How post-translational modifications influence amyloid formation: A systematic study of phosphorylation and glycosylation in model peptides

Article abstract of DOI:10.1002/chem.200902452

A reciprocal relationship between phosphorylation and O-glycosylation has been reported for many cellular processes and human diseases. The accumulated evidence points to the significant role these post-translational modifications play in aggregation and fibril formation. Simplified peptide model systems provide a means for investigating the molecular changes associated with protein aggregation. In this study, by using an amyloid-forming model peptide, we show that phosphorylation and glycosylation can affect folding and aggregation kinetics differently. Incorporation of phosphoserines, regardless of their quantity and position, turned out to be most efficient in preventing amyloid formation, whereas O-glycosylation has a more subtle effect. The introduction of a single βgalactose does not change the folding behavior of the model peptide, but does alter the aggregation kinetics in a site-specific manner. The presence of multiple galactose residues has an effect similar to that of phosphorylation.

Full text of DOI:10.1002/chem.200902452

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FULL PAPER
DOI: 10.1002/chem.200902452
How Post-Translational Modifications Influence Amyloid Formation: A
Systematic Study of Phosphorylation and Glycosylation in Model Peptides
Malgorzata Broncel, Jessica A. Falenski, Sara C. Wagner,
Christian P. R. Hackenberger, and Beate Koksch*[a]
Abstract: A reciprocal relationship be-
tween phosphorylation and O-glycosyl-
ation has been reported for many cellu-
study, by using an amyloid-forming
model peptide, we show that phospho-
tion, turned out to be most efficient in
preventing amyloid formation, whereas
O-glycosylation has a more subtle
AHCTUNGTRENNUNG
G
ryl
E
lar processes and human diseases. The
accumulated evidence points to the sig-
nificant role these post-translational
modifications play in aggregation and
fibril formation. Simplified peptide
model systems provide a means for in-
vestigating the molecular changes asso-
ciated with protein aggregation. In this
folding and aggregation kinetics differ-
ently. Incorporation of phosphoserines,
regardless of their quantity and posi-
effect. The introduction of a single b-
galactose does not change the folding
behavior of the model peptide, but
does alter the aggregation kinetics in a
site-specific manner. The presence of
multiple galactose residues has an
effect similar to that of phosphoryl-
Keywords: aggregation
·
coiled
coils · glycopeptides · protein fold-
ing · protein models
AHCTUNGTRENGNaUN tion.
Introduction
human diseases (cancer or diabetes).[4] The accumulated evi-
dence highlights the role of post-translational modifications
in the protein aggregation that leads to neurodegeneration.[5]
In Alzheimerꢀs disease (AD), for example, abnormally phos-
phorylated tau protein aggregates lead to the formation of
neurofibrillary tangles, one of the hallmark brain lesions re-
sponsible for neuron death.[6] It has been shown that tau is
also modified by O-glycosylation and that this modification
regulates the phosphorylation of tau in a site-specific
manner.[7] The underlying molecular mechanisms, however,
are still largely unknown. Another AD-related protein, the
b-amyloid precursor protein, has also been found to be
modified in its cytoplasmic domain by both phosphorylation
and glycosylation.[8] However, the exact role of these specif-
ic modifications remains to be elucidated.
