Wild-type hen egg white lysozyme aggregation in vitro can form self-seeding amyloid conformational variants

    Wild-type hen egg white lysozyme aggregation in vitro can form self-seeding amyloid conformational variants Vishwanath Sivalingam, Nalla Lakshmi Prasanna, Neetu Sharma, Archana Prasad, Basant K Patel PII: DOI: Reference:

S0301-4622(16)30144-2 doi: 10.1016/j.bpc.2016.09.009 BIOCHE 5924

To appear in:

Biophysical Chemistry

Received date: Revised date: Accepted date:

17 May 2016 6 September 2016 23 September 2016

Please cite this article as: Vishwanath Sivalingam, Nalla Lakshmi Prasanna, Neetu Sharma, Archana Prasad, Basant K Patel, Wild-type hen egg white lysozyme aggregation in vitro can form self-seeding amyloid conformational variants, Biophysical Chemistry (2016), doi: 10.1016/j.bpc.2016.09.009

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TITLE

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Wild-Type Hen Egg White Lysozyme Aggregation In Vitro Can Form Self-seeding Amyloid

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Conformational Variants

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AUTHORS

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Vishwanath Sivalingam, Nalla Lakshmi Prasanna, Neetu Sharma#, Archana Prasad#, and Basant

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K Patel*

AFFILIATIONS

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Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Sangareddy,

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Medak Dist., Telangana-502285, India

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*CORRESPONDING AUTHOR Basant K Patel

Phone: +91-4023016008 Fax: +91-4023016032

Email: [email protected]

# Equal contributions

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ACCEPTED MANUSCRIPT ABSTRACT Misfolded β-sheet-rich protein aggregates termed amyloid, deposit in vivo leading to debilitating diseases

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such as Alzheimer‟s, prion and renal amyloidosis diseases etc. Strikingly, amyloid can induce conversion

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of their natively folded monomers into similarly aggregated conformation via „seeding‟. The specificity of seeding is well documented in vivo for prions, where prion-variants arising from conformationally altered

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amyloids of the same protein, faithfully seed monomers into amyloid displaying the original variant‟s

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conformation. Thus far, amyloid variant formation is reported only for a few non-prion proteins like Alzheimer‟s Aβ42-peptide and β-2 microglobulin, however, their conformational cross-seeding

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capabilities are unexplored. While mutant human lysozyme causes renal amyloidosis, the hen egg white lysozyme (HEWL) has been extensively investigated in vitro as a model amyloid protein. Here we

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investigated if wild-type HEWL could form self-seeding amyloid variants to examine if variant formation

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is more wide-spread. We found that HEWL aggregates formed under quiescent versus agitated conditions, displayed different particle sizes, detergent stabilities & β-sheet content, and they only seeded

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monomeric HEWL under similar incubation conditions, but not under swapped incubation conditions thereby showing amyloid variant formation by HEWL analogous to prion variants. This may have implications to the amyloidosis caused by different mutants of human lysozyme.

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ACCEPTED MANUSCRIPT Keywords

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Amyloid, Hen Egg White Lysozyme, Amyloid Variants, AFM, Dynamic Light Scattering

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Introduction

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Aggregation and deposition of a normally soluble protein into insoluble, self-seeding β-sheet-rich

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aggregates, called amyloid, can manifest as serious clinical disorders [1-3]. Over 30 proteins have been found to be associated with amyloid deposition diseases, including devastating disorders such as

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Alzheimer‟s disease associated with Aβ-42 peptide amyloid deposition, prion diseases linked with protein PrPSc deposition and Parkinson‟s disease where α-synuclein is found aggregated in the nervous system [4,

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5]. Amyloid aggregates share a common structural feature containing cross-β structure, and

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characteristically bind planar dyes Congo Red and Thioflavin T, the respective absorbance and

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fluorescence properties of which are altered upon binding to amyloid [6-8]. Many proteins which may not cause any amyloid diseases, can also be induced in vitro to form amyloid-like aggregates and an extrapolation predicts that the ability to form organized amyloid-like aggregates may be a generic property of all proteins [9-11].

Amyloid aggregates can exhibit polymorphism, which may manifest as alteration in shapes and sizes of aggregates [12-15]. The human prion disease CJD has been shown to manifest with different incubation times and neuropathological lesion profiles in affected individuals, proposedly owing to structural alteration in its amyloid aggregates leading to PrPSc strains (also called: variants) [16, 17]. Likewise, single cell eukaryote yeast can harbor protein-based prions-like elements which follow non-Mendelian genetic inheritance patterns [18]. Prion variant formation has been vividly demonstrated for the yeast prion [PSI+] that can exist as a strong or weak variant which can be de novo induced in vivo by respectively seeding with wild-type Sup35p amyloid aggregates formed in vitro at 4°C or 37°C and also 3

ACCEPTED MANUSCRIPT by variation in the ionic composition during Sup35p aggregation [19-22]. The Sup35p amyloid variants formed at 4°C and 37°C sequester different amino acid lengths in their amyloid core contributing to the

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assumption of altered β-sheet structures [23]. The Alzheimer‟s disease Aβ-42 peptide has been shown to form prion-like self-seeding conformational variants where samples incubated at static and agitated

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conditions, showed different mass per length densities and morphologies [13]. It has been proposed that

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agitation can increase exposure of the protein molecules to water-air interface which can affect their conformation adopted during amyloid aggregation leading to variant formation versus the protein

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aggregated under static incubation [13].

