J. Biol. Macromol., 8 (1), 11-18 (2008)

ArticleEfficient conversion of intact hen lysozyme into amyloid fibrils by seeding
Ken Sasaki, Ken Nakatsuka, Itsuho Hayashi, Buddha R. Shah, Koichi Morimoto, and Kazuyuki Akasaka
Department of Biotechnological Science, School of Biology-Oriented Science and Technology,
Kinki University, 930 Nishimitani, Kinokawa-shi, Wakayama 649-6493, Japan.
Received December 26, 2007; Accepted January 21, 2008
Clarifying the mechanism how intact protein is transformed into amyloid fibrils has central
importance in the study of amyloidosis. When hen lysozyme is kept in an acidic solution (pH 2.2, 80
mM NaCl) at a temperature close to its transition temperature (e.g., 57 ºC), amyloid fibrils are formed
after a lag-time of several to 11 days. These fibrils are mainly composed of peptide fragments producedby acid-degradation of the protein at selective Asp-Gly sites (Mishra et al., J. Mol. Biol. 366, 1029-1044,
2007). We found that, when these fibrils are sonicated into small particles and a fraction is mixed into asolution of fresh intact hen lysozyme (pH 2.2, 80 mM NaCl), the second-generation amyloid fibrils are
rapidly formed from the intact protein in an apparent two-state manner. The rate of the fibril
formation depends critically on temperature in the transition region, showing that the fibrillation
formation initiates by the interaction of the seed with heat-denatured protein. The efficient conversionof α-rich monomer proteins into β-rich amyloid fibrils by seeding with preformed fibrils could be partof a general mechanism of amyloid fibril formation and infection in vivo.
Keywords: hen lysozyme, degraded peptides, amyloid fibrils, sonication, seed-induced fibril formation,
circular dichroism, atomic force microscopy
Introduction
The amyloid fibril formation has been linked totissueororgandysfunctionsuchasneurodegeneration, i.e., Alzheimer’s disease in thecase of amyloid-β peptide, Parkinson’s disease (αsynuclein) and Creutzfeldt-Jakob disease (prion
protein) [1]. Various globular proteins undergo
conformational changes in vivo and in vitro into βsheet-rich structure forming fibrillar aggregates,
generally known as amyloid fibrils [2, 3]. The
mechanism of transformation of intact protein intoamyloid fibrils has central importance in the studyof amyloidosis. In our recent studies using disulfidedeficient mutant (0SS) of hen lysozyme, we haveshown that the protein exists as fully denatured
protein as monomer, but that it forms amyloid
protofibrils spontaneously when certain ionicconcentration is present [4]. Furthermore, we have
Correspondence to Kazuyuki Akasaka.
Tel: +81-736-77-0345 ext. 4110
Fax: +81-736-77-4754
E-mail: akasaka8@spring8.or.jp

also shown that the growth of 0SS takes place by
the “linear polymerization mechanism” in which amonomer is added successively to a growing end of
the protofibril [5, 6]. Here the average rate of
growth of the protofibril will be proportionate to theconcentration of the growing end of the protofibril
rather than to the total concentration of the protein.
In the present work, we use wild-type henlysozyme (4 S-S) (Fig. 1A) as a model to humanlysozyme whose specific mutants are known tocause hereditary systemic amylodosis [7]. Previous
studies reported that hen lysozyme is slowly
degraded into fragments when kept in acidic
solution at elevated temperatures, from which theamyloid fibrils are formed [8, 9]. In addition,
acceleration of the fibril formation by the additionof preformed amyloid fibrils (without sonication orother treatments) was reported [10], but no detailed
study has been made.
_______________________________________
The abbreviations used are: CD, circulardichroism; AFM, atomic force microscopy.

12

Efficient conversion of intact hen lysozyme into amyloid fibrils by seeding

B

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Figure 1. A. Structure of hen lysozyme (ribbon model). The α and β domains are shown withlocations of the four disulfide bonds. B. Thermal transition of hen lysozyme (1.0 mg ml-1 in 80 mM
NaCl, pH 2.2) as monitored by CD absorption at 222 nm. The mid point of transition is at ~ 57 ºC.
In the present work, using circular dichroism(CD) and atomic force microscopy (AFM) alongwith SDS-PAGE analysis, we demonstrate that the
α-rich intact hen lysozyme (in 80 mM NaCl, pH
2.2) is rapidly and efficiently converted into the βrich fibrils by seeding with particles produced fromthe first-generation amyloid fibrils by extensive
sonication.
We will show that the efficientconversion takes place without degradation of intact
hen lysozyme only in the temperature range inwhich thermal denaturation of hen lysozyme issignificant, showing that the fibril forming reaction
is caused by the interaction of the seed with heatdenatured and non-degraded hen lysozyme.

mg ml-1 was chosen for the easier measurement ofCD spectra in an optical cell of 0.1 cm light path.
The fibril forming reaction was qualitatively thesame for the two concentrations.

