In this study, we tested the amyloidogenic properties, if any, of sea bream TTR sbTTR and Escherichia coli transthyretin-related protein ecTRP, which share 52% and 30% sequence identity,
Trang 1Transthyretin (TTR) is a homotetrameric plasma
pro-tein that binds and transports the thyroid hormones
3,5,3¢-triiodo-l-thyronine and
3,5,3¢,5¢-tetraiodo-l-thyr-onine (thyroxine) and retinol by binding to the
retinol-binding protein when it is loaded with retinol [1] TTR
is mainly expressed in the adult liver, the choroid
plexus of the brain, and the retina [2,3] TTR is
involved in three amyloid diseases: familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy (FAC), and senile systemic amyloidosis (SSA) [4,5] Whereas SSA is associated with native TTR, point mutations, of which more than 80 have been identified, cause FAP and FAC [6] TTR mutations associated with familial amyloid diseases display a wide range of
Keywords
amyloid; fibril formation; HIU hydrolase;
transthyretin; transthyretin-related protein
Correspondence
A E Sauer-Eriksson, Department of
Chemistry, Umea˚ University, SE-90187
Umea˚, Sweden
Fax: +46 90 7865944
Tel: +46 90 7865923
E-mail: elisabeth.sauer-eriksson@chem.
umu.se
(Received 7 November 2008, revised 20
January 2009, accepted 26 January 2009)
doi:10.1111/j.1742-4658.2009.06936.x
Human transthyretin (hTTR) is one of several proteins known to cause amyloid disease Conformational changes in its native structure result in aggregation of the protein, leading to insoluble amyloid fibrils The trans-thyretin (TTR)-related proteins comprise a protein family of 5-hydroxyiso-urate hydrolases with structural similarity to TTR In this study, we tested the amyloidogenic properties, if any, of sea bream TTR (sbTTR) and Escherichia coli transthyretin-related protein (ecTRP), which share 52% and 30% sequence identity, respectively, with hTTR We obtained filamen-tous structures from all three proteins under various conditions, but, inter-estingly, different structures displayed different tinctorial properties hTTR and sbTTR formed thin, curved fibrils at low pH (pH 2–3) that bound thioflavin-T (thioflavin-T-positive) but did not stain with Congo Red (CR) (CR-negative) Aggregates formed at the slightly higher pH of 4.0–5.5 had different morphology, displaying predominantly amorphous structures CR-positive material of hTTR was found in this material, in agreement with previous results ecTRP remained soluble at pH 2–12 at ambient tem-peratures By raising of the temperature, fibril formation could be induced
at neutral pH in all three proteins Most of these temperature-induced fibrils were thicker and straighter than the in vitro fibrils seen at low pH
In other words, the temperature-induced fibrils were more similar to fibrils seen in vivo The melting temperature of ecTRP was 66.7C This is approximately 30C lower than the melting temperatures of sbTTR and hTTR Information from the crystal structures was used to identify possible explanations for the reduced thermostability of ecTRP
Abbreviations
AFM, atomic force microscopy; BME, b-mercaptoethanol; CR, Congo Red; DSC, differential scanning calorimetry; ecTRP, Escherichia coli transthyretin-related protein; EM, electron microscopy; FAC, familial amyloidotic cardiomyopathy; hTTR, human transthyretin; rTTR, rat transthyretin; sbTTR, sea bream transthyretin; SSA, senile systemic amyloidosis; ThT, thioflavin-T; TLP, transthyretin-like protein; TRP, transthyretin-related protein; TTR, transthyretin.
