propensity of big- h3 110)122 to form amyloid spontaneously as it displayed the highest ThT signal from day 1 to day 15 and had the fastest aggregation kinetics (Fig. 3). The higher amyloi- 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. These data strongly suggest that hindering hydrogen bond formation decreases the b-pleated sheet structures of the peptides, thereby limiting amyloid fibril formation. Effect of the b-pleated sheet inhibitor on amyloid fibril formation ‘ in vitro ’ The BB1 peptide, specifically designed to hinder amyloid formation, was incubated with C110)131. The presence of the BB1 peptide resulted in a 35% reduction in the yielding of amyloid fibrils in vitro throughout the complete length of the experiment (Fig. 8). Conversely, the BB2 peptide did not interfere with C110)131 amyloid fibril formation. The BB2 ThT fluorescence curve matched the C110)131 ThT fluorescence curve from day 1 to day 14. Therefore, BB1 peptide was able to inhibit amyloid fibril formation, and this inhibition was specific as BB2 control peptide did not modify amyloid yielding. DISCUSSION We previously presented an effective dialysis-based in vitro system to study the formation of amyloid fibrils from TGFBI protein-related synthetic peptides [14]. Based on this system, we have now assessed the influence of the structure of mutant TGFBI protein related peptides centered on codon 124 on amyloid formation. From our data it becomes apparent that (a) the particular amino-acid occupying the position 124, (b) the presence of Val–Val at positions 112–113, and (c) the existence of disulfide and hydrogen bonding, are of utmost importance in determining the tendency of TGFBI protein related peptides to adopt a 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 wide fibrillar structure undistinguishable from amyloid fibrils (· 60 000, bar ¼ 50 nm). 5154 C F. Schmitt-Bernard et al. (Eur. J. Biochem. 269) Ó FEBS 2002 effect on amyloid formation. Although the behavior of a synthetic peptide in vitro is not identical to that of the complete protein in vivo, our findings are in perfect agreement with the phenotype–genotype associations observed in clinical reports and identify some important factors determining the appearance of amyloidosis. Our system also allowed us to test a small peptide specifically designed to prevent amyloid fibril formation from the TGFBI protein related peptides. The results of these experiments were very encouraging. Concerning the importance of codon 124, our data clearly show that of all the peptides analyzed C110)131 is the peptide with the greatest tendency to form amyloid fibrils. The amount of amyloid material produced was the highest and kinetic studies showed that it was the fastest to precipitate into amyloid fibrils. On the contrary, the mutated S110)131 and the native R110)131 proved to have the least tendency to form amyloid fibrils as shown by ThT fluorescence analysis, while H110)131 presented an intermediate degree of amyloid fibril formation. Cysteine has the capacity to form disulfide bonds. As disulfide bonding of the protein may participate in the appearance of aggregates and predispose to the formation of amyloid fibrils, we explored the consequences of preventing disulfide bonding on amyloid transformation. Blocking the Cys residues decreased amyloid formation by approximately 50%, demonstrating that Cys–Cys bond 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 Val112-Val113 hydrophobic sequence in amyloid fibril formation from TGFBI protein derived peptides. Finally, hydrogen bonding also seems to participate substantially in the formation of amyloid fibrils as demon- strated by our studies on the secondary structure of the peptides, both in H 2 O (allowing hydrogen bonding) and D 2 O (preventing from hydrogen bonding) with less b-pleated structures under the milieu preventing hydrogen bonding. Recent clinical reports are challenging some of the classically accepted axioms on corneal dystrophy. Diseased corneas of mixed granular corneal dystrophy type 2 (GCD2), associated with the R124H mutation, contain substantial amounts of amyloid deposits when carefully studied [22]. More surprisingly, granular corneal dystro- phies of early and late onset, with pure granular deposits on clinical examination, have also been found to contain amyloid deposits in the cornea when assessed by electron transmission microscopy [23]. In that report, amyloid deposits were only identified in 1 out of the 2 affected family members [24]. Therefore, amyloid fibrils may be present in the deposits of supposedly pure granular dystro- phies while undetected clinically or with Congo red staining and light microscopy. The results reported in our study are in keeping with these recent clinical reports, as they demonstrate the capacity of forming amyloid fibrils in vitro 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. Fig. 8. Effect of the BB1 and BB2 peptides on amyloid fibril formation. Amyloid fibril formation from C110)131 peptide was significantly reduced in the mix containing BB1 (LPVVD) (35% when compared with the control curve of C110)131 in the absence of BB1). The mix with the nonspecific BB2 peptide (LPFFD) displays a ThT fluores- cence curve matching the C110)131 control curve thus showing no interference of BB2 with C110)131 amyloid fibril formation. Ó FEBS 2002 TGFBI protein Arg124 mutations and amyloid formation (Eur. J. Biochem. 269) 5155 These local factors may also explain the corneal involve- ment in other forms of amyloidosis [26]. In summary, autosomal dominant corneal diseases linked to mutations in the BIGH3 gene are a prominent cause of corneal blindness in adulthood. Conventional therapies remain unsatisfactory due to the high rate and short delay of recurrence after penetrating keratoplasty. Our findings explain at the molecular level the disparities observed among the different types of BIGH3 R124 mutation-linked corneal dystrophies and suggest that designing specific peptides able to inhibit the b-pleated conformational organization of TGFBI protein related peptides is an obvious route to successful therapy. The results of the first molecule with this type of capacity are reported. 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BIGH3 (TGFBI) Arg124 mutations in uence the amyloid conversion of related peptides in vitro Implications in the BIGH3- linked corneal dystrophies Clair-Florent. keratoepithelin; lattice corneal dys- trophy; granular corneal dystrophy; synthetic peptide. Hereditary corneal dystrophies are a cause of blindness. These dystrophies