Materials and Methods
Preparation of the first-generation amyloid fibrils
Lyophilized hen lysozyme obtained from
Sigma was first dissolved in pure water. The protein
concentration was determined by MultiSpec-1500
spectrophotometer (Shimadzu, Kyoto) at λ = 280
nm, using an extinction coefficient of 2.57 (mg)-1
cm2. The solution was mixed with sodium chloride
to give a final protein concentration of 4.0 mg ml-1
in 80 mM NaCl at pH 2.2 (pH adjusted with HCl).
The solution (~ 1.5 ml) was then incubated inplastic tubes (Eppendorf) at 57 ºC for as long as 11days. For the study of temperature dependence ofthe fibril formation, a protein concentration of 1.0

Preparation of the second-generation amyloid
fibrils by seeding
A quantity (150 μl) of the seed solution was
mixed with 1350 μl of the hen lysozyme solution(1.0 or 4.0 mg ml-1) in 80 mM NaCl, pH 2.2, theseed-to-protein ratio being 1/9 by weight. The
mixture solution was then incubated at 57 ºC or atother temperatures for typically 10 h to produce thesecond-generation fibrils.

Preparation of the seeds
The fibrils in the tube (~ 1.5 ml) were
subjected to extensive sonication on ice using a
BRANSON SONIFIER model S-250A, which
operated continuously for 60 min at a frequency of
20 kHz at a maximum power of 20 watts. The
sonicated (first-generation) fibrils were used as“seeds” for producing the second-generation fibrils
without separating non-fibril peptides.

AFM measurements
The first and second generation fibrils were
examined by AFM on an SPI-3800 (Seiko
Instruments Inc.) at 25 ºC. An aliquot (2 μl) each of

K. Sasaki et al.

the fibril solution was dropped on a fresh surface of
mica and washed with 6 ml distilled water, before
AFM images were recorded with the cyclic contact
mode at a frequency of 119 Hz.
CD measurements
The time-dependent change of the CD
spectrum upon the protofibril formation wasrecorded on a J-820 spectropolarimeter (JASCO,Tokyo) using cylindrical quartz cells of 0.01 cm
path length for 4.0 mg ml-1 and 0.1 cm path lengthfor 1.0 mg ml-1, both cells being water-jacketed fortemperature control.
SDS-PAGE
The SDS gel electrophoresis was performed on
second-generation fibrils using an SDS treating
buffer containing 8 M Urea, 1% β-mercaptoethanoland 1% SDS. The regular boiling treatment of thesolution was not employed for feat of degrading theprotein molecule. Instead, the solutions were kept atroom temperature for 1 h before applying them ongels made of 15% SDS polyacrylamide. Reference
markers obtained from APRO Science, Inc. wereused.
Results
Formation of the first-generation fibrils

13

The solution of hen lysozyme (4.0 mg ml-1)
containing 80 mM NaCl, pH 2.2 was kept at 57 ºC,
close to the transition temperature of hen lysozyme(~ 57 ºC) (Fig. 1B), similarly to the procedure in theliterature [8, 9]. The change in mean residue
ellipticity at 222 nm started after a lag time of ~ 5days (data not shown), when protofibrils, consistingof long straight fibers of 2.0 ~ 3.5 nm in height,
started to form on AFM. Finally, after ~ 11 days,some of the protofibrils formed a bundle
omonomeric hen lysozyme into β-amyloid fibrils at
57 ºC. A. CD spectral changes with time ofincubation of hen lysozyme solution (4.0 mg ml-1 in
80 mM NaCl, pH 2.2) in the presencf fibrils of 8 ~12 nm in height, suggesting the formation of
matured amyloid fibrils (Fig. 2A). Examination by
SDS-PAGE showed that the protein was degraded
into fragments with time, which nearly completed in~ 4 days (data not shown), in close agreement withthe literature [8]. The time required for the
degradation nearly coincided with the start of the
fibril formation, suggesting that the degradedfragments, and not the intact protein, are responsible
for the formation of the fibrils. Recently, Mishra etal. showed in their detailed analysis that the fibrils(hereafter called the first-generation fibrils) mainly
consist of fragmented peptides (residues 49-101 and53-101) specifically cleaved in Asp-Gly bonds

