Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission 1–40⁄ 41–123, named Th1-LA or a deletion of a chain segment of 12 amino
Trang 1of a-lactalbumin
Patrizia Polverino de Laureto1, Erica Frare1, Francesca Battaglia1, Maria F Mossuto1,
Vladimir N Uversky2,3,4and Angelo Fontana1
1 CRIBI Biotechnology Centre, University of Padua, Italy
2 Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Russia
3 Department of Biochemistry and Molecular Biology, Medical School, Indiana University, Indianapolis, IN, USA
4 Molecular Kinetics Inc., Indianapolis, IN, USA
Keywords
amyloid; a-lactalbumin; circular dichroism;
infrared spectroscopy; limited proteolysis;
molten globule; protein aggregation
Correspondence
A Fontana, CRIBI Biotechnology Centre,
University of Padua, Viale G Colombo 3,
35121 Padua, Italy
Fax: +39 49 827 6159
Tel: +39 49 827 6156
E-mail: angelo.fontana@unipd.it
(Received 21 December 2004, revised 23
February 2005, accepted 2 March 2005)
doi:10.1111/j.1742-4658.2005.04638.x
a-lactalbumin (LA) in its molten globule (MG) state at low pH forms amyloid fibrils Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission (1–40⁄ 41–123, named Th1-LA) or a deletion of a chain segment of 12 amino acid resi-dues located at the level of the b-subdomain of the native protein (1–
40⁄ 53–123, named desb-LA) We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA Th1-LA and desb-LA aggregate at pH 2.0 much faster than the intact protein and form long and well-ordered fibrils Furthermore, in contrast
to intact LA, the LA derivatives form regular fibrils also at neutral pH, even if at much reduced rate In acidic solution, Th1-LA and desb-LA adopt a MG state which appears to be similar to that of intact LA, as given by spectroscopic criteria At neutral pH, both Th1-LA and desb-LA are able to bind the hydrophobic dye 1-anilinonaphtalene-8-sulfonate, thus indicating the presence of exposed hydrophobic patches It is concluded that nicked Th1-LA and gapped desb-LA are more relaxed and expanded than intact LA and, consequently, that they are more suitable protein spe-cies to allow the large conformational transitions required for the poly-peptide chain to form the amyloid cross-b structure As a matter of fact, the MG of LA attains an even more flexible conformational state dur-ing the early phases of the aggregation process at acidic pH, as deduced from the enhancement of its susceptibility to proteolysis by pepsin Our data indicate that deletion of the b-subdomain in LA does not alter the ability of the protein to assemble into well-ordered fibrils, implying that this chain region is not essential for the amyloid formation It is proposed that a proteolytic hydrolysis of a protein molecule at the cellular level can trigger an easier formation of amyloid precipitates and therefore that lim-ited proteolysis of proteins can be a causative mechanism of protein aggregation and fibrillogenesis Indeed, a vast majority of protein deposits
in amyloid diseases are given by protein fragments derived from larger protein precursors
Abbreviations
[h], mean residue ellipticity; ANS, 1-anilinonaphtalene-8-sulfonate; apo-LA, calcium-depleted form of LA; desb-LA, protein species with excision of the 41–52 chain segment; E ⁄ S, enzyme to substrate ratio; LA, a-lactalbumin; MG, molten globule; RP, reverse-phase; TFA, trifluoroacetic acid; Th1-LA, disulfide-crosslinked, nicked protein species of LA with the peptide bond 40–41 cleaved; ThT, thioflavin T.
Trang 2The propensity to form amyloid fibrils appears to be a
general property of polypeptide chains, as under
speci-fic experimental conditions a variety of proteins can be
induced to aggregate into highly ordered protein
aggre-gates [1–10] Considering that all protein fibrils,
inde-pendently of the native structure of the given
amyloidogenic protein, adopt the common cross-b
structure motif, a large conformational rearrangement
has to occur prior to fibrillation [10] Such changes
cannot develop within a tightly packed native protein
and thus destabilization of the protein structure and
formation of a partly folded state is required In fact,
experimental conditions that favor the partial
unfold-ing of the protein are known to trigger the fibrillation
process [5–7,10] It has been proposed that specific
interactions between amino acid residues or chain
regions of a polypeptide with a high propensity to
acquire a b-sheet secondary structure lead to the kernel
that determines aggregation of other molecules into
the final, well-ordered fibrils [1–3] However, as even
small unfolded peptides are able to form amyloid
fibrils, protein conformational parameters are likely to
be insufficient to explain the molecular mechanism(s)
of fibrillation [11,12] In a number of cases, a
