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 circu
Trang 1Amyloidogenic nature of spider silk
John M Kenney1, David Knight2, Michael J Wise3and Fritz Vollrath2,4
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
E X P E R I M E N T A L P R O C E D U R E S
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 20C up to 75 C using a Jasco 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: kenney@ifa.au.dk
(Received 27 March 2002, revised 12 June 2002, accepted 12 July 2002)
Trang 2structure 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 (NRMSDundiluted> 0.2) than
for the diluted ones (0.1 < NRMSDdiluted<
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
R E S U L T S A N D D I S C U S S I O N
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 35C to an enriched b-sheet state that persists to at least 70C 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 above35 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
Trang 3genic 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 31helix [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 theSWISSPROT 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, CA24CANFA), 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) and 55 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).
Trang 4occurs at position 638 in the list (Pr¼ 0.0032), while the
avian prion protein, PRIO-CHICK, is at position 974
(Pr¼ 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-NH2, n¼ 3–8, were found to self assemble into fibril-like structures [26] Both of these are examples of low complexity peptides
C O N C L U S I O N
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
A C K N O W L E D G E M E N T S
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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