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Human lysozyme which is an anti-bacterial enzyme found in several body secretions when mutated is found to be associated with familial non-neuropathic systemic amyloid deposition in kidney, liver and

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gastrointestinal tract. So far, variations in disease symptoms with different mutants in human lysozyme

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have not been examined [24]. Wild-type human lysozyme and familial mutants can also aggregate as amyloid in vitro [2, 25]. Hen egg white lysozyme (HEWL) which has around 60% sequence similarity

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with human lysozyme can also form amyloid aggregates in vitro at acidic pH, when briefly boiled and then incubated at 37°C for over 7 days [26]. HEWL aggregates display the key amyloid properties such as β-sheet-rich structure, fibrillar morphology and self-seeding abilities [26-29]. A peptide fragment of HEWL (amino acids 49-101) which resulted due to protein hydrolysis caused by heating at the low pH 2.0, has been identified to be the amyloidogenic core for HEWL [30, 31]. Although HEWL can also form amyloid aggregates under a few other incubation conditions, whether these aggregates represent any prion-like conformational variants remains to be investigated. Here we examined whether wild-type HEWL protein has the capability of forming faithfully self-seeding amyloid variants similar to prion variants. We tested aggregation of HEWL at acidic pH without preboiling followed by incubation under either agitated or quiescent conditions. To test variant formation, aggregates formed under these altered incubations were examined for stability & conformational 4

ACCEPTED MANUSCRIPT differences and also for inability to conformationally cross-seed the aggregation of monomers incubated

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under swapped incubation conditions.

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EXPERIMENTAL METHODS

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Materials

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Hen egg-white lysozyme (HEWL), Thioflavin T, 3-aminophenol (3-AP), 4-amonophenol (4-AP), Pepsin (porcine), sarkosyl, sodium dodecyl sulfate (SDS) and tricine were purchased from Sigma-Aldrich

were obtained from SPI Supplies (USA).

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Aggregation of HEWL

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(USA). Congo red were procured from HiMedia Labs (India). Mica sheets (Grade V) for AFM imaging

HEWL can form amyloid aggregates when incubated at pH 2.0 at elevated temperatures due to release of

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amyloidogenic peptide by acid hydrolysis which can readily aggregate [26, 30, 31]. Aggregation of HEWL was performed using method described in Krebs et al.(2000) with minor modifications [26]. A one mM solution (14 mg/ml) of HEWL in 0.5 M Glycine-HCl buffer, pH 2.0 was kept at 65°C for 2 days. Also, 1 mM solution of HEWL pH 2.0 was incubated at 37°C for 15 days under quiescent state (static) or for 7 days with agitation. For a control, 1 mM HEWL pH 2.0 prepared similarly was incubated at 4°C to prevent any aggregation. Protein concentration of HEWL was determined using its extinction coefficient: =2.63 [32]. Amyloid detection assays Thioflavin T binding assay- Thioflavin T (ThT) associates specifically with amyloid aggregates, which shifts its fluorescence excitation maximum from 385 nm to 450 nm and enhances its emission intensity at 485 nm [6]. Aliquots of 100 μM of monomeric, agitated or static HEWL samples were incubated with 50 5

ACCEPTED MANUSCRIPT μM ThT and the ThT fluorescence was recorded using Molecular Devices SpectraMax M5e Multi-mode plate reader. Upon excitation at 450 nm, fluorescence emission intensity was either recorded directly at

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485 nm or emission spectra were recorded from 460-560 nm to assess the amyloid aggregation of HEWL.

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Another 1 mM solution of HEWL incubated at 65°C for 2 days, was used for comparison. Congo Red binding assay- Binding of Congo red to amyloid aggregates induces a characteristic red shift

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in its maximum absorbance wavelength from 490 nm towards 540 nm [33]. Congo red solution was

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added to soluble or aggregated HEWL solutions at 10:1 (protein:Congo red) ratio followed by incubation for thirty minutes at room temperature. Absorbance was recorded from 300-700 nm to examine amyloid

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nature of the HEWL samples.

Turbidimetry- Upon amyloid aggregation, perceivable increase in turbidity of the solution occurs.

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Aliquots (100 μl) of HEWL samples pre-incubated at 4°C, under agitated or static conditions were

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examined for turbidity by recording absorbance at 405 nm [34].

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Inhibition of HEWL aggregation- In a previous study, 4-aminophenol (4-AP) has been shown to inhibit in vitro aggregation of HEWL [37]. Although, the mechanistic details of inhibition by amino phenol has not been reported, in the view that several aromatic compounds with phenol groups are found to inhibit several amyloid protein‟s aggregations [35, 36], a combination of hydrogen bonding abilities of the -OH & -NH2 functional groups and steric hindrance by the aromatic ring towards beta-sheet formation, may have the inhibitory contributions. Here we examined the effect of 4-aminopehnol (4-AP) and a related compound 3-aminophenol (3-AP) on the aggregation of HEWL. For this, 1 mM HEWL was incubated in the aggregation buffer with four-fold molar excess of 4-AP or 3-AP and the samples were allowed to undergo aggregation under quiescent or agitated incubations. ThT binding assay was carried out to estimate the amyloid aggregation. Samples incubated similarly but without 4-AP or 3-AP were used as controls for comparison.