Trang 2diversity in age of onset, penetrance, and tissues affected
[7,8] SSA is a geriatric disease affecting approximately
25% of the European Caucasian population over
80 years of age [4] Like FAC, SSA is characterized by
heavy deposits of amyloid fibrils in the heart
Structures of TTRs from different species have been
studied [9], including human transthyretin (hTTR)
[10–12], rat TTR (rTTR) [13], chicken TTR [14], and sea
bream TTR (sbTTR) [15,16] Within the TTR family,
fish TTR has the lowest sequence identity with hTTR
(e.g sbTTR 52% [16,17], and lamprey TTR 47% [18])
The transthyretin-related proteins (TRPs) comprise
a family of proteins recently shown to function as
5-hydroxyisourate hydrolases in the purine degradation
pathway [19–23] These proteins are also referred to in
the literature as transthyretin-like proteins (TLPs)
However, to separate this family, whose members have
the characteristic sequence motif YRGS at their
C-ter-minal end, from other protein sequences listed as
TTR-like, we prefer to refer to them as TRPs [19,24]
Sequence analysis of representative TTRs, TRPs and
TLPs suggests that the three protein groups are not
functionally related (Fig 1)
The sequence identity between TRPs and TTRs is
relatively low; Escherichia coli TRP (ecTRP) shares
30% and 35% sequence identity with hTTR and
sbTTR, respectively (Fig 1) [19] Structures of TRP
from several species have been determined, and despite
their low sequence identity, the TTR and TRP
struc-tures were found to be very similar [24–27]
In TTR amyloidoses, the normally folded, secreted
protein cannot assemble into amyloid fibrils unless a
preceding partial unfolding event occurs [28]
Muta-tions that destabilize the native structure of TTR are known to lead to disease [29], but thermodynamic stability alone does not reliably predict the severity of the disease [30] Instead, thermodynamic data, combined with kinetic data, more reliably explain why only some mutations lead to severe pathologies [31]
To understand the mechanism behind hTTR dissoci-ation, misfolding, and amyloid formdissoci-ation, studies from other species have provided valuable information Like hTTR, rTTR forms amyloid-like fibrils in vitro after partial acid denaturation [32] rTTR shares 85% sequence identity with hTTR, which raises the question
of how important tertiary similarities are, as opposed
to sequence identity, for the ability of the protein to form fibrils In an attempt to answer this question, we have investigated the fibril-forming properties of sbTTR and ecTRP in vitro, and compared the results with those of hTTR
Our results showed that hTTR, sbTTR and ecTRP can form fibrillar structures, but under different solu-tion and temperature conditions Furthermore, depending on the conditions used, fibrils of different morphology were obtained Recent studies have shown that sbTTR binds thioflavin-T (ThT) at low pH, sug-gestive of amyloid [33] In our study, we verified that sbTTR forms fibrillar structures at low pH that are similar in shape to those of hTTR We also found that, even though 70% of the amino acids of ecTRP are different from the respective amino acids in hTTR and sbTTR, ecTRP has the ability to form Congo Red (CR)-positive fibrils in vitro if the temperature is increased sufficiently Similar findings for ecTRP were published while this work was in progress [34] The
Fig 1 Multiple sequence alignment of representatives of TRPs, TTRs, and TLPs There are 57 gene clusters in Caenorhabditis elegans, referred to as TTR-1 to TTR-57 in WORMBASE Some of these sequences were originally identified as being structurally TTR-like by Sonnham-mar & Durbin [68] They seem to be functional and to influence aging in C elegans, and are referred to as TRPs [69] TTR-1 to TTR-57 are, however, not related to the YRGS TRP family [19], which is why we prefer to refer to them as TLPs The 57 TLP sequences present in
C elegans are nematode-specific and share low sequence identity with each other; however, sequences TTR-18 to TTR-31 seem to comprise a subgroup that is also found in other nematodes (A) In this sequence alignment, TLP representatives from six nematodes have been aligned with representatives from TTR and TRP Identical and homologous residues within the three subgroups are marked in red and pink, respectively The first residue after signal sequence cleavage is highlighted in black Sequence numbering refers to mature proteins Similarity is defined as amino acid substitutions within one of the following groups: FYW, IVLMF, RK, QDEN, GA, TS, or HNQ Identical and similar residues within the TRP family are shown in dark and light green, those within the TTR family in dark and light blue, and those within the TLP subgroup in dark and light gray The secondary structure elements are based on hTTR [12] Residues lining the substrate-binding channel in TTRs [13] and TRPs [26] are marked with blue stars The assignment (*.:) shown below the sequences is directly from CLUSTALW 2 [75] and refers to alignment of the six TLP sequences only Only two amino acids (proline and glycine) are conserved throughout the three groups Whereas residues at the ligand-binding sites are almost completely conserved in the TTR and TRP families, they are not conserved within the TLP group In contrast, the most conserved regions are found at sites corresponding to b-strands B and E in TLPs, and to a-helix E
in TTRs and TRPs (B) Phylogenetic tree of TRPs, TTRs, and TLPs The tree was based on the multiple sequence alignment from (A) Caenor-habditis elegans TRP-R09H10.3 (GI:115532920); C elegans TRP-ZK697.8 (GI:115534555); Mus Musculus TRP (GI:81916776); Bacillus subtilis TRP (GI:3915561); Escherichia coli TRP (GI:3915454); Petaurus breviceps TTR (GI:1279636); Sminthopsis macroura TTR (GI:1279727); Gallus gallus TTR (GI:45384444); Homo sapiens TTR (GI:114318993); Sparus aurata TTR (GI:6648602); Heterodera glycines TLP (GI:8571913); Rado-pholus similis TLP (GI:145279861); Brugia malayi TLP (GI:170583879); Xiphinema index TLP (GI:55724912); C elegans TLP (TTR-30, GI:25153261); Caenorhabditis briggsae TLP (TTR-18 GI:187037056).