proteo-lytic event of a precursor protein appears to be
required for amyloidogenesis [13–16]
Lysozyme and a-lactalbumin (LA) belong to the
same protein superfamily, being homologous proteins
displaying similarity in their amino acid sequence and
overall 3D structure ([17] and references cited therein)
Human lysozyme has been shown to form amyloid
fibrils in individuals having point mutations in the
lyso-zyme gene [18] Recently, in order to understand better
the mechanism of lysozyme fibrillation, the aggregation
processes of lysozymes from different sources have been
investigated in detail (see [19] for references] Overall, it
seems appropriate to study the aggregation phenomena
of proteins belonging to the same protein superfamily
Probably, by studying similar proteins that form fibrils
in vitro it will be possible to highlight some peculiar
structural features that dictate the overall process of
protein fibrillogenesis Indeed, bovine LA is also able
to form amyloid fibrils if the protein is induced to
adopt the molten globule (MG) state at low pH [20]
However, fibril formation at pH 2.0 is much more
rapid if the protein, upon partial reduction oif its four
disulfide bridges, adopts a more open conformation
than that of the classical MG in acid solution [21,22]
This finding correlates with the observation that the
conformational features of apomyoglobin leading to
amyloid precipitates are more expanded than those of a
well defined and compact partly folded state of the
pro-tein [23] Similarly, the aggregation process of a SH3
domain at low pH implies significant structural rear-rangements, requiring a more flexible protein species that subsequently forms well-ordered fibrillar structures [24] Also a partly folded state has been shown to be involved in the fibrillogenesis of a-synuclein, a ‘natively unfolded’ protein [25–27] known to be involved in the pathogenesis of several neurodegenerative disorders [28] It has been found that a-synuclein undergoes fibrillation under experimental conditions that stabilize
a partly folded state, named premolten globule [29] These various observations can be interpreted as indi-cating that partly folded, but substantially open and dynamic states of proteins are those required for trig-gering the process of fibrillogenesis [10,20]
In this work, we analyse the effects of the dissection
of the LA molecule on its conformational features and aggregation processes Two protein species are herewith examinated, those given by the N-terminal fragment 1–40 covalently linked by the disulfide bridges of the protein to the C-terminal fragment 41–123 (Th1-LA) or fragment 53–123 (desb-LA) (Fig 1) These species have been prepared by limited proteolysis of LA by thermo-lysin (E.C 3.4.24.27) in 50% trifluoroethanol at neutral
pH or by pepsin (E.C 3.4.23.1) in acid solution [30–33] LA is not associated to any specific disease, but several observations have suggested that LA can evoke a variety of physiological effects [34] Besides its role in modulating the activity of the lactose synthase, new intriguing properties have been evidenced, such as the apoptotic activity in tumor cells of a partly folded variant of LA bound to oleic acid [35,36], the ability of the protein to bind histones [37,38] and the bactericidal activity of some of its chymotryptic peptides [39] Here,
we show that nicking of the 123-residue chain of LA at
a single peptide bond (Th1-LA) or removing a 12-resi-due segment (desb-LA) leads to protein species that easily form amyloid fibrils at pH 2.0 It is concluded that the inherent flexibility of these LA derivatives allows the large conformational changes required to form the cross-b-structure of the amyloid fibrils Of interest, it is also shown that the initial stages of fibril-lation of intact LA at low pH involve protein inter-mediate(s) characterized by enhanced chain flexibility This study emphasizes that the precursor structures of amyloid fibrils require a more unfolded and⁄ or flexible state than that of the MG [21,22]
Results Molecular features of Th1-LA and desb-LA Far-UV CD measurements indicate that, in acidic solution at pH 2.0 or at neutral pH in the presence of
Trang 3EDTA, the LA variants Th1-LA and desb-LA adopt
a conformation similar to the MG state displayed by
intact LA at low pH, retaining a native-like a-helical
content (Fig 2A,B) Furthermore, Th1-LA and
desb-LA bind the fluorescent dye
1-anilinonaphtalene-8-sulf-onic acid (ANS), which is considered diagnostic of a
protein MG state [40] While at pH 2.0 the three LA
species bind ANS in a similar way, at pH 7.4 the nicked or gapped LA species bind ANS more signifi-cantly than the intact protein, indicating that a larger hydrophobic surface is exposed to the solvent in the