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ACCEPTED MANUSCRIPT ANS binding assay ANS (1-Anilinonaphthalene-8-sulfonic acid) dye is widely used to measure exposed hydrophobic patches

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on proteins upon their unfolding or aggregation. Upon binding with hydrophobic regions in proteins,

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ANS fluorescence intensity rises sharply at 480 nm after excitation at 380 nm. Aliquots (50 μM) of soluble HEWL, agitated HEWL, static HEWL or the 65 oC aggregated HEWL samples, all at pH 2.0,

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were incubated with ANS (200 μM) and ANS fluorescence emission spectra were recorded from 400 to

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600 nm upon excitation at 380 nm.

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Dynamic Light Scattering

Dynamic Light Scattering (DLS) was monitored using Delsa Nano C Particle Analyzer (Beckman

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Coulter) using 3 ml cuvette with the sample chamber pre-maintained at 25 °C and the data were processed

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using Delsa Nano 3.7 software. The agitated and static HEWL aggregates were diluted 20-fold in the aggregation buffer pH 2.0 before recording the scattering. The samples were illuminated with 658 nm

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laser and the scattering intensities were measured at 165°. Autocorrelation functions were also measured simultaneously. Effects of pH shift to 7.0 and addition of 2% sarkosyl detergent on the preformed HEWL aggregates were also monitored using DLS. For comparsion, 14 mg/mL samples of soluble HEWL pH 7.0, with or without 2% sarkosyl, were also examined by DLS as above. For a control, a sample with only 2% sarkosyl pH 7.0 was also examined to eliminate any contributions to the HEWL aggregate sizes from micelle formation by sarkosyl. Pepsin digestion of HEWL aggregates Amyloid aggregates may display partial resistance to digestion by proteases like proteinase K or pepsin [31]. As proteinase K has been previously reported to be inefficient at the proteolysis of HEWL [38], we employed pepsin digestion, which also has the advantage that it functions at pH 2.0, the same pH as that of the aggregation buffer used for HEWL. The soluble and agitated HEWL samples (70 µg) were 7

ACCEPTED MANUSCRIPT incubated with pepsin (0.7 µg) at 50°C in protease assay buffer (50 mM Glycine-HCl buffer, pH 2.0) at the pepsin: HEWL ratio of 1:100 w/w. To terminate the pepsin activity, reaction mixtures were

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immediately transferred to -20oC. Proteolytic digests were then separated on 16% Tricine-SDS-PAGE

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under reducing conditions and were visualized using Coommassie staining [39].

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Atomic Force Microscopy

AFM images were obtained using a Nanoscope V scanning microscope (Bruker Instruments) in tapping

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mode. A 5 μl aliquot of the sample was drop-casted on a freshly cleaved mica sheet and incubated for 10 minutes. The surface was then rinsed with deionised water and allowed to dry for 15 min before scanning.

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For scanning, OTESPA tip (Bruker AFM Probes) with a nominal radius of ~7 nm and a spring constant of 42 N/m was used. Scan rate was set at 1 Hz and the imaging was performed as 256-lines scan in 5 mm x 5

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mm or 1 mm x 1 mm area. Images were further analyzed & processed using WSxM software [40]. Circular Dichroism Spectroscopy

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Increase in β-sheet conformation upon amyloid aggregation can be monitored by recording circular dichroism (CD) spectra from 190-260 nm (far-UV range). Aggregates of HEWL were examined for secondary structural changes by far-UV CD spectroscopy and compared with monomeric HEWL. The CD spectra were acquired on a Jasco J-1500 spectropolarimeter with scan speed of 50 nm/min using 1 mm path length quartz cuvette. The protein samples were diluted to 0.1 mg/mL in aggregation buffer for the soluble, agitated, & 65 oC HEWL, and to 0.7 mg/mL for the static HEWL samples, just before recording the spectra. The spectra were corrected for baseline contributions and then expressed as mean residue ellipticity [θ] using the following equation: [θ] = (θobs* MRW)/(10*c*l) where, θobs, c, and l respectively represent the observed ellipticity in milli-degrees, protein concentration in g/mL and the path length of the cuvette. Mean residues weight (MRW) of amino acids was taken as 8

ACCEPTED MANUSCRIPT 112 Da. Using the respective far-UV CD spectra, the relative secondary structural contents were estimated with the online server BeStSel [41]. Standard deviations were calculated from the results

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obtained from three independent samples and depicted as error bars.

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Amyloid Seeding Assay

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Amyloid seeds were generated by brief sonication (three pulses of 10 seconds with intermittent 5 seconds incubation on ice) of the HEWL aggregates pre-formed under static or agitated conditions

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(Supplementary Figure 3S). For self-seeding, 5μl of sonicated seed from the static amyloid was added to monomeric HEWL solution to obtain 5% (v/v) final seed followed by static incubation at 37°C for 96

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hours without any agitation. Similarly, 5% (v/v) of agitated seed was added to monomeric HEWL and seeding was assayed by incubation at 37°C for 96 hours with agitation. For assessing conformational

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cross-seeding, monomeric HEWL was added with 5% (v/v) of static HEWL amyloid seeds followed by

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incubation at 37°C under agitated conditions. Likewise, 5% (v/v) seeds from the agitated HEWL amyloid

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samples were added to monomeric HEWL and aggregation was assayed at static incubation. Amyloid formation was continuously monitored using Spectramax M5e multimode reader by examining ThT binding. RESULTS