Figure 2. Amyloid fibrils from hen lysozyme monitored by AFM. A. The first-generation
amyloid fibrils formed by incubating the acidic solution of hen lysozyme (4.0 mg ml-1 in 80 mM
NaCl, pH 2.2) at 57 ºC for 11 days. B. Fragmented first-generation amyloid fibrils formed byextensive sonication (used as seeds to produce the second-generation amyloid fibrils). C. The
second-generation amyloid fibrils formed by incubation of hen lysozyme solution (4.0 mg ml-1
in 80 mM NaCl, pH 2.2) in the presence of 10% (w/w) seeds at 57 ºC for 24 h.

14Efficient conversion of intact hen lysozyme into amyloid fibrils by seeding

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Time (min)Figure 3. Seed-induced transition of intact e of 10% (w/w) seeds. B. Plot of ellipticity at
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besides some non-fibril-forming peptides [9].
Preparation of seeds from the first-generation
fibrils
The solution, containing the first-generation
fibrils as main component, was extensively sonicatedaccording to the procedure described in Materials
and Methods. The AFM image showed that the
fibrils were degraded into much smaller roundshaped particles (Fig. 2B), which were used as seeds
for the formation of the second-generation fibrils.
Preparation of the second-generation fibrils using
seeds
The extensively sonicated solution (termed
here as “seeds”), which contains the first-generation
fibrils as main component, was added to thesolution of intact hen lysozyme (4.0 mg ml-1, 80
mM NaCl, pH 2.2) at 57 ºC to the ratio of 1/9 (10%seed against 90% hen lysozyme, weight by weight).
Fig. 3A shows CD spectral changes during theformation of the second-generation fibrils at 57 ºC.
The spectral changes depict a transition of the αrich hen lysozyme (constituting 90% of the proteinmaterials in the solution) into a β-rich structure
without a lag time, almost like a two-state transition
with an effective isodichroic point at 209 nm. The
plot of the mean residue ellipticity at 216 nm withtime (Fig. 3B), considered to represent the growthof the β-sheet, shows that the growth of the β-sheet
is largely (~ 90%) complete within 10 h.
Examination by AFM indicates that by this reaction
protofibrils are formed (Fig. 2C). Thus the seeded
reaction, forming the β-sheet structure (Fig. 3),

Figure 4. The SDS PAGE analysis of the second-generation amyloid fibrils produced
by incubation of hen lysozyme solution (4.0 mg ml-1) in 80 mM NaCl, pH 2.2 in thepresence of 10% (w/w) seeds for different incubation times. See Materials andMethods for experimental details. Lane M: molecular weight markers, Lane C: intact
hen lysozyme, Lanes 0 to 10: the second-generation amyloid fibrils with increasingtime of incubation (0-10 h).

K. Sasaki et al.

represents the monomer to protofibril transition, i.e.,
the formation of the second-generation amyloid
fibril. It is clear that the growth of the secondgeneration fibril is much faster than that of the firstgeneration fibrils requires ~ 11 days under the same
solution condition and temperature.
To examine whether or not the degradation of
intact hen lysozyme occurs similarly in the firstgeneration fibrils, the SDS-PAGE analysis (Fig. 4)
was performed on the second-generation fibrils.
The result confirms clearly the presence of a mainband at 14 kDa attributable to intact, non-degraded
hen lysozyme and diffuse bands below 14 kDa,
attributable to the fragmented peptides in seeds
from the first-generation fibrils. The origin of therelatively sharp band below the main 14 kDa band,
found also in the control lane, is not known. The
fact that the SDS-PAGE pattern is nearly invariant
over 10 h of incubation, along with the AFM and
CD results in Figs. 2 and 3, gives clear evidence
that the second-generation fibril is formed before
the acid degradation of the protein takes place,
namely a successful conversion of intact hen
lysozyme into amyloid fibrils.
The extremely efficient conversion of intact
hen lysozyme rich in α-helix (the major componentof the reactants) in the monomeric state into theamyloid fibril state rich in β-structure is quite
interesting in view of the fact that a similar
conversion from α-monomer into β-oligomer isconsidered as a key event governing the infection ofprion disease.
Such a clear two-state likeconversion of α-rich structure into β-rich structure
as observed here (Fig. 3A) may shed new light on
the infection mechanism of prion diseases and,furthermore, suggests the possibility of infection inamyloidosis.
In an effort to understand the mechanism of theconversion, we examined the fibril forming reaction
as a function of temperature. Figure 5A-D shows
the time-dependent CD spectral changes at different
temperatures of hen lysozyme solutions (1.0 mg ml-1
in 80 mM NaCl, pH 2.2) with a seed-to-protein ratioof 1/9 by weight. Figure 5E gives the plot of
ellipticity at 215.5 nm as a function of time atrespective temperatures. Figure 6 shows the AFM
images of fibrils from the above reaction after 24 h.
We notice that the development of the β-oligomer

