Tài liệu Báo cáo khoa học: Amyloidogenic nature of spider silk ppt

Amyloidogenic nature of spider silk
John M. Kenney
1
, David Knight
2
, Michael J. Wise
3
and Fritz Vollrath
2,4
1
Institute for Storage Ring Facilities, University of Aarhus, Denmark;
2
Department of Zoology, University of Oxford, UK;
3
Department of Genetics, University of Cambridge, UK;
4
Department of Zoology, University of Aarhus, Denmark
In spiders soluble proteins are converted to form insoluble
silk fibres, stronger than steel. The final fibre product has
long been the subject of study; however, little is known about
the conversion process in the silk-producing gland of the
spider. Here we describe a study of the conversion of the
soluble form of the major spider-silk protein, spidroin,
directly extracted from the silk gland, to a b-sheet enriched
state using circular dichroism (CD) spectroscopy. Combined
with electron microscopy (EM) data showing fibril forma-
tion in the b-sheet rich region of the gland and amino-acid
sequence analyses linking spidroin and amyloids, these
results lead us to suggest that the refolding conversion is
amyloid like. We also propose that spider silk could be a
valuable model system for testing hypotheses concerning

b-sheet formation in other fibrilogenic systems, including
amyloids.
Keywords: spider silk; spidroin, amyloids; CD spectroscopy;
low complexity peptides.
Spiders have evolved sophisticated systems not only to
produce and store proteins at high concentrations (‡ 30%)
in solution, but also to control the conversion of these
soluble proteins to form insoluble silk fibres [1]. The
conversion process appears to rely on refolding of one or
two members of the spidroin family of major silk proteins
[2,3], but is poorly understood. Although the structure of
the insect silkworm (Bombyx mori) silk has been well
characterized [4] and a model [5] has been proposed for
insect silk formation based on regenerated fibroin (the
major protein in silkworm silk), the structure of spider silk is
less well understood and appears to be different from insect
silk in several respects [6]. Though progress has also been
made in spinning fibres from recombinant spider silk
produced in mammalian cells [7], the formation process in
the spider has remained largely unexplored. Here, we
describe the conversion of spidroin in the spider (Nephila
edulis) using circular dichroism spectroscopy, electron
microscopy and amino-acid sequence analyses. The results
reveal a striking molecular-level similarity between spider-
silk processing and amyloid-fibril formation. We conclude
that the spider-silk production process, particularly the
mechanisms that the spider employs to secrete, store and
manipulate silk protein, could prove to be a valuable model
system for exploring fibrilogenesis of amyloid, prion and
other related proteins.

EXPERIMENTAL PROCEDURES
Spider and sample preparation
Prior to dissection, Nephila edulis female spiders were kept
in plastic boxes (10 · 40 · 40 cm) and exclusively fed flies
for several weeks. Under these conditions the spiders do not
make webs thus ensuring a large accumulation of spidroin
in their silk glands. The major-ampullate dragline-produ-
cing silk gland of a freshly decapitated spider was removed
and the epithelium of the ampulla was carefully stripped off
under spider Ringer solution (pH 7.4) [8]. The highly
viscous remainder containing concentrated spidroin was
separated into fractions derived from the two morphologi-
cally distinct regions of the gland known as the A and B
zones [9]. Freshly prepared samples from individual glands
of undiluted solute and solute diluted (1 : 4) in spider
Ringer solution were used for CD analysis.
Circular dichroism spectroscopy and secondary
structure prediction
The samples were carefully loaded into a 0.01 mm light path
quartz cell (Hellma 124-QS) to avoid shearing. Prior to CD
analysis they were examined by polarized light microscopy
to confirm that loaded samples did not exhibit birefringence
and therefore had not suffered from shear-induced poly-
merization. Spectra were recorded for each sample, two raw
(undiluted) and three diluted from each zone, over a series
of temperatures from 20 °Cupto75°CusingaJasco
J720 CD UV spectrometer equipped with a water-bath
temperature controller. Each spectrum is the average of 16
scans (each 3 min in duration) from 260 nm to 190 nm with
0.5 nm steps. The samples were allowed to equilibrate for at