Agitated and static HEWL aggregates show amyloid-like features Formation of HEWL amyloid aggregates has been previously achieved under prolonged incubation at pH 2.0 preceded by boiling at high temperatures [26]. We have attempted here the amyloid aggregation of HEWL without boiling to check if HEWL could form conformationally distinct variants. The frequently used protocol where boiling at acidic pH is utilized, leads to hydrolysis of certain peptide bonds thereby releasing the amyloidogenic core peptide of HEWL, which then forms amyloid aggregates [30]. In this study, HEWL solubilized at 14 mg/ml (1 mM) concentration at pH 2.0 was incubated at 37°C either under 9

ACCEPTED MANUSCRIPT static or agitated conditions and amyloid-like aggregation was then evaluated and compared with HEWL amyloid aggregates formed at 65 oC pH 2.0 using the previously established method [26].

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The agitated and static samples were examined after seven and fifteen days respectively for amyloid

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aggregation by monitoring amyloid-like ThT & Congo red binding affinities and also by turbidity measurements. As expected of amyloid aggregates, both type of samples whether incubated with or

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without agitation exhibited high affinity to ThT (Figure 1a and Supplementary Figure 1S-a) and Congo

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red (Supplementary Figure 2S) as well as increased turbidity (Figure 1b). Interestingly, the levels of some of the amyloid-specific properties seemed to show consistently distinct patterns. While the ThT

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fluorescence intensity at 485 nm of the static samples showed ~10-fold increase as compared with that of a freshly prepared soluble HEWL sample (pH 2.0), the agitated HEWL aggregates showed over 40-fold

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increase (Figure1a). Similarly, the turbidity measurement at 405 nm showed ~4-fold increase for the static

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HEWL aggregates whereas ~12-fold increase for the agitated samples was observed relative to the soluble HEWL sample (Figure 1b). As a control, when we analyzed HEWL amyloid aggregates formed after two

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days of incubation at pH 2.0 at 65oC, its ThT fluorescence emission was markedly higher than that of the static as well as the agitated HEWL aggregates. However, the turbidity of the pH 2.0 65oC HEWL sample was comparable to the static aggregates while the agitated HEWL aggregates exhibited the highest turbidity (Figures 1a & b).

Furthermore, when effects of small organic compounds 3-aminophenol (3-AP) & 4-aminophenol (4-AP) on HEWL aggregation was examined, they showed inhibition of aggregation of the static, agitated as well as the 65oC HEWL samples (Figure 1c and Supplementary Figure 1S-b). This observation is similar to as previously reported for 4-aminophenol (4-AP) which is known to inhibit HEWL amyloid aggregates formed at pH 2.0 with pre-boiling [37]. This inhibitory effect of 3-AP & 4-AP under both static and agitated incubations further supports amyloid-like nature of these HEWL aggregates. Conformational differences between the static and agitated HEWL aggregates was also probed by ANS dye which binds 10

ACCEPTED MANUSCRIPT to exposed hydrophobic patches. High ANS fluorescence intensity at ~480 nm of soluble HEWL at pH 2.0 indicates presence of relatively unfolded state of HEWL at pH 2.0 which would expose hydrophobic

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patches with affinity to ANS. The static and agitated HEWL aggregates, also present at pH 2.0, displayed even higher ANS fluorescence thereby indicating further conformational changes during the process of

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aggregation (Figure 1d). The HEWL control amyloid aggregates formed at pH 2.0 65 oC, also displayed

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increased ANS fluorescence, however, comparatively less than the static and agitated HEWL aggregates, possibly due to loss of ANS binding sites (hydrophobic patches) upon fragmentation of the HEWL

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protein at pH 2.0, 65 oC. The relatively higher intensity of ANS fluorescence in presence of static

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aggregates compared to the agitated aggregates may be further indicative of differential exposure of hydrophobic surfaces and presence of conformational differences between these aggregates.

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In the view of these observed differences, further analyses were undertaken to check whether the

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aggregates formed under the two incubation conditions represented any bonafide self-propagating amyloid variants similar to as documented for prion variants [19, 20]. For this, differences in the

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stabilities, secondary structures, self-seeding and conformational cross-seeding of the agitated & static HEWL aggregates were further examined and compared. Agitated and static HEWL aggregates exhibit stability and conformational differences When analyzed by dynamic light scattering, the agitated aggregates showed higher hydrodynamic radii (~700-800 nm) compared to the static aggregates (~ 100 nm) (Figure 2a & 2d). Also the comparative hydrodynamic radii of the aggregates estimated at pH 7.0 (Figure 2b & 2e) and in presence of ionic detergent 2% sarkosyl (Figure 2c & 2f) further suggested relatively higher stabilities for the static HEWL aggregates (Rh ~ 60-80 nm) than the agitated HEWL samples (Rh ~ 25-35 nm). Additionally, the particle size estimation for soluble HEWL pH 7.0 in the absence or presence of 2% sarkosyl respectively showed hydrodynamic radii of ~2.8±1.2 nm & ~1.74 nm thereby suggesting the presence monomeric HEWL, in the view that a hydrodynamic radius of ~1.9 nm has been previously reported for monomeric HEWL 11