Trang 3TLP H glycines
TLP B malayi
TLP C elegans TTR-30 TLP C briggsae TTR-18 TTR P breviceps
TTR S macroura TTR G gallus
TTR H sapiens TTR S aurata TRP E coli
TRP C elegans R09H10.3 TRP C elegans ZK697.8 TRP M musculus
TRP B subtilis
TLP X index TLP R similis
Trang 4results emphasize the potential for amyloid formation
as a common property of all proteins, a feature that
can sometimes even bring new functionality [35–37]
The thermal stability of ecTRP was found by
differen-tial scanning calorimetry (DSC) to be approximately
30C lower than that of sbTTR and hTTR
Compara-tive studies of structures from homologous thermophiles
and mesophiles have revealed several factors that
gener-ally contribute to the intrinsic thermal stability of
pro-teins [38–40] These include tighter hydrophobic packing
of the protein core [41–43], increased electrostatic
inter-actions on the surface of the protein [44,45], more
pro-lines and alanines [46,47], and increased hydrogen
bonding of the polypeptide chain [48–50] In addition,
improved intersubunit contacts within oligomeric
pro-teins contribute to protein stability [51,52] Here, we
analyze the structural basis for the differences in
ther-mostability of hTTR, sbTTR, and ecTRP
Results
Partial acid denaturation generates fibrils of hTTR
and sbTTR
Partial acid denaturation combined with turbidity assays
is a method frequently used to induce and monitor the
degree of protein aggregation and fibril formation of
hTTR [28,53] Application of this method to the three
proteins in our study showed that hTTR displayed an
increase in turbidity at low pH, with a turbidity
maxi-mum at pH 4.5 (Fig 2) This is in agreement with
previ-ous results [28,53] For sbTTR, we measured only minor
turbidity increases at the low pH range of 4.0–5.5, and
for ecTRP, no effect was apparent over the entire pH
range (2–12) (Fig 2) It should be noted that the pI of
ecTRP is estimated to be 8.2, in contrast to those of
hTTR and sbTTR, which are estimated to be 5.5–6.0
The samples treated at low pH were visualized with
atomic force microscopy (AFM) to determine the
morphologies of the protein aggregates Both hTTR and sbTTR displayed fibrils at pH 2.0–3.5 that were very similar in structure (Fig 3) For hTTR, small amounts
of fibrillar structures were also formed at pH 4.0 The thickness of these structures was found to be 1.3 nm, which means that they were thinner than amyloid fibrils Their curved morphology agreed with previous observa-tions of both hTTR and rTTR in vitro fibrils, suggesting that they most likely represented protofibrils [32,54–56] hTTR in vitro fibrils are reported to have widths varying between 2.8 [55] and 10 nm [54], and the thicker in vitro fibrils are believed to consist of up to five intertwined protofilaments [54] At the pH interval 4.0–5.5, predomi-nantly amorphous aggregates, rather than fibrillar struc-tures, were observed in the hTTR samples (Fig 3A) It
is, however, not possible to quantitatively estimate the ratio of aggregates to fibrillar structures from the AFM images The hTTR and sbTTR samples were also visual-ized with electron microscopy (EM) The EM images were consistent with the morphologies that we eluci-dated from the AFM images, and verified that fibrillar structures were present in the protein samples at pH 4.5 (Fig 3B) To determine whether the lack of fibrillar structures in the hTTR and sbTTR samples at pH 4.5 could be an effect of the technique used for analysis (that is, the fibrils are unable to bind to mica gels at this pH), fibril-containing samples of hTTR formed at
pH 2.0 were adjusted to pH 4.5 and incubated for vari-ous lengths of time AFM images of this material showed that the fibrils formed at pH 2.0 persisted at
pH 4.5, thereby verifying that these fibrillar forms can bind to mica gels even at higher pH (data not shown)
Tinctorial properties of fibrillar structures formed
at low pH The protein fibrils and aggregates obtained by the partial acid denaturation experiments were tested for ThT, which is a fluorescent dye commonly used to
Fig 2 Turbidity assays for hTTR, sbTTR, and ecTRP The turbidity was measured at
330 nm after incubation of protein samples
at 37 C for 72 h.