least 10 min at each temperature before the CD spectrum
was recorded. Several times the spectrum was repeatedly
recorded at the same temperature to ensure that it was stable
in time. The spectra of five (three diluted and two undiluted)
different samples of A zone were essentially identical,
though the spectra of the undiluted samples exhibited a
lower signal-to-noise ratio, probably due to light losses
resulting from absorption by the high protein concentration
and scattering by inhomogeneous distribution of the sample
material in the cell. However, the similarity of the spectra of
diluted and undiluted samples confirmed that the diluted
samples spectra are valid measures of the in vivo molecular
Correspondence to J. M. Kenney, Institute for Storage Ring Facilities,
University of Aarhus, 8000 Aarhus C, Denmark.
Fax: + 45 8612 0740, Tel.: + 45 8942 3721,
E-mail:
(Received 27 March 2002, revised 12 June 2002, accepted 12 July 2002)
Eur. J. Biochem. 269, 4159–4163 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03112.x
structure. The same is true of the spectra of B zone samples.
Background (spider Ringer solution) reference spectra were
recorded before and after each sample spectrum was
recorded. Background-corrected spectra of the samples
were analyzed using the SUPER3 algorithm [10], courtesy
of B. Wallace (Birkbeck College, London, UK). This is the
only published concentration independent method for
analysing CD spectra and is the only one that could be
used because the protein concentration of each sample is not
precisely known. For other CD analysis algorithms to
provide reliable results the protein concentration must be
known to approximately 1%. The normalized standard

deviation (NRMSD) [11] measure of the accuracy of the
undiluted samples was worse (NRMSD
undiluted
>0.2)than
for the diluted ones (0.1 < NRMSD
diluted
< 0.2); how-
ever, the estimate of secondary structure content was similar
to that of the diluted sample spectral analysis, again
confirming the validity of the analysis of the diluted samples
spectra. Their analyses showed a systematic variation
(attributed to a systemic error in background correction)
in the predicted secondary structure content (by 5–15%)
from one sample to the next, however, the trends were
identical in all cases; e.g. increasing b-sheet content with
temperature for the A zone samples. The reproducibility of
the spectra and the moderately low NMRSD suggest that,
though indicative rather than exact, the secondary structure
predictions are valid and support the proposition that the
spidroin at the downstream end of the B zone is rich in
b-sheet and that heating converts the A zone secreted
spidroin to a b-sheet enriched state.
Electron microscopy
Major ampullate silk glands and ducts were dissected from
Nephila edulis spiders, fixed in a modified Karnovsky
fixative, embedded in Spurr’s resin and sectioned on a
diamond knife [9]. The sections were stained with uranyl
acetate and lead citrate. Images were recorded at 40 000
times on a Philips 400 transmission electron microscope.
RESULTS AND DISCUSSION

Circular dichroism shows a b sheet transition
We used CD spectroscopy to characterize the molecular
structure of spidroin [12] in the major ampullate silk gland
of the spider Nephila Edulis and its conversion along the silk
production pathway. This gland has two morphologically
distinct zones. Upstream in the silk production pathway the
A zone secretes material that makes the silk fibre core and
downstream the B zone secretes a thin coating [9]. Analyses
of the CD spectra show that there is a remarkable difference
in the initial as well as temperature-induced refolding of the
secondary structure of the protein extracted from two
different zones in the silk gland (Fig. 1). At ambient
temperature the solute extracted from the A zone has a
stable structure poor in a helix and b sheet, while from the
downstream end of the B zone the extracted sample,
composed of a mixture of A- and B zone secretions, is
primarily b sheet. As the temperature is increased the
B zone protein sample secondary structure remains relat-
ively stable. With heating, however, the A zone protein
sample undergoes a dramatic refolding conversion at
approximately 35 °Ctoanenrichedb-sheet state that
persists to at least 70 °C. This b-sheet enriched state remains
stable upon return to ambient temperature and is also seen
to be irreversible by modulated differential scanning calori-
metry. This shows that energy is required to convert the
b-sheet poor state to the b-sheet rich state of spidroin. It is
unlikely that the spider uses heat to facilitate the conversion,
though it is interesting that the spiders were observed not to
spin silk above 35 °C. The spiders take advantage of these
two states to control the production, transportation and