LA derivatives (Fig 2C,D) (see also [32])
The molecular features of LA, Th1-LA and desb-LA
at pH 2.0 have been further analysed by limited proteo-lysis, urea-induced unfolding and hydrogen⁄ deuterium (H⁄ D) exchange measurements Limited proteolysis has been conducted at pH 2.0 using pepsin as protease and
LA and Th1-LA as substrates [41] Desb-LA was not analysed, because this species is a product of proteo-lysis of LA at pH 2.0 [30–32] The comparative suscep-tibility to proteolytic attack by pepsin of LA and Th1-LA is shown in Fig 3A The extent of proteolysis was calculated from the amount of residual protein determined by reverse phase (RP)-HPLC analysis of aliquots of the reaction mixtures at various times of proteolysis After 10 min’ incubation of the proteins with pepsin at pH 2.0, Th1-LA has been 80% cleaved, while LA only 50% After 20 min’ reaction, the proteolysis is complete for Th1-LA, whereas some intact LA is still present in the proteolysis mixture even after 50 min’ reaction (Fig 3A)
To evaluate further the molecular differences between
LA and its derivatives, the effect of urea on the protein tertiary structure was analysed by near-UV CD spectros-copy (Fig 3B) Earlier it was shown [32] that these LA derivatives retain some tertiary interactions at pH 2.0, as given by a near-UV CD spectrum characterized by a band centered at about 290 nm due to the contribution
of tryptophan residues [42] As shown in Fig 3B, the increase in urea concentration at pH 2.0 was accompan-ied by a gradual reduction of the CD signal at 291 nm for the three LA species, implying that the urea-medi-ated denaturation is not a cooperative process Even if the overall processes are quite similar for the three pro-tein species, it seems that intact LA retains some residual structure in the presence of 4 m urea, whereas desb-LA appears to be completely unfolded under the same conditions In the case of Th1-LA, a complete disappear-ance of the CD signal at 291 nm occurs in 6 m urea The flexibility of the polypeptide chain of LA and its gapped and nicked species was also analysed by
H⁄ D measurements, i.e monitoring by ESI-MS the change in the protein mass that results from the replacement of labile protons by deuterium The fea-tures of the H⁄ D exchange of the low-pH MG of LA have been already examined [43–46], showing that only the amides in the a-domain are most protected from
H⁄ D exchange [43] The protein species herewith inves-tigated are characterized by a nicking or a removal of the b-subdomain (Fig 1) and therefore a significant
N
C
Ala40-Ile41 Leu52-Phe53
5-11 h1b 23-34H2 S1 S2 S3
h2 86-98H3 105-110H4 h3c
Gln-Ala-Ile-Val-Gln-Asn-Asn-Asp-Ser-Thr-Glu-Tyr-Gly-Leu-Phe-Gln
Th1-LA
desβ-LA
Fig 1 (Top) Schematic representation of the 3D structure of LA.
The diagram was drawn using the PDB file 1HFZ using the program
WEBLAB VIEWER PRO 4.0 (Molecular Simulations Inc., San Diego, CA).
The chain segment 41–52 encompassing the antiparallel b-sheet is
colored in red The four disulfide bonds are represented by yellow
sticks and the calcium atom by a solid sphere in green (Middle)
Scheme of the secondary structure of the 123-residue chain of LA
[58] The four a-helices (H1–H4) along the protein chain are
indica-ted by major boxes and below them the corresponding chain
seg-ments are given The three b-strands (S1, 41–44; S2, 47–50; S3,
55–56) and the 310helices (h1b, 18–20; h2, 77–80, h3c, 115–118)
are indicated by small boxes The amino acid sequence of the chain
region 39–54 of LA is explicitly shown and the sites of proteolysis
by pepsin and thermolysin are indicated by arrows (Bottom)
Sche-matic structure of the LA derivatives Th1-LA and desb-LA These
protein species are given by the N-terminal fragment 1–40
cova-lently linked by the four disulfide bridges of the protein to the
C-ter-minal fragment 41–123 (Th1-LA) or fragment 53–123 (desb-LA).
The connectivities of the four disulfide bridges (thin lines) of LA are
6–120, 28–111, 61–77 and 73–91.
Trang 4difference in their H⁄ D behavior is not expected In
fact, Fig 3C shows that the time-courses of H⁄ D
exchange for the three LA species are only slightly
dif-ferent However, the lower and the higher extent of
exchanged protons by LA and desb-LA, respectively,
are consistent with a more compact and a more
flex-ible structure in LA and desb-LA, respectively
Aggregation properties of LA derivatives
To induce amyloid formation, Th1-LA and desb-LA
were dissolved (5 mgÆmL)1) in 10 mm HCl pH 2.0, or
in 50 mm Tris⁄ HCl buffer pH 7.4, containing 0.1 m
NaCl, and incubated at 35C for up to 20 days The
aggregation process was followed by thioflavin T (ThT)
binding assay [47,48] and electron microscopy (EM)
CD and FTIR measurements have been used to
mon-itor the conformational changes during aggregation