15(Fig. 5) and the formation of fibrils (Fig. 6) barely
proceed at 40 ºC, i.e., at a temperature below theonset of thermal transition of hen lysozyme (cf. Fig.
1B). However, the rate of the reaction increases
dramatically at higher temperatures (Fig. 5E) at
which thermal transition begins and denaturation
becomes prominent. The results indicate that theconversion of the α-rich structure to the β-richoligomer takes place only when the thermallydenatured fraction of hen lysozyme is significantly
present in the system. This suggests that the βoligomer formation is initiated by the interaction of
the seed with denatured hen lysozyme. Namely,
Native lysozyme ⇄ Denatured lysozyme + seed
➯ β-oligomer (Scheme 1).
Once the first β-oligomer is formed, it will growinto a protofibril (as observed in Fig. 2C) by
attaching another denatured intact hen lysozyme toits end repeatedly according to the linear
polymerization mechanism [6, 11].
Native lysozyme ⇄ Denatured lysozyme +β-oligomer ➯・・・➯ protofibril (Scheme 2).
Scheme 1 and 2 correspond to the initiation andelongation reactions, respectively. As “seed” here
actually consists of a mixture of the fragmented
fibrils and non-fibril forming peptides, only thelatter would participate in the proposed reactions
above. Further studies on details of the above
schemes along with the structural information on“seeds” themselves may shed new light on thegeneral mechanism of amyloidosis and priondisease.
Discussion
We have confirmed that amyloid fibrils areformed from hen lysozyme after ~ 11 days of
incubation at pH 2.2 at 57 ºC, which consist of thinfilaments (protofibrils) and bundles of thin filaments(matured amyloid fibrils) and further that these
fibrils consist of peptide fragments specificallycleaved at Asp-Gly bonds. Although the firstgeneration amyloid fibrils (without further
treatments) were reported to accelerate the amyloid
fibril formation of the second generation [10], the16Efficient conversion of intact hen lysozyme into amyloid fibrils by seeding

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Time(min)Wavelength(nm)Figure 5. A-D: Seed-induced changes of the CD spectrum of hen lysozyme at various temperatures (A: 40ºC, B: 47 ºC, C: 55 ºC, D: 58 ºC). Hen lysozyme solution (1.0 mg ml-1) was incubated in 80 mM NaCl,
pH 2.2 in the presence of 10% (w/w) seeds. E: Plot of ellipticity at 215.5 nm against time of incubation.

A

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Figure 6. AFM images of fibrils producedin hen lysozyme solution (1.0 mg ml-1) byincubation in 80 mM NaCl, pH 2.2 in thepresence of 10% (w/w) seeds at various
temperatures (A: 40 ºC, B: 47 ºC, C: 55 ºC,
D: 58 ºC).