ACCEPTED MANUSCRIPT using DLS (Figure 2g & 2h) [54]. Using the 1.74 nm average hydrodynamic radius of HEWL in the presence of 2% sarkosyl, the residual static HEWL aggregates in presence of 2% sarkosyl were estimated

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to be made up of ~23-47 monomers whereas the agitated HEWL aggregates comprised of ~14-21 HEWL

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monomers, thereby indicating their differential structural stabilities against sarkosyl. To further compare the relative stabilities of the static and agitated HEWL amyloid aggregates, effect of

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change in pH from the aggregation buffer pH 2.0 to neutral pH 7.0, was also assessed by ThT binding

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assay. When shifted to pH 7.0, the static and agitated HEWL aggregates respectively showed about 20% and 80% decrease in their ThT emission fluorescence suggesting relatively higher stability of the static

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aggregates (Figure 2j). This further indicates presence of conformational differences between the static and agitated HEWL aggregates and suggests assumption of amyloid variant like-features.

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Furthermore, when limited proteolysis of the agitated & static HEWL aggregates was examined using

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digestion by pepsin protease, a prominent ~10 kDa digestion product was observed from the agitated

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HEWL aggregates but not from the static HEWL aggregates thereby further supporting presence of conformational differences between these two kinds of aggregates (Figure 2k). Furthermore, when these HEWL aggregates were visualized by atomic force microscopy, the agitated HEWL samples appeared to have straight fibrils whereas the static samples manifested curvi-linear fibrils (Figure 3a & 3b). Also, the static HEWL aggregates displayed higher population of relatively thicker fibrils (~2.5-3 nm) compared to the agitated HEWL aggregates (~2-2.5 nm) thereby further indicating possible differences in their structural organization. The control 65 oC pH 2.0 amyloid aggregates displayed relatively thinner fibrils of ~1.5-2 nm height, possibly due to being composed of fragmented HEWL peptides (Figure 3c). To further examine the underlying conformations, when secondary structure was analyzed by far-UV circular dichroism, both the static and agitated HEWL aggregates exhibited more negative CD ellipticities at 215 nm (ϴ215nm) compared with a freshly prepared HEWL sample at pH 2.0, suggesting increment in β12

ACCEPTED MANUSCRIPT sheet content as expected upon amyloid-like aggregation (Figure 4a). Furthermore, secondary structural feature predictions using the tool BeStSel, which is preferred for structural predictions for aggregated -

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sheet-rich proteins [41], showed that the agitated HEWL aggregates contained relatively higher β-sheet content compared to that of the static HEWL aggregates thereby further supporting the presence of

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conformational differences (Figure 4b). The control 65oC pH 2.0 HEWL aggregates also displayed

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markedly increased β-sheet structural content compared to soluble HEWL pH 2.0, which corroborates their amyloid nature ((Figure 4a & b). Possibly, the length of amino acids assuming the β-sheet

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conformation and thus contributing to the amyloid core of the aggregates may be different in the two

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HEWL aggregate forms. Previously, for amyloid aggregates of Sup35 yeast prion protein, higher β-sheet content and relatively longer amyloid core have also been documented for the Sup35 amyloid aggregates

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formed at 37°C versus those formed at 4°C [23].

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Lack of conformational cross-seeding confirms prion-like amyloid variant formation

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Amyloid aggregates can grow by nucleation wherein after an initial lag period, a rapid acceleration of the aggregation process ensues. This lag period can however be shortened upon addition of “seeds” which are fragmented pre-made amyloid aggregates of the same protein by a process termed as “self-seeding” [34, 42]. While self-seeding is an efficient process, cross-seeding of amyloid aggregation which would involve two different proteins is expectedly rare and inefficient due to conformational incompatibility arising out of the different amino acid sequences [43]. By similar analogy, it would be expected that even amyloid aggregate variants of a single protein could have difficulty in conformational cross-seeding. As expected, self-seeding was efficiently observed when pre-formed static HEWL aggregates (5% v/v) were added to monomeric HEWL incubated under static conditions, as evident by reduction of the lag phase to ~ 20 hours (Figure 5a). Likewise, the sonicated pre-formed agitated HEWL aggregates also seeded the HEWL monomers incubated with agitation, as observed by the increase in the ThT fluorescence without any lagphase as compared with the unseeded controls (Figure 5c). The relatively rapid seeding observed under 13

ACCEPTED MANUSCRIPT agitated incubation over the static incubation may be due to shearing effect of the agitation to efficiently produce amyloid fragment ends amenable to faster recruitment of monomers, as also previously observed

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for Rnq1 yeast prion amyloid aggregation in vitro [44]. The observed self-seeding behaviours of the static and the agitated HEWL aggregates further confirm their amyloid-like natures. If the amyloid

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aggregates obtained upon static and agitated incubations were truly conformationally distinct alike prion

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amyloid variants, they would be expected to be inefficient at seeding the monomers incubated under swapped incubation conditions. Indeed, even up to 70 hours of incubation, no seeding was observed by

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the agitated seeds to the static HEWL monomers (Figure 5b). Likewise, the aggregation trend of the

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agitated samples incubated with 5% static seed or without any seed showed similar lag periods (~ 20 hours) thereby suggesting lack of any perceivable seeding (Figure 5d). This absence of conformational cross-seeding strongly supports that the static and agitated HEWL aggregates are indeed prion-like

DISCUSSION

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amyloid variants bearing conformational distinction.