The nicked or gapped LA derivatives aggregate very
fast (Fig 4) and form long and well-defined fibrils
(Fig 5) The sigmoid curve of the ThT fluorescence
emission at 485 nm vs time of aggregation (Fig 4)
observed with intact LA is characterized by a lag time
and is consistent with a nucleation-dependent
elonga-tion model of fibrillogenesis [20] On the other hand,
the lag time is almost not observed with both Th1-LA and desb-LA incubated at pH 2.0 (Fig 4) Further-more, with the LA derivatives a larger increase in ThT fluorescence is observed upon prolonged incubation at low pH and the intensity of fluorescence reaches a plat-eau after about 70–80 h Assuming that the ThT fluor-escence enhancement is proportional to the population
of well-ordered protein aggregates [47,48], we can con-clude that the amount of fibrils formed by Th1-LA and desb-LA is decidedly larger than that formed by intact
LA under similar experimental conditions EM reveals that the fibrils formed by Th1-LA and desb-LA after
70 h incubation at pH 2.0 are typical amyloid, with a filamentous aspect, unbranched and with a diameter of
10 nm (Fig 5), quite similar to those formed by intact LA under the same experimental conditions [20] Aggregation experiments of the LA derivatives have been conducted also at pH 7.4 While intact LA does not form fibrils at neutral pH [20], both Th1-LA and desb-LA after 230 h of incubation do form ordered aggregates In fact, EM reveals the presence of spher-ical aggregates with dimensions of 4–8 nm (Fig 5, right panels) and some of them show the typical mor-phology of protofibrils [49] In the case of Th1-LA, the initial aggregates are rare, if compared to those
250 240 230 220 210 200 190
.m
1-)
-15 -10 -5 0 5
pH 2.0
pH 7.4 fibrils
Wavelength (nm)
250 240 230 220 210 200 190 -15 -10 -5 0 5
pH 2.0
pH 7.4 fibrils
0 200 400 600 800
1000
LA Th1-LA
pH 2.0
0 10 20 30 40 50
LA
Th1-LA desβ-LA
pH 7.4
Fig 2 Conformational characterization of
Th1-LA and desb-LA by CD (A, B) and ANS
binding (C, D) Far-UV CD spectra of Th1-LA
and desb-LA were recorded in 10 m M
Tris ⁄ HCl ⁄ 0.1 M NaCl buffer pH 7.4,
contain-ing 1 m M EDTA or in 0.01 M HCl ⁄ 0.1 M NaCl
pH 2.0, at a protein concentration of 0.05–
0.1 mgÆmL)1 The far-UV CD spectra of
fibrils of Th1-LA and desb-LA refer to those
of samples obtained by aggregation of the
protein at 35 C pH 2.0, for 48 h and 65 h,
respectively Fluorescence emission spectra
(C, D) of 20 l M ANS in the presence of
10 l M of Th1-LA or desb-LA The spectra
are recorded at 20–22 C from 390 to
650 nm after excitation at 370 nm in 10 m M
HCl pH 2.0, or in 10 m M Tris ⁄ HCl pH 7.4.
Trang 5observed with desb-LA Moreover, after a prolonged time of incubation, fibrils with a more defined mor-phology develop in the case of desb-LA, but more slowly in the case of Th1-LA
Analysis of the protein secondary structure has been carried out by far-UV CD measurements [50] on aliqu-ots taken at intervals (40–70 h) from a solution of Th1-LA and desb-LA incubated at 35 C pH 2.0 An extensive conformational rearrangement takes place during the aggregation process, as given by the disap-pearance of the a-helix bands at 208 and at 220 nm and by the development of the band near 217 nm, typ-ical of b-secondary structure (Fig 2A,B) On the other hand, a sample of intact LA incubated under similar solvent conditions does not develop significant changes
of CD spectra (data not shown), even after prolonged time of incubation and thus even if aggregated (Fig 4) This could be explained by considering that the helical secondary structure, even if present in mod-erate percentages in respect to other types of secondary structures (random coil, b-sheet), shows a CD signal that prevails on the others [50]
To verify if under the acid conditions used to induce fibril formation the protein samples are chemically modified or hydrolyzed, the fibrillar material produced after 10 days of incubation has been analysed by MS, following essentially procedures previously described
Time (min)
0
20
40
60
80
100
Urea concentration (M)
]θ0
0.0
0.2
0.4
0.6
0.8
1.0
Time (min)
0
20
40
60
80
B
C
Fig 3 Molecular features at pH 2.0 of LA (d), Th1-LA (s) and
desb-LA (.) probed by limited proteolysis (A), urea-induced
unfold-ing (B) and H ⁄ D exchange rates (C) (A) Susceptibility to proteolysis
by pepsin at pH 2.0 of LA and Th1-LA The percent of undigested
protein was calculated from the area of the protein peaks in the
RP-HPLC chromatograms of protein samples analysed at different
time intervals (B) Urea-induced unfolding of the tertiary structure
of LA, Th1-LA and desb-LA monitored by CD measurements at
291 nm at 20–22 C pH 2.0 (C) H ⁄ D exchange profiles of LA,
Th1-LA and desb-LA at pH 2.0 as monitored by MS.