conversion of spidroin in the gland so that the enrichment
for b sheet can take place just prior to spinning the silk fibre.
These results illuminate three important aspects of the
conversion of the soluble spidroin as it flows through the
silk gland on its way to becoming an insoluble fibre: (a)
there is a refolding of the structure to enrich for b-sheet
structure; (b) that the refolding can be induced by adding
energy (e.g. heat); and (c) that the enriched b-sheet state is
stable and irreversible.
Fibrils seen by electron microscopy and structural
similarities to amyloids
Moderated conversion to a stable b-sheet enriched state is
also a signature of amyloid fibril formation [13] and other
related disease-associated proteins, e.g. prions [14]. In the
spider the conversion of silk solute to solid fibre occurs
during the spinning process. Using electron microscopy
(EM) we examined the silk gland and duct to determine the
site of fibril formation. No fibrils could be seen in the EM of
the luminal contents of the A zone part of the gland where
the CD spectroscopy analysis shows that the protein
exhibits a b-sheet poor structure. Fibrils, however, are
found downstream of the B zone (Fig. 2), where the CD
spectroscopy analysis shows that the extracted samples are
rich in b sheet. This is close to the previously proposed site
of b-sheet formation [15]. Here, the fibrils nucleate within
the A zone secreted material. The fibrils are approximately
10 nm wide and are similar in aspect to amyloid, prion and
other amyloidogenic fibrils, such as a-synucleins [16].
The appearance of fibrils at the end of the silk production
pathway implies that a b-sheet enriching structural conver-

sion of spidroin plays a role in their formation.
Both amyloids and spider silk contain b sheets with
antiparallel strands [6,13]. In the final dragline silk fibre
spun by the spider the strands are found to be aligned with
the fibre axis making a parallel-b structure [6], this should
not be confused with a parallel-b sheet which refers to the
relative (parallel) orientation of adjacent strands. On the
other hand, in amyloid fibrils the strands are perpendicular
to the fibril axis forming a cross-b structure [17]. However,
when cross-b silk fibres are stretched they assume the
parallel-b structure characteristic of Group 3 silk proteins
(including spider silk) [6]. Therefore, it is possible that the
fibrils we see appearing in the b sheet enriched region of the
silk gland could have a cross-b structure (like amyloid
protein fibrils) which is converted to the parallel-b structure
observed in the final silk fibre product due to the extension
of the fibre during the spinning process. This is supported by
the observation that the orientation function (i.e. alignment
of the strands with respect to the axis) increases with the
speed that the silk is drawn from the spider [18]. Addition-
ally, there are other structural similarities between amyloido-
4160 J. M. Kenney et al.(Eur. J. Biochem. 269) Ó FEBS 2002
genic and spider silk proteins. Specifically, there is a link
between the prion octapeptide repeats and the spidroin Pro-
Gly repeats which is evident in the similar structures that
have been calculated for them in the context of prion fibrils
and dragline silk, respectively. The octapeptide repeat has
been found to have a polyproline Form II structure [19]
while the Pro-Gly repeat is a 3
1