Self-aggregation of proteins into amyloids is implicated in pathogenesis of several debilitating diseases where they are found to accumulate in vital organs such as brain, kidneys, liver or spleen [1, 5]. The ability to form amyloid-like structures is regarded as a general property of proteins [9], however, whether all amyloid proteins can form self-seeding prion-like amyloid variants from their single primary amino acid sequence remains unestablished. Formation of prion-like strains can have important clinical manifestations such as: prion variants in the transmissible spongiform encephalopathies resulting in distinct disease incubation times and deposition patterns of PrPSc in the brains of the affected CJD individuals and likewise pathological heterogeneity in the Alzheimer‟s disease [45, 46].

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ACCEPTED MANUSCRIPT In this study, we investigated if the model amyloid forming protein, HEWL could produce self-seeding prion-like conformational variants under different aggregation conditions. Incubating HEWL either at

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quiescence or with agitation at 37°C under acidic conditions (pH 2.0) that induces its partial unfolding, as seen by increased binding to the hydrophobic probe dye ANS, exhibited formation of amyloid-like

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aggregates. As the HEWL protein was not fragmented at this temperature, in contrast to the otherwise

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observation in the commonly used method of HEWL aggregation using acid hydrolysis with heating at pH 2.0 [26], investigation of formation of amyloid conformational variants could be undertaken. The rate

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of aggregation of HEWL was relatively rapid upon incubation with agitation as compared to the static HEWL incubation. The mechanical agitation has also been reported to accelerate the aggregations of

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other amyloid proteins like Aβ-40, prion protein, insulin, glucagon and β-2 microglobulin supposedly by breaking of the matured longer amyloid aggregates into small fragments thereby providing new ends for

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faster recruitment of the monomers into the aggregates [13, 44, 47-50]. Furthermore, the agitated HEWL aggregates displayed higher ThT fluorescence than the static HEWL aggregates, possibly due to more

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efficient amyloid formation caused by mechanical shearing and thus providing more ThT binding sites. Likewise, the agitated HEWL also displayed higher affinity for Congo red than the static HEWL aggregates. Furthermore, consistent with greater aggregation and higher turbidity, the agitated HEWL samples were found to exhibit significantly higher light scattering than the static HEWL samples. However, the ThT fluorescence intensities of both the agitated & static aggregates were much less than that of the aggregates formed using previously reported method involving acid hydrolysis of HEWL (pH 2.0, 65 oC). Whether these altered incubation conditions (static or agitated) only induced different levels of aggregation or imparted underlying conformational variations between these aggregates was further examined. Indeed, the presence of conformational differences between these two HEWL aggregates were suggested by different relative stabilities against a detergent sarkosyl, a denaturant urea and shift to neutral pH conditions. This presence of differential stability is in fact analogous to that of the yeast prion Sup35 where variant-specific SDS-stable aggregate with different sizes have been reported, and it thus 15

ACCEPTED MANUSCRIPT possibly suggests induction of HEWL amyloid variants here [51]. The structural differences in the two forms of HEWL aggregates were also supported by CD spectroscopy. Far-UV CD mean residue

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ellipticity at 215 nm for the agitated HEWL aggregates was more negative than that of the static HEWL aggregates, thereby suggesting relatively higher β-sheet content in the agitated aggregates which was also

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corroborated by secondary structural predictions by the BeStSel algorithm. Possibly, agitation induces

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more efficient misfolding thus HEWL assumed a longer amyloid core with relatively more number of amino acids being in the β-sheet conformation compared to that in the static aggregation. Notably, for

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amyloid aggregates of yeast Sup35 prion protein, an increased β-sheet content and a longer amyloid core

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has also been previously documented for amyloid aggregates formed at 37°C versus those formed at 4°C [23].

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Previously, morphological characterizations of HEWL amyloid fibrils obtained at acidic conditions with

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pre-heating (60-65°C) have been found to display elongated and slender fibrils [52, 53]. Also, HEWL proto-fibrillar aggregates obtained previously by heating manifest hydrodynamic radii of ~22 nm [54]

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whereas the HEWL mature aggregates obtained with heating (56°C for upto 10 hours) have been shown to produce particles with ~35-70 nm radii [55]. However, the HEWL aggregates obtained here at acidic pH but without heating, appeared relatively bigger in dimensions possibly due to being composed of the full-length HEWL protein rather than only the amyloidogenic core peptide that is generated by acid hydrolysis at the elevated temperatures. Additionally, the greater dimensions of the aggregates may also be in part due to lateral association of the full-length individual amyloid fiberils. Further supporting the formation of HEWL amyloid variants, the static and agitated aggregates also displayed differences in the particle sizes and heights of the amyloid aggregate species as monitored respectively by DLS and AFM. We found that self-seeding of both static HEWL and agitated HEWL aggregates increases the aggregation rates of HEWL monomers in vitro, which is analogous to prion-like self-seeded propagation. However, the agitated HEWL and the static HEWL aggregates failed to seed the soluble protein under swapped 16

ACCEPTED MANUSCRIPT incubation conditions possibly due to the conformations attained in the agitated aggregates being significantly different from the static amyloid aggregates thereby resulting in conformational

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incompatibility of the monomers to associate to pre-formed fragemented aggregates obtained by sonication & used as the seed. Notably, weak [PSI+] and strong [PSI+] prion variants and their variant-

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specific in vitro made Sup35p amyloid aggregates are also conformationally incompatible and do not

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cross-seed each other [56].