Time of Incubation (h)
0 20 40 60 80
100 desβ-LA
Th1-LA
LA
Fig 4 Time course analysis of aggregation at 35 C pH 2.0, of LA, Th1-LA and desb-LA monitored by thioflavin T (ThT) binding Aliqu-ots (7 lL) were taken from the protein solution after the indicated time and added to a 25 l M solution (500 lL) of ThT in 25 m M phos-phate buffer pH 6.0 The excitation wavelength was at 440 nm and the emission was measured at 485 nm The protein concentration
of each protein sample was 0.4 m M
Trang 6[19] In the case of LA, some protein degradation
( 10%) has been actually observed (data not shown)
Instead, the desb-LA and Th1-LA fibrils are composed
by intact protein molecules This could be due to the
more rapid aggregation process of the LA derivatives
in respect to that of the intact protein, likely rendering
the aggregated desb-LA and Th1-LA somewhat
pro-tected from degradation
Analysis of fibrillogenesis of intact, nicked and
gapped LA by FTIR
To evaluate the conformational rearrangements of
LA and its derivatives during the aggregation process,
FTIR measurements [51–53] were conducted on
mono-meric and aggregated proteins Aggregation was
moni-tored by analysing the second derivative of the FTIR
spectra of protein samples incubated for varying length
of time at 35C pH 2.0 The second derivative of the
FTIR spectrum of LA at pH 2.0 before aggregation
(Fig 6) shows three major bands, one centered at
1648 cm)1 characteristic of a-helix and⁄ or random
structure and two centered at 1632 and 1675 cm)1
related to the antiparallel b-structure [51–53] These
spectral characteristics are in agreement with those
reported for intact LA exposed to low pH [20] After
6–8 h incubation, the main band at 1648 cm)1shifts to
a lower wavenumber (1643 cm)1), indicating a preval-ence of random coil and therefore a further structural unfolding of the protein After 48 h, the bands due to the b-sheet structure (1632 and 1675 cm)1) are evident and, upon prolonged incubation (300 h), a band cen-tered at 1616 cm)1 develops, together with a shift of the band at 1675 cm)1 to higher wavelengths These last bands have been related to fibril formation, as the association of b-sheets cause a splitting of the antipar-allel b-sheet bands [20]
Figure 6 (middle panel) shows the evolution of the FTIR spectrum of Th1-LA during aggregation at
pH 2.0 Initially, the spectrum of Th1-LA is quite similar to that of intact LA under the same solvent conditions A structural rearrangment of Th1-LA appears to take place before initiation of the aggrega-tion process (Fig 6, 1h) In fact, the FTIR spectrum recorded after 1 h shows a main band at 1645 cm)1, which is indicative of an enhanced content of dis-ordered structure The spectrum remains unmodified
up to 8–10 h of incubation After 24 h of Th1-LA incubation, the band at 1643 cm)1 is no more present and a new band at 1616 cm)1 (aggregation band) appears Upon prolonged incubation, there is a preval-ence of the bands characteristic of protein fibrils (1616
des -LA, pH 7.4, 235 h
Th1-LA, pH 7.4, 235 h
des b -LA, pH 2.0, 70 h b b
Th1-LA, pH 2.0, 70 h
des -LA, pH 7.4, 720 h
Th1-LA, pH 7.4, 800 h
Fig 5 Electron micrographs of Th1-LA and desb-LA fibrils obtained after 70 h incubation of the LA variants at 35 C pH 2.0 The morphology
of the initial aggregates produced after 235 h of incubation of the protein samples at 35 C pH 7.4, and of those produced after a much pro-longed incubation time are also reported.
Trang 7and 1680 cm)1), whereas those of the regular structure
reduce their intensity Overall, the FTIR data indicate
that the aggregation processes of LA and Th1-LA are
similar, but that Th1-LA aggregates significantly
fas-ter The aggregation process of desb-LA, followed by
FTIR analysis, shows a similar behavior to that
dis-played by Th1-LA both in terms of rate of appearance
and type of FTIR bands (Fig 6, right panel)
Early stages of LA aggregation followed
by limited proteolysis
In previous studies we have shown that proteolytic
probes can be used to analyse structure and dynamics
of proteins in their partly folded or MG states [41]
and during their aggregation process [24] Here, we
have applied this technique for analysing the early
sta-ges in the aggregation process of intact LA To this
end, aliquots of the protein sample of LA incubated at
35C pH 2.0, were withdrawn from the reaction
mix-ture at various times (from 0 to 200 h) and treated
with pepsin in order to test the susceptibility to
proteo-lysis of LA during the aggregation process Fig 7
shows the reverse phase–HPLC analyses of the
proteo-lysis reactions conducted on LA after 0, 8, 24 and 72 h
incubation The pattern of LA proteolysis with pepsin
(panel A) is similar to that reported previously [30–32],
with the major products being the fragment species
1–40⁄ 53–123 (desb-LA), 1–40 ⁄ 104–123 and 53–103
The protein sample left to aggregate for 8 h at pH 2.0 appears to be more sensitive to the protease action, because more fragments and less intact protein are found in the mixture (Fig 7B) The analysis of the proteolysis of a sample of LA incubated for a pro-longed time (panels C and D) reveals that the protein becomes more and more resistant to proteolysis In Fig 7 (bottom), the percent hydrolysis of LA by pep-sin as a function of aggregation time is reported It is seen that LA at the initial stages of the aggregation process is more easily digested by proteolysis and thus is more unfolded and⁄ or flexible, a feature that appears to render the polypeptide chain more prone to fibril formation [24]
Discussion Here, we have studied the aggregation processes of LA species with a nick (Th1-LA) or a chain deletion (desb-LA) at the level of the b-subdomain of the 123-residue chain of the protein (Fig 1) We have also characterized the early stages of protein aggregation in order to get insights into the molecular mechanisms of fibrillogenesis At pH 2.0, the overall features of the partly folded or MG states formed by Th1-LA and desb-LA appear to be quite similar to that formed by intact LA at low pH under equilibrium conditions, as judged from CD measurements (see also [32]) How-ever, there are some differences in stability and⁄ or
h 0
h 6
h 8
Wa v e u m e ( c m - 1 )
0 6 0 6 0 6 0 6 0 1 0 1
h 0 3
5 1
8 1 2 1
6 1
3 1
0 1
5 1
5 1 8 1 2 1
h 0
h 1
h 4
a
W v nu b e m er ( cm - 1 )
0 6 0 1 0 6 0 6 0 6 0 7
h 6
2 1 9 1 5 1
5 1
9
1 1 2
6 1
0 1
5 1
5 1
s
eβ- A L
h 0
h
h 4
Wave u m b r e ( cm - 1 )
0 6 0 1 0 6 0 6 0 6 0 7
h 6
5 1 8
1 1 2
6 1
5 1
0 1
5 1
5
1 1 8 1 2
Th1-LA t
c a t n I
Fig 6 Aggregation process of LA, Th1-LA and desb-LA monitored by FTIR The panels show the time-evolution of the FTIR spectra of the amide I region (continuous line) of LA (left panel), Th1-LA (middle panel) and desb-LA (right panel) during fibril formation at 35 C pH 2.0 The second derivative spectra (dashed lines) are also shown.