Phosphorylation and glycosylation are major post-transla-
tional modifications of cellular proteins. These reversible,
covalent modifications have an impact on diverse properties
of a protein, ranging from subcellular localization and bind-
ing to interactions with other proteins. Because phospho
ACHTUNGTNERrNUNG yl-
ACHTUNGTRENNUNG
taining amino acids, a reciprocal relationship between these
modifications has been suggested.[1] Many reports document
that phosphate and O-linked b-N-acetylglucosamine (O-
GlcNAc) alternatively occupy the same or adjacent hydroxyl
moieties.[2,3] This finding led to the hypothesis that this sugar
may be a temporary site-blocker or a competitor of phos-
phorylation. In fact, a dynamic interplay between O-glyco-
A
E
Despite the great number of studies conducted to date,
neurodegeneration is still one of the least understood pro-
cesses, which is mostly due to the complex nature of molec-
ular interactions as well as the challenging physicochemical
properties of naturally aggregating systems. In particular,
the low solubility of the proteins involved, their tendency to
aggregate, and their difficult synthesis often preclude de-
tailed structural characterization. To overcome these obsta-
cles, the application of simplified peptide model systems is
of great interest.[9] Because it has been shown that amyloid
formation is a general feature of peptides and proteins,[10] in-
cluding various non-disease-related proteins, many groups
merous cellular processes (transcription and translation) and
[a] M. Broncel,+ J. A. Falenski,+ Dr. S. C. Wagner,
Dr. C. P. R. Hackenberger, Prof. Dr. B. Koksch
Institut fꢁr Chemie und Biochemie
Freie Universitꢂt Berlin
Chem. Eur. J. 2010, 16, 7881 – 7888
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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have directed their efforts towards developing simplified
models for the studying of aggregation in response to di-
verse stimuli, including pH,[11,12] temperature,[13] metal
ions,[14] and hydrophobic defects.[15] However, to our knowl-
edge, only a few reports describe the influence of phospho-
ACHTUNGTRENNUNGrylACHTUNGTRENNUNGation and glycosylation by using the same model
system.[16–18] Liang et al. used a 38-residue a-helical hairpin
peptide (a-helix/turn/a-helix), which transforms from its ini-
tial monomeric state to an amyloid at elevated temperature,
as a model system to explore how post-translational modifi-
cations affect the conformational properties and kinetics of
amyloid formation.[16] They found that the principal effect of
glycosylation and phosphorylation is to slow down the pep-
tide fibrillogenesis, with the greater effect induced by glyco-
sylation. However, the conformational differences induced
by a single sugar or phosphate group introduced into the
flexible loop region (Thr19) of the helix–turn–helix model
peptide did not have a great effect on the overall structure.
In contrast with the above-mentioned strategy, our group
has recently shown that structural changes in amyloid-form-
ing model peptides can be initiated without thermal activa-
tion.[11,19,20] We have explored the a-helical coiled coil fold-
ing motif, which is very common in nature and has been ex-
tensively studied in recent years, proving its application as a
reliable model system.[9,11,19,20,41] Furthermore, the design
principles of coiled coils are very well understood,[21] and
folding is based on oligomerization, which is also the foun-
dation of amyloid formation. Coiled coils consist of at least
two a-helices wrapped around each other in a slight super-
helical twist. The primary structure is characterized by a pe-
riodicity of seven residues, the so-called heptad, which is
commonly denoted (a-b-c-d-e-f-g)n (Figure 1). Positions a
and d are typically occupied by nonpolar residues that form
the first recognition domain by hydrophobic core packing
(“knobs-into-holes”). Charged amino acids at positions e
and g form the second recognition motif and engage in in-
terhelical electrostatic interactions. Polar residues are often
found at the remaining heptad repeat positions b, c, and f,
which are solvent exposed.
Figure 1. Helical wheel representation of the PP. Black arrows indicate
the phosphorylation and glycosylation sites.
linked b-galactose was used as a general phosphorylation-
site blocker.
The key design features of our model peptide (Figure 1)
are as follows: 1) hydrophobic leucine residues at positions a
and d in combination with interhelical electrostatic attrac-
tions between glutamate and lysine at positions e and g
ensure stability of the a-helical coiled-coil structure and
2) amino acids at positions b, c, and f have a minor effect on
the stability of the coiled coil; therefore, these positions
have been used to incorporate three valine residues that
make the system prone to amyloid formation. The resulting
peptide contains elements of two competing secondary
structures: a helix and b sheet.[20] The effect of post-transla-
tional modification of the parent peptide (PP) has been in-
vestigated by the introduction of phosphorylation and O-
glycosylation into position f of the coiled coil (Ser10, 17, and
24; Table 1). Various complementary spectroscopic and ana-
lytical techniques have been used to fully characterize the
behavior of our model system.
Herein, we have used a de novo designed coiled-coil pep-
tide that can form amyloids in a time-dependent fashion as
a simplified model for deciphering the impact of two ubiqui-
tous post-translational modifications, phosphorylation and
O-linked glycosylation, on the amyloid formation process.