Trang 5assess amyloid fibril formation [57,58], including hTTR
amyloid [59,60] Pronounced emission at 482 nm, as a
result of ThT binding, was seen for the human and sea
bream samples incubated at pH 2.0 and 2.5,
respec-tively (Fig 4) This is in agreement with the presence
of the thin fibrils seen with AFM (Fig 3A) At a pH
value around 3.0, both hTTR and sbTTR showed
significantly reduced ThT binding as compared to that
at lower pH However, at the slightly higher pH range
of 3.5–4.5, hTTR formed structures that bound to
ThT (Fig 4) This ThT-binding pattern correlates well
with the increase in turbidity of hTTR observed at
pH 4.5 (Fig 2) The sbTTR samples did not display
increased ThT binding at pH 3.5–4.5, and
demon-strated only a very small increase in turbidity at this
pH range ecTRP did not react with ThT at any pH range tested
The partially denaturated protein samples were also tested for CR staining, visualized with EM Generally, the ability of fibrils to bind CR and to display a char-acteristic apple-green birefringence under polarized light are the two most important criteria for detection
of amyloid fibrils in vivo Such fibrils are called CR-positive, but the specificity of this test has recently been questioned [61,62] The material from hTTR was CR-positive at pH 4.0–4.5 (Fig 3C), in agreement with previous studies [28,53] Aggregates from sbTTR, on the other hand, were not found to be CR-positive at
B
C
Fig 3 (A) AFM images of hTTR (left) and sbTTR (right) The samples were incubated at 37 C for 72 h Fibrils were present in samples incu-bated at pH 2.0–3.5, whereas aggregates were predominantly present in samples incuincu-bated at pH 4.5–5.5 No fibrils or aggregates were detected in the ecTRP samples, at any of the pH intervals tested (pH 2.0–12.0; data not shown) The white scale bar is 500 nm (B) EM images of fibrils of hTTR (a) and sbTTR (b) incubated at pH 4.5 (C) CR staining of hTTR incubated for 3 days at pH 4.5 (a) shows fluores-cence (at 594 nm) and (b) shows the characteristic apple-green birefringence with polarized light.
Trang 6any pH range Also, the large amounts of small
fibril-lar hTTR and sbTTR structures, observed with AFM
(Fig 3A) at pH 2.0–3.0, did not stain with CR
Apparently, ThT is more promiscuous than CR in
binding to thinner immature fibrillar structures of
TTR
To determine whether the thin and curved protofi-brils formed at pH 2.0 by hTTR and sbTTR could be induced to form amorphous aggregates or thicker fila-ments, the samples were readjusted to a pH of 4.5 Interestingly, even after 6 weeks at 8C, these samples contained the same type of protofibrils This implies that the low-pH-induced and ThT-binding fibrils are not readily converted to CR-positive fibrillar aggre-gates or thicker filaments, or at least not under these conditions
Fiber formation induced by heating Fibrillar structures of hTTR, sbTTR and ecTRP were obtained by heating the protein samples for 72 h with-out stirring at different temperatures Fibrils of hTTR have previously been reported at 75C [63] In this study, we obtained thick fibrils at 55C for hTTR and
65C for sbTTR and ecTRP (Fig 5A) Of these, only fibrils from ecTRP showed a strong ThT response (data not shown) These fibrils were verified to be CR-positive (Fig 5B)
Protein stability measured by SDS/PAGE and DSC
The propensity for TTR amyloid formation is coupled
to tetramer dissociation The stability of the tetrameric structures of hTTR, sbTTR and ecTRP was analyzed
by gel electrophoresis according to the method of Lai
et al [53] (Fig 6) Unboiled samples from the acid denaturation experiment were run on gel electrophore-sis in the presence of SDS and b-mercaptoethanol (b-ME) Whereas tetrameric hTTR dissociated at pH
Fig 4 ThT-binding assays Relative intensity of emission at
482 nm for samples incubated for 72 h at different pH The
excita-tion wavelength was 440 nm The standard deviaexcita-tions between
triplet samples for pH intervals from 7.0 to 2.0 and between double
samples for pH intervals from 12.0 to 7.5 are shown Strong ThT
binding is seen for hTTR and sbTTR at the lowest pH values, 2.0–
3.0 For hTTR, weaker ThT binding is also detected between
pH 3.5 and pH 4.5 As expected, neither hTTR nor sbTTR samples
incubated at pH 2.0 or 4.5 produce increased emission at 482 nm if
ThT is not added, which means that the increase in emission is
due to actual ThT binding A local ThT-binding minimum is seen at
pH 3.0 for hTTR, even though fibrils are detected with AFM The
reason for this is unknown The fiber thickness at pH 3.0 generally
seems to be the same as for the fibers seen at lower pH ecTRP
does not bind ThT at any pH level.