helix [20]. Both of these are
left-handed helices with exactly three residues per turn.
Sequence low complexity: spidroin and amyloids
The similarity between spidroin and fibril-forming amyloido-
genic proteins suggested by our CD and EM results was
further investigated by amino-acid sequence analyses. We
were first struck by the obvious low complexity (highly
nonrandom with substantial regularities) [21] of the spi-
droin 2 sequence, SPD2-NEPCL; repetitions of the peptide
PGGYGPGQQG (a low complexity sequence itself) are
interleaved with polyalanine stutters. When the
SWISSPROT
database (Version 40, as at March 15, 2002) was probed
with SPD2-NEPCL using the SCANPS implementation of
the Smith Waterman sequence alignment algorithm [22]
many other low complexity sequences were returned. High
on the list are the sequences of elastomers [23] such as
collagen (e.g. CA1-BOVIN, CA24 °CANFA), elastin
(ELS-RAT), silk-moth fibroin (FBOH-BOMMO), glutenin
(GLT5-WHEAT) and mucin (MUC1-HUMAN). Some-
what down the list is the trinucleotide disease protein
DRPL-HUMAN (dentatorubral-pallidoluysian atrophy
protein). The first of the prion sequences, PRIO-MUSVI,
Fig. 1. Typical CD spectra and predicted molecular secondary structure of silk protein are shown. (A) CD spectra of A zone sample at 20 °C (black),
30 °C(blue),35°C(green),40 °C (orange), 45 °C(red)and55 °C (pink); (B) fraction of predicted secondary structure of A zone sample for a helix
(solid), b sheet (dashed) and other (dotted); (C) CD spectra of downstream B zone sample at 20 °C(black),30°C (blue), 40 °C (orange) and 50 °C
(red); (D) fraction of secondary structure of B zone sample for a helix (solid), b sheet (dashed) and other (dotted).
Ó FEBS 2002 Amyloidogenic nature of spider silk (Eur. J. Biochem. 269) 4161
occurs at position 638 in the list (Pr ¼ 0.0032), while the
avian prion protein, PRIO-CHICK, is at position 974

(Pr ¼ 0.068). However, it is inevitable that this search will
also encompass proteins such as the fish antifreeze protein
ANP-NOTCO, that match the polyalanine stutters, so an
alternate search was undertaken using just the octapeptide
repeats from PRIO-HUMAN. As expected, mammalian
prion proteins fill the top of the list, but PRIO-CHICK is
found at position 199 with a probability of 2.71. SPD2-
NEPCL is at position 543. The extremely low probability
scores occur because the query sequence is very short and, in
general, the statistical model used in scoring alignments is
better suited to globular proteins (which are predominantly
high complexity sequences) rather than these structural
proteins.
The common theme among all of the proteins discussed
above is that they contain low complexity peptide
sequences. Interestingly, if a set of 32 known amyloidogenic
sequences is examined using 0j.py [24], a tool specifically
designed for demarcating low complexity protein sequences,
we find that 20 (i.e. > 60%) have scores greater than or
equal to 3 vs. 41 607 out of 104 939 for SwissProt as a
whole, which gives rise to a one-sided binomial-distribution
probability of 0.0075. In other words, the amyloidogenic
sequences have significantly lower complexity than proteins
generally. The set of sequences includes proteins such as
SAA5-MESAU, amyloid protein A5 and SYUA-MOUSE,
alpha-synuclein protein, both of which also appear in the list
returned by the similarity search using SPD2-NECLP but
not the prion proteins discussed above.
We hypothesize therefore that low sequence complexity
may be a property of many, if not most, fibril-forming

proteins, including spidroin. Experimental evidence of such
a link is already present in the literature. Proteins created
from alternating polar and nonpolar residues were found to
form fibril-like structures while at the same time being rare
in nature [25]. In another study, water soluble peptides of the
form DPKGDPKG-(VT)
n
-GKGDPKPD-NH
2
, n ¼ 3–8,
were found to self assemble into fibril-like structures [26].
Both of these are examples of low complexity peptides.
CONCLUSION
Elucidating the details of the spidroin conversion process in
the spider is vital to understanding silk fibre formation.
Using CD spectroscopy, electron microscopy and sequence
analysis we have revealed similarities in the nature of spider
silk and amyloidogenic fibril formation. This suggests that
spider-silk processing, not the least for the large quantities of
material produced in vivo in the spider, may also promise to
be beneficial for studying amyloid-like fibril formation in
general. Significant applications could include the develop-
ment of genetically modified spidroin to test hypotheses on
the formation and inhibition of pathogenic fibrils.
ACKNOWLEDGEMENTS
We thank Karen Wise (Zoology, Oxford, UK) for assistance with EM;
Cait MacPhee, Klaus Doering and Michael Gross (OCMS, Oxford,
UK) for help with CD experimental design and interpretation; and
Bonnie Wallace (Birkbeck College, London, UK) for guidance in
quantitative analysis and the use of the SUPER3 algorithm. The work

was funded in part by a grant from the Danish Natural Science
Research Council and supported by the European Science Foundation.
Michael Wise is supported by a grant from Bristol-Myers Squibb,
which is gratefully acknowledged.
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