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The findings here suggest that agitation imparts a significant change in the structural features of the HEWL aggregates in comparison with aggregates formed without agitation, which is reflected in their

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relative stabilities against denaturing agents and marked differences in the seeding capabilities. While the Aβ-42 peptide was previously shown in vitro to form distinct amyloid aggregates under different

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incubation conditions, a more recent in vivo finding likewise further demonstrated an analogous formation

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of prion-like variants from the Swedish and arctic Aβ-42 point mutations in Alzheimer‟s patients thereby corroborating the variant hypothesis for Aβ-42 amyloid aggregation [13, 46]. The finding here of

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amyloid variant formation by HEWL could possibly in future have similar implications to the understanding of human lysozyme amyloidosis where different familial mutations have been documented in different renal amyloidosis patients. CONCLUSIONS

In this study, we examined whether self-seeding prion-like amyloid variant formation is a more widespread phenomena, using hen-egg white lysozyme (HEWL) protein as a model. Aggregates of HEWL formed at pH 2.0, 37°C under static and agitated incubations showed amyloid-like Thioflavin-T binding & turbidities, however, to different extents. These aggregations were also inhibited by addition of 4aminophenol which is already known as an inhibitor of HEWL aggregation. The static and agitated HEWL aggregates manifested different levels of stabilities against pH change and also displayed different sarkosyl detergent-stable molecular aggregates sizes when examined by dynamic light scattering. Limited 17

ACCEPTED MANUSCRIPT proteolytic digestion by pepsin also indicated presence of conformational differences. Furthermore, differences in the aggregate morphologies when examined by AFM and differences in their secondary

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structural features when probed by Far-UV circular dichroism spectroscopy, were also documented. Although the seeds from the agitated and static HEWL aggregates were very efficient at self-seeding, they

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showed conformational incompatibility and did not efficiently cross-seed HEWL monomers incubated

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under swapped incubation conditions thereby establishing the agitated and static aggregates as true prion-

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like amyloid variants of HEWL.

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ACKNOWLEDGEMENT

Senior research fellowship (SRF) from University Grants Commission (UGC), Govt. of India to Mr.

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Vishwanath S. and SRF from Ministry of Human Resource Development (MHRD), Govt. of India to

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Neetu Sharma & Archana Prasad, are duly acknowledged. Nalla Lakshmi Prasanna thanks MHRD for research assistant fellowship. We thank IIT-Hyderabad funded by MHRD, Govt. of India for research

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consumable and infrastructural support. We sincerely thank Dr. C. S. Sharma, Dr. R. Ramadurai, Mr. J. Joseph, & Mr. K. Miriyala from IIT-Hyderabad, for help with AFM imaging. We also thank Dr. S. Mazumdar and Mr. U. Bhutani from Dept. of Chemical Engineering, IIT-Hyderabad, for help with the DLS data acquisition.

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Amyloid-like aggregation of HEWL at pH 2.0, 37°C under static and agitatedincubations.

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a. HEWL samples at 1 mM concentration were incubated for aggregation at pH 2.0 37°C for seven days

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with agitation or for fifteen days without agitation and also at 65°C for 2 days. Aliquots (100 μl) were subsequently mixed with 50 μM Thioflavin-T and examined for ThT fluorescence emission intensity

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at 485 nm upon excitation at 450 nm using Molecular Devices SpectraMax M5e multi-mode plate

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reader. ThT emission intensity of a freshly solubilized HEWL sample at pH 2.0 was used as a control. b. Aliquots (100 μl) of the static, agitated, 65°C or freshly solubilized HEWL as in (a) were examined

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for turbidity by recording absorbance at 405 nm.

c. Inhibition of amyloid-like aggregation of HEWL under static or agitated incubation and at 65°C was

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examined by pre-incubating HEWL in the aggregation buffer along with 4 mM of 3-aminophenol (3-

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AP), and 4-aminophenol (4-AP) (HEWL:inhibitor molar ratio 1:4). ThT emission intensity at 485 nm for agitated and static HEWL samples lacking the inhibitor were considered as 100%, to estimate the

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inhibition of HEWL aggregation.

d. 50 μM aliquots of soluble HEWL, agitated, static and 65°C samples were were incubated with 200 μM ANS. Fluorescence emission spectra were recorded from 400 nm to 600 nm upon excitation at 380 nm. The relatively higher intensity of ANS fluorescence in presence of static aggregates compared to the agitated aggregates and 65°C HEWL, may be indicative of differential exposure of hydrophobic surfaces and thus suggesting presence of conformational differences between these aggregates. Figure 2. Assessment of relative stabilities and sizes of HEWL aggregates. a-i. Comparison of particle size distributions of agitated and static HEWL samples by Dynamic Light Scattering (DLS) measurements. The agitated HEWL sample shows large particle sizes at pH 2.0 of ~700-800 nm (a) probably due to bigger agglomerate formation as compared with the observed ~ 100 22

ACCEPTED MANUSCRIPT nm sizes for the static samples (d). When these aggregates are shifted to pH 7.0, the agitated aggregates are broken to ~280-300 nm particle sizes (b) whereas the dimensions of the static

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aggregates (~ 90-100 nm) do not change significantly (e). Also, addition of 2% sarkosyl further disaggregates the agitated HEWL sample which manifests particle sizes of ~30 nm (c), whereas the