Trang 8flexibility between the three protein species, as inferred
from proteolysis experiments, urea-mediated
denatura-tion and H⁄ D exchange measurements (Fig 3) The
major conclusion of this study is that the
conforma-tional features of Th1-LA and desb-LA, which are
more relaxed and expanded in comparison to intact
LA at low pH, are more suitable for triggering the
protein fibrillation process Importantly, the deletion
of the b-subdomain in LA does not alter the ability of the protein to assemble into well-ordered fibrils In this context, it is interesting to recall that in previous studies it has been proposed that the b-domain is of particular significance in triggering the fibril formation
in the case of lysozyme ([54] and references cited therein), a protein belonging to the same structural superfamily of LA Instead, here it is shown that a LA derivative such as desb-LA, lacking the three b-strands
of the protein (Fig 1), is able to aggregate even more readily than the intact protein, implying that the b-sheet region of LA is not required for fibrillogenesis
It may be proposed that the exposure of the hydro-phobic interior of the protein at low pH is sufficient for promoting the aggregation phenomenon, leading ultimately to well-ordered fibrils
An important aspect of this study is the analysis of the molecular features of LA and its derivatives at the initial stages of protein aggregation at pH 2.0 During the lag time, intact LA undergoes significant conform-ational changes, as evidenced from FTIR (Fig 6) and limited proteolysis experiments (Fig 7), leading to an increase in the amount of disordered structure in LA
at the early stages of fibrillation (after 6–10 h incuba-tion) In fact, less intact LA remains in the proteolysis mixture with pepsin (Fig 7), indicating that the pro-teolytic degradation increases after incubation of LA
at pH 2.0 for 6–10 h We may infer that LA, at the initial stages of aggregation, exists in a more expanded and flexible conformational state and, for this reason,
is more sensitive to proteolytic attack [41] Import-antly, also Th1-LA and desb-LA appear to initially reach a more unfolded⁄ flexible state (from FTIR data, see Fig 6), but faster and in about 1 h only This is likely due to the fact that Th1-LA and desb-LA are more open and flexible protein species than intact LA
By analogy, similar arguments can be used also to explain why, at variance from the LA derivatives, the intact protein does not aggregate at neutral pH [20] Clearly, the intact protein is native and rigid at neutral
pH and does not populate the partly folded state required for fibrillation
An interesting question here is the apparent discrep-ancy between FTIR data and far-UV CD measure-ments FTIR data provide evidence of an initial phase
in which the three proteins herewith studied develop
an increase in the disordered structure and, at longer incubation times, the band characteristic of the b-sheet structure of the amyloid is clearly observed (Fig 6) The FTIR data are in agreement with the fact that aggregated Th1-LA and desb-LA show a far-UV CD spectrum which is typical of the b-sheet secondary structure (see Fig 2A,B) On the other hand, far-UV
Retention Time (min)
24 h
41-52 1-40/104-123
LA
1-40/53-123 1-40/53-123
LA
1-40/104-123
41-52
1-40/53-123 LA
1-40/104-123
41-52
53-103 53-103
Time of Incubation (h)
0
20
40
60
80
100
Fig 7 Early stages of aggregation of intact LA at pH 2.0 as
monit-ored by limited proteolysis LA was allowed to aggregate at 35 C
pH 2.0 Aliquots (50 lL) were taken at intervals from the LA
solu-tion and mixed with a pepsin solusolu-tion for 45 min at 20–22 C (Top)
A sample of the proteolysis mixture was analysed by RP-HPLC
using a Vydac C18 column (150 · 4.6 mm), eluted with a linear
gra-dient of acetonitrile ⁄ 0.1% TFA from 5% to 34% in 4 min and from
34% to 50% in 18 min (Bottom) Time-course of the susceptibility
of LA to proteolysis by pepsin during aggregation at 35 C pH 2.0.
The extent of cleavage was calculated by integration of the area of
the peaks in the RP-HPLC chromatograms of protein samples
ana-lysed at different time intervals.