K. Sasaki et al.

phenomenon has not been examined in detail. We
found that when the first-generation fibrils are madeinto small particles by extensive sonication and an
aliquot is added to the solution of intact hen
lysozyme at 57 ºC or other temperatures above 40ºC, β-oligomers start to form immediately without alag time as monitored by CD and grow into
protofibrils as monitored by AFM. The CD spectralchanges without a lag time suggest that intact hen
lysozyme can be transformed into β-rich protofibrils
by interacting with the seed before it is fragmented
into peptides by acid degradation. This expectationis confirmed by the SDS-PAGE analysis of Fig. 4.
The efficient conversion, almost like a two-statetransition from the intact protein into amyloid fibrils
is a process in which the intact protein is “infected”
in shape by the sonicated amyloid fibril.
We have shown that the extent and the rate of
the conversion, as observed by CD, is criticallydependent on temperature. The reaction occurs onlyin the temperature range for thermal transition, in
which a significant fraction of denatured protein ispresent in solution (Fig. 1B). This observation
indicates that the presence of denatured fraction of
the protein is essential for the reaction, meaning thatthe transformation of intact hen lysozyme into the
β-oligomer will take place only by the interaction of
the seed with the denatured hen lysozyme (Scheme1). Once the initial β-oligomer is formed, its
growing end will react further with anotherdenatured protein and the reaction will be repeated
to form longer oligomers or protofibrils according to
the linear polymerization mechanism [6, 11](Scheme 2). After a long run, the protofibrils may
eventually grow into matured fibrils with somebundle structures.
The present result, showing a clear conversionof the wild-type protein conformation from one toanother by contacting with a preformed seed, may
have general implication for amyloid disease. Ifsuch seeds are produced continuously in living cellsby the degradation of pre-formed amyloid fibrils bysome stress or environmental effects, it may resultin an efficient conversion of intact proteins into thesecond-generation amyloid fibrils.
The cycle,
represented by Scheme 2, may continue autocatalytically to finally convert a large quantity of
intact proteins into amyloid fibrils, leading toamyloidosis. The growth mechanism of amyloid

17

fibrils studied in vitro will continue to give clues tothe mechanisms of growth of amyloid fibrils in vivo.
Acknowledgements
This work was supported by a Grant-in-Aid for
Scientific Research No. 16370054 from Ministry of
Education, Culture, Sports, Science and Technology
and by the Bovine Spongiform Encephalopathy
Control Project from Ministry of Agriculture, Forest
and Fishery. Buddha R. Shah is a recipient of the
JSPS RONPAKU fellowship.
References
1. Obici, L., Perfetti, V., Palladini, G., Moratti, R.,
and Merlini, G. (2005) Clinical aspects ofsystemic amyloid diseases. Biochim. Biophys.
Acta 1753, 11-22.
2. Dobson, C. M. (1999) Protein misfolding,evolution and disease. Trends Biochem. Sci. 24,
329-332.
3. Koo, E. H., Lansbury, P. T., and Kelly, J. W.
(1999) Amyloid disease; abnormal protein
aggregation in neurodegeneration. Proc. Natl.
Acad. Sci. USA. 96, 9989-9990.
4. Niraula, T. N., Konno, T., Li, H., Yamada, H.,
Akasaka, K., and Tachibana, H. (2004)
Pressure-dissociable reversible assembly ofnaturally denatured lysozyme is a precursor for
amyloid fibrils. Proc. Natl. Acad. Sci. USA 101,4089-4093.
5. Kamatari, Y. O., Yokoyama, S., Tachibana, H.,
and Akasaka, K. (2005) Pressure-jump NMR
study of dissociation and association of amyloid
protofibrils. J. Mol. Biol. 349, 916-921.
6. Abdul Latif, A. R., Kono, R., Tachibana, H.,
and Akasaka, K. (2007) Kinetic analysis ofamyloid protofibril dissociation and volumetricproperties of the transition state. Biophys. J. 92,
323-329.
7. Morozova-Roche, L. A, Zurdo, J., Spencer, A.,
Noppe, W., Receveur, V., Archer, D. B, Joniau,
M., and Dobson, C. M. (2000) Amyloid fibril
formation and seeding by wild-type humanlysozyme and its disease-related mutationalvariants. J. Struct. Biol. 130, 339-351.
8. Frare, E., Polverino De Laureto, P., Zurdo, J.,
Dobson, C. M., and Fontana, A. (2004) A
highly amyloidgenic region of hen lysozyme. J.
Mol. Biol. 23, 1153-1165.

18

9.

Efficient conversion of intact hen lysozyme into amyloid fibrils by seeding

Mishra, R., Soergjerd, K., Nystroem, S.,
Nordigarden, A., Yu, Y. -C., andHammarstroem,P.
(2007)
Lysozyme
amyloidogenesis is accelerated by specificnicking and fragmentation but decelerated byintact protein binding and conversion. J. Mol.
Biol. 366, 1029-1044.
10. Krebs, M. R. H., Wilkins, D. K., Chung, E. W.,
Pitkeathly, M. C., Chamberlain, A. K., Zurdo,J., Robinson, C. V., and Dobson, C. M. (2000)

Formation and seeding of amyloid fibrils fromwild-type hen lysozyme and a peptide Fragmentfrom the β-domain. J. Mol. Biol. 300, 541-549.
11. Oosawa, F. and Asakura, S. (1975)
Thermodynamics of the polymerization ofprotein. Academic Press, London.

Communicated by Masayuki Oda