A
B
0.2 µm
Fig 5 (A) AFM images of hTTR, sbTTR and ecTRP heated at 55 C (hTTR) or 65 C (sbTTR and ecTRP), respectively, for 72 h The fibril heights were estimated to be
2.8 nm for hTTR, 3.5 nm for sbTTR, and 4.0 nm for ecTRP The white scale bar is 500 nm (B) Left: EM image of ecTRP from the same sample as in (A) The mate-rial shows fluorescence (at 594 nm) (middle) and apple-green birefringence (right) after visualization with polarized light.
Trang 7values lower than 5.0, as shown previously [53], sbTTR
and ecTRP did not completely separate into their
monomeric units until the pH values were below 4.6
and 3.0, respectively (Fig 6) In agreement with the
partial acid denaturation experiments, the ecTRP
tetra-meric structure seems to be more resistant to
varia-tions of pH However, the SDS⁄ b-ME treatment
generally dissociated a larger fraction of the ecTRP
protein material than of hTTR and sbTTR into
mono-meric structures This behavior is likely to reflect
differences in the chemical composition of the proteins
SDS is a detergent that readily dissolves hydrophobic
molecules, whereas acid denaturation affects
electro-static interactions rather than the hydrophobicity of
molecules Gel filtration chromatography of ecTRP in
the presence and absence of 0.5% SDS in the running
buffer produced monomers only when the SDS was
included (data not shown)
The thermal stability of hTTR, sbTTR and ecTRP
was further studied by DSC (Fig 7) The measured
thermal melting point (Tm) for hTTR was close to values previously reported [64,65] Even though tetra-meric sbTTR seems to be more stable than hTTR at lower pH, we found that it was less stable than hTTR
at physiological pH, with the Tm value for sbTTR being approximately 4.5C lower than that for hTTR Gilthead sea bream is an ectotherm whose normal habitat is the Mediterranean Sea One feature that gen-erally defines cold-adapted proteins and distinguishes them from their mesophilic and thermophilic counter-parts is their lower thermal stability [66] This could therefore be one explanation for the reduced stability that we observed for sbTTR as compared to hTTR More unexpected, however, was the low thermo-stability of the ecTRP protein, with a Tmvalue approx-imately 26C below the values for both sbTTR and hTTR (Fig 7) The inability of ecTRP to form fibrils
at low pH can therefore not be directly correlated with the thermostability of its tetrameric and monomeric structures
Thermostability and protein structures
We have analyzed the structures of hTTR (Protein Data Bank code: 1F41 [12]), sbTTR (Protein Data Bank code: 1SN2, [16]) and ecTRP (Protein Data Bank code: 2G2N [24]) in an attempt to identify factors that contribute to their profound differences in stability The results are summarized in Table 1 Introduction of alanines, prolines and aromatic resi-dues can contribute to protein stability [46,47,52] hTTR and sbTTR have more alanine residues than ecTRP, 12 versus eight and 13 versus eight, respec-tively, which might contribute to entropic stabilization
On the other hand, ecTRP has more aromatic residues than hTTR and sbTTR, 13 versus 12 and 13 versus
Fig 6 Analysis of tetramer stability by SDS ⁄ PAGE The samples
were not boiled The tetrameric structures of sbTTR and ecTRP
show increased stability at lower pH as compared to hTTR
Nota-bly, both hTTR and sbTTR are unaffected by the SDS ⁄ BME
treat-ment, and remain either in a monomeric or a tetrameric state,
depending on the pH of the protein buffer In contrast, SDS ⁄ b-ME
treatment alone dissociates a fraction of the tetrameric ecTRP into
the monomeric state at all pH values.
Fig 7 DSC profiles of hTTR, sbTTR, and ecTRP The melting temperatures (Tm) were 97.8 C for hTTR, 93.0 C for sbTTR, and 66.7 C for ecTRP.