Trang 9CD spectra measured for intact LA during the entire
aggregation process show minor changes in their shape
and characteristics mostly of a-helical structure (data
not shown) This can be explained by considering that
it is likely that residual a-helical structure is still
pre-sent in the aggregated mixture of intact LA and that
the intensity of the CD signal of the helical structure is
much higher than that of other secondary structures,
including b-sheet [50]
The key result of this study is that limited
proteo-lysis of LA leads to protein species that are much
more prone to fibrillogenesis than the intact protein
Therefore, it is shown here, with the model protein
LA, that amyloid fibril formation can require
proteoly-sis This is in line with the fact that a large proportion
of protein deposits associated with amyloid diseases is
made by protein fragments derived from proteolysis of
larger protein precursors [2,11,12] First of all, we may
mention here that the prototypic fibril-forming
frag-ment Ab in Alzheimer’s diseases is derived from the
amyloid precursor protein by a combination of
pro-teolytic cleavages given by b- and c-secretase [55]
Caspase-cleavage of the cytoskeletal tau protein is an
early event in Alzheimer’s diseases tangle pathology
[16] Human gelsolin is expressed as an 81-kDa
intra-cellular protein and its limited proteolysis at the level
of peptide bonds 172–173 and 243–244 leads to a
71-residue fragment that is found in protein deposits
in individuals with familial amyloidosis [13] The
34-residue ABri peptide is derived from a putative
transmembrane precursor and is found in plaques of
familial British dementia [56] These and other
obser-vations [2,11,12], in line with the results of this study,
provide an evidence that proteolysis can be a critical
prefibrillogenic event
Protein fragments, originating by proteolysis of
pro-tein precursors, are particularly vulnerable to
aggrega-tion, because they can usually adopt, at most, partly
folded states and cannot establish the long-range
inter-actions that stabilize the native intact protein In
par-ticular, protein fragments may contain hydrophobic
clusters of residues that can trigger protein
aggrega-tion In the case of the LA derivatives Th1-LA and
desb-LA, it may be well that the nicking of the
123-residue chain polypeptide chain causes an untighting
of the structural domains of LA (see Fig 1),
determin-ing exposure of hydrophobic patches Furthermore,
the fact that the LA derivatives aggregate much more
easily at low pH can be explained by considering that
a minimization in acid solution of the negative charges
of the carboxylates of the Asp and Glu residues in the
calcium-binding region of LA can produce a marked
decrease in the charge-to-charge repulsions and a
concomitant enhancement of the protein association process by favoring intermolecular hydrophobic inter-actions
In recent years, numerous studies have been conduc-ted on the low pH MG state of LA, nowadays consid-ered a prototype MG in protein folding studies [21,22]
A consensus view is that the MG of LA retains signifi-cant native-like structure in the a-domain, while the b-domain (approximately region 50–90) is disordered From this and previous [32] study, it seems that the
MG state at pH 2.0 retains substantial native-like structure that does not allow the protein to form the amyloid precipitate On the other hand, the low pH MGs adopted by Th1-LA and desb-LA are similar to the MG of intact LA, but they are more open and flexible and thus more prone to aggregation There-fore, a limited proteolysis phenomenon of a protein can lead to an enhanced population of a rather unfol-ded and⁄ or flexible protein species that triggers the fibrillation process
Conclusions Substantial data are available to support a model for the amyloid formation in which intermolecular inter-actions between hydrophobic patches in partly folded
or MG states of proteins are responsible for protein aggregation [6–10] The intermediates are more prone
to aggregate than the unfolded state because they retain clusters of hydrophobic side chains, which have
a strong propensity for aggregation [6] However, a conformation that is more expanded than typical MG appears to be required before protein assembly into an ordered amyloid-like structure [10,20] Our observa-tions are in agreement with a general view that a more flexible conformation or a moderately unfolded struc-ture could be the key species triggering fibril forma-tion In particular, the results of this study highlight the possible role of limited proteolysis as a causative event of fibrillogenesis It seems possible to propose that a proteolytic attack of a protein at the cellular level can shift the equilibrium between the different protein conformational states towards a species that is more prone to aggregate
Experimental procedures Materials
Bovine a-lactalbumin, porcine pepsin, thermolysin and ThT were from Sigma (St Louis, MO, USA) All other chemi-cals were of analytical reagent grade and were obtained from Sigma or Fluka (Basel, Switzerland)
Trang 10Isolation of nicked and gapped LA
The LA derivatives were obtained by limited proteolysis of
the protein, as described previously [30–33] Proteolysis of