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static HEWL sample shows particle sizes of ~60-80 nm thereby suggesting comparatively higher

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resistance to disaggregation by the detergent (f). Freshly solubilized HEWL pH 7.0 samples in the absence (g) or presence (h) of 2% sarkosyl respectively displayed hydrodynamic radii of ~2.8±1.2 nm

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& ~1.7 nm. Only sarkosyl (2%) pH 7.0 samples, lacking any protein, displayed particles of < 1 nm

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sizes (i) thereby eliminating the possibility of any significant contribution to the particle size estimates of the agitated & static aggregates examined in presence of 2% sarkosyl in the panels c & f. Comparative stability of the agitated & static aggregates to pH 7.0. Aliquots (20 μl) of static and

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agitated HEWL aggregates at pH 2.0 were brought to neutral pH using 0.6 M Tris-HCl buffer (pH 7.5). The residual stability of the aggregates was then monitored by ThT fluorescence assay.

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k. Comparative proteolysis of the agitated & static HEWL aggregates by Pepsin. Soluble and agitated HEWL samples (70 µg) were incubated with Pepsin (0.7 µg) at 50°C in protease assay buffer (50 mM Glycine-HCl buffer, pH 2.0) at pepsin: HEWL ratio of 1:100 w/w. To terminate pepsin activity, the reaction mixtures were immediately transferred to -20 oC. The soluble lysozyme samples were subjected to proteolysis at pepsin: HEWL ratio of 1:100 for 1, 5 and 10 min at 50°C. Proteolytic digests were separated on 16% Tricine-SDS-PAGE under reducing conditions and were visualized using Coomassie staining. Agitated samples show a distinguishable digestion product with an additional band of ~ 10 kDA compared to the static HEWL, indicating presence of conformational difference. Figure 3. Comparison of agitated and static HEWL aggregates morphologies by AFM

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ACCEPTED MANUSCRIPT AFM images of agitated & static HEWL and 65°C HEWL aggregates were obtained by ambient tapping mode using Nanoscope V atomic force microscope. Height profiles were estimated using the

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software WSxM and after analyzing 74, 64 & 25 fibrils respectively from agitated, static & 65 oC HEWL fibrils, height versus fibril percentages were plotted. The static HEWL aggregates (b)

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displayed relatively thicker fibrils compared to the agitated (a) and the 65°C HEWL samples (c).

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Figure 4. Secondary structure analysis of agitated and static HEWL aggregates by Far-UVcircular dichroism.

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a. Pre-formed agitated and static HEWL aggregates were diluted in aggregation buffer and far-UV CD spectra were collected from 190-260 nm usingJasco J-1500 spectropolarimeter and 1mm path length

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quartz cuvette. Data were expressed as mean residue ellipticity (MRE). A freshly prepared soluble HEWL at pH 2.0 (dashed line) was used to record spectrum representing the non-aggregated HEWL

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reference amyloid conformation.

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conformation. Likewise, far-UV CD spectrum of 65°C HEWL sample was recorded to represent a

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b. Using the far-UV CD spectra from the panel (a), the relative secondary structural contents were predicted using online server BeStSel. The relative contents of -helix, -sheet, turns and random coils are shown for comparison. The agitated, static & 65 oC HEWL aggregates exhibited relatively higher content of -sheet conformation compared to the monomeric soluble HEWL at pH 2.0. Notably, similar -sheet content for the 65 oC HEWL aggregates has also been previously reported by other workers as well [57]. Error bars represent standard deviations of secondary structure predictions obtained from three independent samples. Figure 5. Assessment of conformational compatibility of static and agitated HEWL aggregates by amyloid seeding assay. a. Self-seeding of static HEWL aggregates was examined by adding 5% pre-formed and briefly sonicated static aggregates to monomeric HEWL solubilized in the aggregation buffer. Samples were

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b. Capability of conformational cross-seeding by pre-made agitated HEWL aggregates was examined by adding briefly sonicated agitated aggregates (5% final) to monomeric HEWLdissolved in aggregation

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buffer and incubated without agitation (solid line). Kinetics of amyloid growth was monitored by ThT

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binding assay. An unseeded (dotted line) sample was included and incubated similarly for comparison.

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c. Self-seeding of agitated HEWL aggregates was monitored by adding 5% pre-formed and briefly

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sonicated agitated aggregates to HEWL monomers solubilized in the aggregation buffer. Samples were incubated with agitation and kinetics of seeded growth of amyloid (solid line) was monitored as before. An unseeded sample was also used incubated as a reference (dotted line).

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d. Ability of conformational cross-seeding by pre-made static HEWL aggregates to monomeric HEWL incubated under agitated conditions was examined by adding 5% briefly sonicated static HEWL

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aggregates (solid line). ThT fluorescence change was recorded to monitor amyloid growth. An unseeded sample was also monitored similarly as a reference (dotted line).

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Graphical abstract

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ACCEPTED MANUSCRIPT Highlights: 1. Full-length HEWL forms amyloid-like aggregates at pH 2.0, 37 oC under quiescent and

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agitated conditions.

2. Agitated and static HEWL aggregates display differences in AFM morphologies and

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stability

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3. Amyloid seeds from agitated and static HEWL amyloid aggregates do not efficiently cross-seed HEWL incubated under swapped conditions suggesting conformational

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