Trang 811, respectively The backbone flexibility of the TTR
structures is probably also reduced because of a higher
proline content, which decreases the entropy of
unfold-ing [46] hTTR and sbTTR have eight proline residues
each, whereas ecTRP has only five
The formation of salt bridges is another important
contributor to the temperature stability of proteins
[67] In agreement with this, there is a higher number
of charged residues in the more thermostable TTR
protein structures (Table 1) There are five salt bridges
in hTTR and sbTTR, but only three in ecTRP
Thermostable proteins are generally more tightly
packed than their less thermostable homologs [42] The
structural homologs hTTR, sbTRP and ecTRP have
no internal cavities However, hTTR and sbTRP have
two polar residues, Thr75 and His88 (hTTR
number-ing), buried within the hydrophobic core of the
mono-mers The side chains of these residues form, together
with the Ne1 atom of Trp79, hydrogen bonds with
three or four buried water molecules [12,16] This
probably increases packing density and contributes to
stability [49] In the ecTRP structure, two
phenylala-nines occupy the same position as His88 and Trp79 in hTTR, and consequently only one water molecule can bind at this site in ecTRP [24]
The protein volumes of single subunits and tetra-meric structures for the three proteins were investi-gated Our calculations show that there is a decrease in the protein volume of each monomer that is related to
an increase in thermal stability Furthermore, there is
a clear correlation between thermostability and molec-ular volume occupied by the tetramers (Table 1) The difference between the tetrameric volume and the volume of the corresponding number of monomeric units, DV, is negative in all cases, demonstrating that the protein density increases slightly upon tetrameriza-tion
Analysis of the number of hydrogen bonds formed within monomers and dimers revealed pronounced dif-ferences between the three proteins As previously mentioned, the hTTR and sbTTR structures contain buried polar residues and water molecules These waters allow the formation of 10 more hydrogen bonds within their monomeric units, and about 20 more hydrogen bonds within their dimeric units, than in the ecTRP protein structure (Table 1) In addition, 14 and
15 hydrogen bonds are formed at the monomer–mono-mer interface of sbTTR and hTTR, whereas only eight are formed in ecTRP Five hydrogen bonds are formed across the dimer–dimer interface of both hTTR and sbTRP, whereas three are formed in ecTRP (Table 1)
Discussion TTRs and TRPs are two protein families with similar structures but different functions, due to divergent evolution The TTRs, found only in vertebrates, func-tion as retinoic acid and thyroid hormone carriers, whereas the TRPs, found predominantly in lower eukaryotes and prokaryotes, are enzymes that hydrolyze 5-hydroxyisourate [19,21–23] in the purine catabolic pathway The structural similarity between several TTR and TRP representatives has been verified by crystallo-graphic studies [11,12,24–27] Members from both protein families have their active site positioned in the hydrophobic channel formed at the dimer–dimer inter-face of their homotetrameric structure The four amino acid sequence motif YRGS at the C-terminal end of the TRP sequences distinguishes them from other proteins annotated as TTR-like or TTR-related [19] These resi-dues are involved in binding to substrate analogs [26] Other proteins with sequence homology to TRPs and TTRs exist, and we refer to these as TLPs TLPs have so far been found only in nematodes Some of these were identified as being structurally TTR-like in 1997 by
Table 1 Structural factors implicated in the thermostabilities of
hTTR, sbTTR, and ecTRP Protein volume calculations were
obtained using VOIDOO [72], and hydrogen bonds were calculated
using WHATIF [73].
Amino acid composition
Protein volume (A˚3 ) b
Hydrogen bondsc
a A distance less than or equal to 4 A ˚ between charged groups
defines an ion pair [74] b The molecular volumes were calculated
using a probe with radius 0 A ˚ , in order to obtain the protein volume
per se [52] Before calculation, alternate conformations were
removed, and the structures were truncated at their N-termini and
C-termini, so that they structurally start and end at the same
posi-tion Residues included from the four chains, A, B, C, and D, of the
tetrameric structures were: hTTR, A10–A122, B10–B122, A¢11–
A¢122, and B¢10–B¢122; sbTTR, A10–A122, B10–B122, C11–C122,
and D10–D122; and ecTRP, A4–A114, B4–B114, C5–C114, and
D4–D114 c The numbers in parentheses are the numbers of buried
water molecules included in the calculation.