bovine LA with thermolysin was performed in a 1 : 1 (v⁄ v)
mixture of 50 mm Tris⁄ HCl pH 7.0, containing 5 mm CaCl2
and trifluoroethanol using an enzyme⁄ substrate (E ⁄ S) ratio
of 1 : 20 (w⁄ w) [30,32] The reaction was conducted at 20–
22C for 6 h and then proteolysis was quenched by
acidifi-cation The proteolysis of LA with pepsin was performed in
0.01 m HCl⁄ 0.1 m NaCl pH 2.0, for 45 min at 20–22 C
and at an E⁄ S ratio of 1 : 750 (w ⁄ w) [32] All digestions
of LA were carried out a protein concentration of
1 mgÆmL)1 Proteolysis was stopped by alkalinization
of the solution with aqueous ammonia (pepsin) or by
acidifi-cation with 4% (v⁄ v) trifluoroacetic acid (TFA) in water
(thermolysin) The LA derivatives (Th1-LA and desb-LA)
were purified by micropreparative RP-HPLC on a Vydac
C18column (150· 4.6 mm, 5 lm; The Separations Group,
Hesperia, CA), eluted at a flow rate 0.6 mLÆmin)1 with a
gradient of acetonitrile containing 0.1% TFA and
monitor-ing the effluent by absorbance measurements at 226 nm
The proteolysis experiments by pepsin conducted in parallel
on LA and its derivative Th1-LA were performed under the
same conditions as above described for LA
Fibril formation and characterization
The amyloid fibrils of LA and its fragments were prepared
by incubating protein samples (5 mgÆmL)1) in 10 mm HCl
pH 2.0, and in 10 mm Tris⁄ HCl pH 7.4, at 35 C for up to
30 days To confirm the presence of protein
aggre-gates⁄ fibrils, aliquots of the samples were examined by the
ThT fluorescence assay [47,48] and by EM The ThT
bind-ing assay was performed usbind-ing a freshly prepared 25 lm
ThT solution in 25 mm sodium phosphate pH 6.0 Protein
samples from suspensions containing aggregates were
dilu-ted into the ThT buffer (final volume 500 lL) Fluorescence
emission measurements were conducted at 25C, using an
excitation wavelength of 440 nm and recording the ThT
fluorescence emission at 485 nm EM pictures were taken
on a JEOL model JEM-1010 instrument operating at
80 kV Samples were diluted 20-fold and a drop of the
solution was placed on a Formvar-coated nickel grid
(400-square mesh, Agar Scientific, Stansted, UK) A drop
of uranyl acetate solution (2%, w⁄ v) was placed on the grid
and after a few seconds the grid was washed with deionized
water (MilliQ, Millipore, Billerica, MA, USA)
Spectroscopic measurements
Protein concentrations were determined by absorption
measurements at 280 nm on a double-beam Lamda-20
spec-trophotometer from Perkin Elmer (Norwalk, CT, USA)
Extinction coefficients (e mgÆmL)1) at 280 nm for LA and its derivatives were evaluated on the basis of their amino acid composition [57] and were 2.01 for LA and Th1-LA (1–40⁄ 41–123) and 2.23 for desb-LA (1–40 ⁄ 53–123) CD spectra were recorded on a Jasco J-710 (Tokyo, Japan) spectropolarimeter equipped with a thermostated cell holder Far-UV CD spectra were recorded using a 1 mm pathlength quartz cell and a protein concentration of 0.05– 0.1 mgÆmL)1 The mean residue ellipticity [h] (degÆcm2Æ dmol)1) was calculated from the formula [h]¼ (hobs⁄ 10)Æ(MRW lc)1), where hobsis the observed ellipticity
in deg, MRW is the mean residue molecular weight (molecular weight of the protein divided by the number of amino acids), l the optical pathlength in cm and c the pro-tein concentration in mgÆmL)1 The urea-mediated unfold-ing at pH 2.0 of LA, Th1-LA and desb-LA was monitored
by following the near-UV CD signal at 291 nm at 20–
22C The measurements were made after equilibrating the protein samples for 10 min A protein concentration of
25 lm and a cuvette of 0.5 cm pathlength were used Fluorescence measurements were performed using a Perkin-Elmer model LS-50 spectrofluorimeter, utilizing a cuvette with 0.1-cm pathlength An excitation wavelength
of 370 nm was used for ANS binding experiments and the emission spectra scanned from 390 to 650 nm [40] All spec-tra were recorded at 20–22C using a 20 lm solution of ANS and a 10 lm solution of protein ANS-binding experi-ments were conducted with protein derivatives dissolved
in 0.01 m HCl⁄ 0.1 m NaCl pH 2.0, or in 10 mm TrisÆHCl ⁄ 0.1 m NaCl buffer pH 7.4 The concentration of the ANS stock solution was determined using a molar absorption coefficient of 5· 103m)1Æcm)1at 350 nm
FTIR spectra were recorded at 20–22C using a Perkin Elmer 1720X spectrometer, purged with a continuous flow
of N2gas Solutions of 0.35 mm LA in D2O were acidified
to the desired pH using DCl Protein solutions were placed between a pair of CaF2 windows separated by a 50 lm Mylar spacer For each protein sample, 50 interferograms were accumulated at a spectral resolution of 2 cm)1 Buffer spectra were recorded under identical conditions to those of the protein samples and subtracted from the protein spec-tra The second derivative of the amide I band was used to identify the different spectral components
H/D exchange measurements
H⁄ D exchange measurements [43–46] were conducted
by recording the spectra on a Q-Tof Micro (Micromass, Manchester, UK) at a capillary voltage of 3 KV and a cone voltage of 40 V To perform the H⁄ D exchange, lyophilized samples of LA, Th1-LA and desb-LA were dissolved in
10 mm HCl pH 2.0, and diluted 35-fold in D2O at the same
pH to give a final concentration of 10 lm The deuterium content was deduced from the increase in molecular mass of