Specifically, we have examined the effect of site-specific and
multiple phosphorylation to mimic the pattern observed in
vivo. Furthermore, b-galactose was incorporated into the
same sequence positions. Al-
Results
Circular dichroism spectroscopy: To investigate structural
changes associated with post-translational modifications,
Table 1. Sequences of the coiled-coil-based model peptides.
though b-galactose is not direct-
ly linked to serine in eukary-
ACHTUNGTRENNUNGotes, it is commonly occurring
at the end positions in gly-
cans.[22] Here, we used b-galac-
tose as an easily accessible resi-
due that serves as a model to
study the impact of O-linked
sugars on folding. Therefore, O-
Peptide
Sequence
AHCTUNGTRENNUNG
PP
p10
p17
p10/17
g10
g17
g10/17
g10/17/24
Abz-LKVELEKLKSELVVLKSELEKLKSEL
Abz-LKVELEKLKpSELVVLKSELEKLKSEL
Abz-LKVELEKLKSELVVLKpSELEKLKSEL
Abz-LKVELEKLKpSELVVLKpSELEKLKSEL
Abz-LKVELEKLK
ACHTUNGTRENNUNG
Abz-LKVELEKLKSELVVLKACHTUGNTRENNNUG
Abz-LKVELEKLK
Abz-LKVELEKLK
N
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
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Amyloid Formation
FULL PAPER
time-dependent circular dichroism spectroscopy (CD) mea-
ACHTUNGTRENNUNGsureACHTUNGTRENNUNGments were performed at pH 7.4 and peptide concentra-
single glycosylation in terms of position in PP sequence. The
folding behavior of peptide g17 turns out to be similar to
that of g10, with the initial helical structure and time-depen-
dent transition to a b-sheet. However, the kinetics of the
conformation switch is faster than for peptide g10. Here,
structural conversion is complete within 48 h (see the Sup-
porting Information). Peptides g10/17 and g10/17/24, con-
taining two or three Gal units, respectively, were mostly un-
folded. No conformational transition was observed for these
multiply glycosylated peptides (Figure 2b–d).
The helical content of all peptides was calculated by using
the characteristic CD signal at l=222 nm (Figure 3). PP has
a mean residue ellipticity of ꢀ27440, which corresponds to a
helical content of 77%. The helical content of g10 is slightly
higher, which further points to sugar-induced stabilization of
the initial fold. This effect, however, is evidently site specific
because the helical content of g17 is reduced to 46%. Multi-
ple glycosylation in peptide g10/17 and g10/17/24 clearly de-
stabilizes the helical structure, resulting in only 22 and 12%
helicity, respectively. As described above, phosphorylation
promotes global unfolding and leads to a dramatic reduction
in helical content to 12 and 15% for p10 and p17, respec-
tively. Peptide p10/17 shows the lowest helical content
(7%).
tions of 100 mm at room temperature. A disaggregation assay
using hexafluoroisopropanol (HFIP) was performed with all
peptides prior to use to ensure a comparable starting condi-
tion. Thus, all aggregates formed during peptide purification
were disrupted. As shown in Figure 2a, the PP at the initial
time point (0 h) shows two minima at l=208 and 222 nm
that are characteristic for a-helical coiled coils. Within 24 h,
the peptide undergoes a structural conversion to a b-sheet,
identifiable by the presence of a single minimum at l=
216 nm. Concomitantly, a white precipitate was observed,
suggesting fibril formation. This structural transition con-
firms that the PP is a suitable model for studying the effect
of phosphorylation and glycosylation on protein conforma-
tion.
Dramatic changes in peptide structure are induced by
phosphorylation. Regardless of the number and position of
the substitution, peptides p10, p17, and p10/17 show com-
plete unfolding. Furthermore, a random-coil conformation is
observed for the duration of the experiment (Figure 2b–d).
In contrast, incorporation of a single Gal (peptide g10) ap-
parently does not perturb the initial helical structure. The
time-dependent behavior of g10 resembles that of PP; how-
ever, the structural a-to-b transition is significantly delayed,
which might suggest an increase in the stability of the initial
a-helical structure. White precipitate was observed after
10 d of incubation. We have also analyzed the influence of a
ThT binding assay: Because CD spectroscopy does not pro-
vide information about amyloid fibril formation, we per-
formed a Thioflavin T (ThT) binding assay to investigate
Figure 2. Time-dependent CD spectra of peptides (100 mm) in tris(hydroxymethyl)aminomethane (Tris)/HCl buffer (20 mm; pH 7.4), with NaN3 (0.025%)
!