Trang 9acid and thermal denaturation Some differences were
apparent Analysis with SDS⁄ PAGE showed that hTTR
is less stable than the other proteins under acidic
condi-tions, and dissociates into monomers when the pH falls
below 5.0 sbTTR shows similar behavior, and is only
marginally less sensitive to acidic pH, dissociating at a
pH below 4.5 Interestingly, we found that ecTRP
main-tains its tetrameric structure even at very low pH values
Different results were obtained when the protein
samples were analyzed with DSC The melting point for
ecTRP was determined to be 66.7C, which is more
than 26C lower than those of both hTTR and sbTTR
(Fig 7) Therefore, whereas previous SDS⁄ PAGE
analysis suggested that the tetrameric structure of
ecTRP is more stable than that of hTTR [34], the DSC
results showed that ecTRP is significantly less
thermo-stable than either hTTR or sbTTR
Comparison of the crystal structures of hTTR,
sbTTR and ecTRP highlights a number of structural
differences that are consistent with the current
explana-tions of thermal stability in proteins Noteworthy is the
reduced number of negatively charged residues in the
ecTRP structure This could possibly also explain its
structural stability at low pH Furthermore, the
ther-mostable TTR proteins have more hydrogen bonds and
ion pairs, and their structures are more densely packed
than that of ecTRP Thus, it seems that the differences
in thermostability are mainly due to the presence of
specific polar and charged residues in hTTR and
sbTTR, which form additional hydrogen bonds that
stabilize their monomeric subunits as well as their
monomer–monomer and dimer–dimer interfaces
In agreement with previous reports, TRP is
amyloi-dogenic; fibrils do form upon heating of the protein
sample ([34] and this study) Whereas the previous study
reported an amyloid-inducing protocol for TRP that
involves heating at 24C at pH 5.8 with stirring, our
protocol involves heating at 65C at pH 7.4 for 3 days
without stirring This temperature was chosen because
it was shown by DSC to be the Tmof the protein These
fibrils are amyloidogenic, as determined from their
formation Interestingly, misfolded and aggregated ecTRP material has been shown to be toxic for neuro-blastoma cells, although the soluble protein is not [34]
In conclusion, sbTTR has properties similar to those
of hTTR in terms of tetramer dissociation and fibril formation Generally, where fibrils were observed for hTTR, fibrils of similar morphology were observed also for sbTTR, after some minor adjustments of the fibrillization protocol We did not detect any CR-posi-tive fibrils of sbTTR at pH 4.5–5.5 This suggests that hTTR forms amyloid fibrils by partial acid dissociation more readily than sbTTR The result does not exclude the possibility that sbTTR can form CR-positive fibrils
at low pH, but more samples need to be examined, or the concentration of protein needs to be increased ecTRP shares 30% sequence identity with hTTR In agreement with previous reports [34], we detected CR-positive fibrils of ecTRP induced by heating These fibrils are similar both in shape and in dimension to the fibrils of hTTR and sbTTR formed by heating The thick and straight morphology of heat-induced fibrils of hTTR, sbTTR and ecTRP is similar to that
of amyloid fibrils in vivo We have so far not been able
to convert thin and ThT-positive protofibrils of hTTR and sbTTR, formed at low pH, to thicker and CR-positive structures, suggesting that the kinetics are very slow This suggests that the TTR amyloid archi-tecture is not the result of only one highly stringent assembly of structures
In the past, the propensity of proteins to form fibrillar structures has most often been associated with disease Recently, however, examples have been presented where conformational changes and fibril formation are associated with an advantageous gain of function [37] It
is not clear whether the fibril formation properties of the TRP family are associated with any gain of function or whether they even have any biological implications whatsoever In vivo fibrils have only been reported with hTTR and rTTR, and it would be interesting to know whether TTR from other species, as well as members of the TRP family, can form fibrils in vivo
Trang 10Experimental procedures
Protein expression and purification
hTTR was expressed as previously described [70] In brief,
competent E coli BL21 cells were transformed with the
pET3a vector containing the hTTR construct, and plated
onto LB agar plates containing 50 lgÆmL)1 carbenicillin