!
!
&
&
*
*
at 208C: Folding behavior of a) the parent peptide ( : 0 h; : 6 h; : 24 h; &: 2 weeks) and b–d) peptides PP ( ), p10 ( ), p10/17 ( ), g10 ( ), and g10/
*
17 ( ) immediately after dissolution (b), after 1 week of incubation (c), and after 2 weeks of incubation (d). For CD spectra of peptides p17, g10/17/24,
and detailed spectra of g10 and g17 see the Supporting Information.
Chem. Eur. J. 2010, 16, 7881 – 7888
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B. Koksch et al.
expected, phosphorylated peptides p10, p17, and p10/17, as
well as glycosylated peptides g10/17 and g10/17/24, do not
show any increase in ThT fluorescence over the course of
the experiment (Figure 4). These results are in good agree-
ment with the CD spectroscopy data.
Seeding: Partial and full unfolding have been postulated to
initiate the conformational transitions associated with amy-
loid formation.[24–26] However, the ThT experiment did not
detect any amyloid formation for the unfolded peptides
(p10, p17, p10/17, g10/17, and g10/17/24) in our case. The
presence of a lag phase in the observed amyloid formation
of PP and g10 indicates a nucleation-dependent polymeri-
zation pathway.[27] Nucleus formation is usually a rate-limit-
ing step in amyloid formation. It is followed by a fast elon-
gation phase, which results in mature amyloids. To exclude
the possibility of a prolonged lag phase, seeding experiments
were conducted. The addition of preformed fibers is known
to shorten or even eliminate the lag phase,[28] and the effi-
ciency of this process seems to correlate with the amino acid
sequence similarity between the fibril seed and the protein
of interest.[29] Preformed aggregates of PP were added to
p10, p17, p10/17, and g10/17. Peptide conformation was
characterized by CD spectroscopy, and amyloid formation
was investigated by the ThT binding assay as described
above. Our results show that all peptides maintain their ini-
tial random coil structure over two weeks. Additionally, no
increase in ThT fluorescence was observed, therefore no
amyloid formation occurred (see the Supporting Informa-
tion).
Figure 3. Calculated helical content of peptides used in this study.
whether the observed b-sheet structures are amyloidogenic.
ThT exhibits enhanced fluorescence upon binding to amy-
loid fibrils, and is frequently used for determination of the
kinetics of fibril formation.[23] As depicted in Figure 4, inset,
ThT in the presence of PP shows a slow increase in fluores-
cence intensity during the first 6 h of incubation. This initial
phase is followed by a rapid increase until 24 h, after which
no further increase is observed (Figure 4). After 150 h, a
slight decrease in fluorescence intensity and a white precipi-
tate are observed. Similarly to PP, peptide g10 follows a
clearly sigmoidal increase in ThT fluorescence. The curve in-
dicates a slow amyloid formation process. The lag phase per-
sisted for about 70 h, and the plateau is reached after two
weeks of incubation. The sigmoidal increase in ThT fluores-
cence is also observed for peptide g17, but here the kinetics
of amyloid formation is much faster than for g10 and resem-
bles more that of PP (see the Supporting Information). As
Transmission electron microscopy: To characterize the mor-
phology of amyloid fibrils formed by PP and g10, two-week-
old samples were stained with 1% phosphotungstic acid
(PTA) and investigated by transmission electron microscopy
(TEM; Figure 5). For the PP, helically twisted ribbons were
!
&
Figure 4. ThT binding assay with peptides (100 mm) PP ( ), p10 ( ), p17
~
!
*
* ^
(
), p10/17 ( ), g10 ( ), g10/17 ( ), and g10/17/24 ( ) in Tris/HCl buffer
(20 mm, pH 7.4) with NaN3 (0.025%) at 208C; the ThT concentration
was 10 mm. One representative data set is shown; experiments were con-
ducted in triplicate. For the ThT assay of peptide g17 see the Supporting
Information. Inset: expanded view at short incubation times.
Figure 5. TEM micrographs of matured peptides (100 mm) a) PP and
b) g10 prepared in Tris/HCl buffer (20 mm, pH 7.4) with NaN3 (0.025%).
Samples were stained with 1% phosphotungstic acid.
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Chem. Eur. J. 2010, 16, 7881 – 7888