Bacteria were grown in LB medium supplemented with
50 lgÆmL)1 carbenicillin at 37C At A600 nm= 0.4, cells
were induced with 0.2 mm isopropyl thio-b-d-galactoside
for 3 h, harvested by centrifugation for 20 minutes at
2800 g, and stored at)20 C The sbTTR gene placed in a
pET24d vector [16] was expressed using a similar protocol
as for hTTR, but with 50 lgÆmL)1kanamycin as the
antibi-otic After induction with isopropyl thio-b-d-galactoside,
the cells were grown overnight at 30C
Similar purification protocols were used for hTTR and
sbTTR Frozen cells were thawed in 20 mm Tris⁄ HCl
(pH 8.0) and 50 mm NaCl, and lysed by sonication in the
presence of DNase I Cell debris was removed by
ultra-centrifugation (120 000 g for 40 min) at 4C The lysate was
filtered through a 0.2 lm syringe filter (Millipore
Corpora-tion, Bedford, MA, USA), and purified on a Q-Sepharose
Fast Flow anion exchange column (GE Healthcare,
Uppsala, Sweden) equilibrated with 20 mm Tris⁄ HCl
(pH 8.0) and 50 mm NaCl, and eluted with a linear gradient
(0.1–1 m NaCl in 20 mm Tris⁄ HCl, pH 8.0) TTR fractions
were pooled and concentrated (Centriprep-10; Amicon
Inc., Beverly, MA, USA), and then further purified by gel
fil-tration on a HiLoad 16⁄ 60 Superdex-75 (GE Healthcare)
column with buffer containing 20 mm Tris⁄ HCl (pH 6.8)
and 50 mm NaCl Pure TTR fractions were pooled,
concen-trated to 5 mgÆmL)1(Centriprep-3; Amicon), and stored at
)20 C ecTRP was cloned, expressed and purified as
previously described [19], using 50 mm Tris⁄ HCl (pH 7.0)
and 200 mm NaCl as buffer in the final gel filtration step
The pure ecTRP fractions were pooled and stored at)20 C
Partial acid denaturation
Denaturation studies were performed according to a
previ-ously described protocol for hTTR [53] hTTR, sbTTR and
ecTRP were dialyzed against 2 mm NaH2PO4⁄ Na2HPO4
(pH 7.4) and 20 mm NaCl, and mixed to a final
concentra-tion of 0.2 mgÆmL)1( 3.5 lm tetramer), corresponding to
the TTR concentration in human plasma, at pH 2.0–12.0,
at intervals of 0.5 pH units The buffers used gave a final
concentration of 50 mm glycine–HCl (pH 2.0–2.5), or
50 mm sodium acetate (pH 3.0–5.5), or 50 mm sodium
phosphate (pH 6.0–8.0), or 50 mm Hepes (pH 8.5–9.0), or
50 mm CAPSO (pH 9.5–10.0), or 50 mm CAPS (pH 10.5–
12.0) All buffers included 100 mm potassium chloride,
1 mm EDTA, and 1 mm dithiothreitol After 72 h at 37C,
all samples were thoroughly vortexed to disperse aggregated material before analysis by absorbance measurements at
330 nm in a standard UV cell
Visualization AFM
Following the turbidity measurements, the protein samples were examined by AFM Samples were diluted 10-fold with H2O, and then 5 lL of the diluted sample solutions was applied to freshly cleaved ruby red mica (Goodfellow, Cambridge, UK) The material was allowed to adsorb for 10 s, washed three times with 100 lL of distilled water, and air dried The surface was analyzed with
a Nanoscope IIIa multimode atomic force microscope (Digital Instruments, Santa Barbara, CA, USA), using Tapping Mode in air A silicone probe was oscillated at around 270 kHz, and images were collected at an opti-mized scan rate corresponding to 1–4 Hz The images were flattened and presented in height mode using nanoscope software (Digital Instruments)
EM
Negative staining for EM was performed on the same sam-ples used in the AFM studies For this purpose, the material was centrifuged at 16 000 g for 30 min, after which most of the supernatant was removed and 200 lL of distilled water was added The material was vortexed, and aliquots of 3–5 lL were applied to Formvar-coated copper grids Contrast was achieved with 2% uranyl acetate in 50% etha-nol, and the material was studied at 100 kV in a Jeol 1230 electron microscope (Jeol, Akishima, Tokyo, Japan)
CR-binding studies
For analysis with CR, 1–2 lL of diluted, vortexed samples were applied to microscope slides and air dried CR stain-ing was performed accordstain-ing to Puchtler et al [71], and examined by light microscopy The presence of amyloid was verified by the green birefringence in polarized light and with red fluorescence in a microscope equipped with filters for wavelengths at 560 nm (excitation) and 590 nm (emission)
ThT-binding studies
Protein samples incubated at 37C for 72 h were vortexed, and 25 lL aliquots were mixed with 173 lL of a buffer containing 100 mm sodium phosphate, 100 mm potassium chloride (pH 7.6), and 2 lL of ThT stock solution (1 mm ThT in 10 mm sodium phosphate, pH 7.4) The samples were excited at 440 nm, and the emission at 482 nm was recorded