Bifunctionality of Iminodiacetic Acid-Modified Lysozyme on Inhibiting

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Biological and Environmental Phenomena at the Interface

Bifunctionality of Iminodiacetic Acid-Modified Lysozyme on Inhibiting Zn2+-Mediated Amyloid #-Protein Aggregation Xi Li, Baolong Xie, Xiaoyan Dong, and Yan Sun Langmuir, Just Accepted Manuscript • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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Bifunctionality

of

Iminodiacetic

Acid-Modified

Lysozyme

2

Inhibiting Zn2+-Mediated Amyloid β-Protein Aggregation

on

3 4

Xi Li,† Baolong Xie, †,‡ Xiaoyan Dong,† Yan Sun*,†

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†Department of Biochemical Engineering and Key Laboratory of Systems

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Bioengineering of the Ministry of Education, School of Chemical Engineering and

8

Technology, Tianjin University, Tianjin 300354, China

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‡Institute of Tianjin Seawater Desalination and Multipurpose Utilization, State

10

Oceanic Administration (SOA), Tianjin 300192, China

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1

ACS Paragon Plus Environment

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ABSTRACT:

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Aggregation of amyloid β-proteins (Aβ) mediated by metal ions such as Zn2+ has been

3

suggested to be implicated in the progression of Alzheimer’s disease (AD). Hence,

4

development of bifunctional agents capable of inhibiting Aβ aggregation and

5

modulating metal-Aβ species is an effective strategy for the treatment of AD. In this

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work, we modified iminodiacetic acid (IDA) onto human lysozyme (hLys) surface to

7

create an inhibitor of Zn2+-mediated Aβ aggregation and cytotoxicity. The

8

IDA-modified hLys (IDA-hLys) kept the stability and biocompatibility of native hLys.

9

Extensive biophysical and biological analyses indicated that IDA-hLys significantly

10

attenuated Zn2+-mediated Aβ aggregation and cytotoxicity due to its strong binding

11

affinity for Zn2+, while native hLys showed little effect. Stopped-flow fluorescence

12

spectroscopy showed that IDA-hLys could protect Aβ from Zn2+-induced aggregation

13

and rapidly depolymerize Zn2+-Aβ aggregates. The research indicates that IDA-hLys

14

is a bifunctional agent capable of inhibiting Aβ fibrillization and modulating

15

Zn2+-mediated Aβ aggregation and cytotoxicity as a strong Zn2+ chelator.

16 17

KEYWORDS: amyloid β-protein; zinc ion; lysozyme; surface modification; chelator;

18

inhibitor

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2


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INTRODUCTION

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Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases

3

causing neuronal loss and dementia.1-3 Pathologically, deposition of amyloid β-protein

4

(Aβ) aggregates in brain tissues is one of the causative factors of AD.4

5

Conformational transitions from soluble disordered Aβ monomers into highly ordered

6

fibrils through toxic oligomers have been proposed to be a crucial step in the

7

progression of AD.5 Hence, researches have focused on the development of

8

anti-aggregation agents to regulate Aβ toxicity.6-9

9

In addition to the self-aggregation, substantial evidences have demonstrated that

10

transition metal ions, such as Zn2+ and Cu2+, affect Aβ aggregation behavior.10-12 The

11

surface charge of Aβ changes due to metal binding, which affects the “colloid-like”

12

stability of the aggregation intermediates.12 Consequently, the deviation induces the

13

formation of off-pathway Aβ aggregates.12 Among metal ions, Zn2+ play an important

14

role in AD and in Aβ aggregation. It has been proven that two His residues (His6 and

15

His13/His14) and two carboxylate side chains (Glu11 and Asp1/Glu3/Asp7, with a

16

slight preference for Asp1) of Aβ involve in the binding of Zn2+.13 The generation of

17

Zn2+-Aβ species has complicated consequences that could lead to the dysfunction of

18

nervous system.14 In order to remove the harmful effects of Zn2+-mediated Aβ

19

aggregation, several metal chelators have been developed to disrupt the binding of

20

Zn2+ for Aβ.14,15 Previous studies have shown that metal chelators such as clioquinol

21

(CQ) and its derivatives are capable of reducing metal-mediated Aβ aggregation and

22

neurotoxicity.16,17 However, the long-term use of CQ with poor biocompatibility is

23

limited by an adverse side effect, subacute myelo-optic neuropathy.18 Besides, these

24

metal chelators showed inappreciable inhibitory effect on Aβ aggregation in the

25

absence of metal ions. Hence, it is highly desired to design bifunctional agents that

26

capable of binding metal ions and possessing potent inhibition effect on Aβ

27

aggregation.

28

In recent years, several bifunctional molecules have been designed to inhibit metal

29

ions-associated Aβ aggregation.19-21 Conjugating metal chelators onto proteins or

30

nanoparticles is a prevalent method to overcome the poor biocompatibility and 3


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insufficient inhibition effect on metal-Aβ aggregation.22,23 Among various

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proteins/nanoparticles, human lysozyme (hLys) is an important natural non-specific

3

immune protein against bacterial and viral infections.24 It has been recognized that

4

lysozyme distinctly inhibits the aggregation of Aβ1-40,25 Aβ17-4226 and Aβ1-42,27 and

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native hLys was found to co-localize with Aβ plaques in AD patients and could

6

decrease the locomotor dysfunction in Drosophila model of AD.26,27 Hence, hLys has

7

the potential as a potent to fight against AD. However, hLys has poor metal-chelating

8

ability for its lack of high-affinity metal binding site.28

9

Hence, we herein developed iminodiacetic acid (IDA)-modified hLys (IDA-hLys)

10

to create a bifunctional agent capable of inhibiting Aβ aggregation and modulating

11

Zn2+-mediated Aβ species. In this study, Aβ40, the most abundant form in AD brain,

12

was used to analyze the inhibition effect of IDA-hLys. The long lag phase of Aβ40

13

aggregation benefits in investigating the inhibition effect of IDA-hLys on the

14

nucleation process of the protein.29 Extensive biophysical and biological assays

15

confirmed the bifunctional feature of IDA-hLys on inhibiting Zn2+-mediated Aβ40

16

aggregation and cytotoxicity. The inhibition mechanism of IDA-hLys was explored to

17

advance the knowledge for the design of bifunctional agents against AD.

18 19

EXPERIMENTAL SECTION

20

Materials. Aβ40 (>95%, lyophilized powder), synthesized by routine solid-phase

21

peptide synthesis and Fmoc chemistry, was purchased from GL Biochem (Shanghai,

22

China).

23

1,1,1,3,3,3-hexafluoro-2-propanol

24

2-[4-(2-hydroxyethyl)-1-piperazine] ethanesulfonic acid (HEPES), sodium borate,

25

boric

26

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were received

27

from Sigma (St. Louis, MO). SH-SY5Y cells were from the Cell Bank of the Chinese

28

Academy of Sciences (Shanghai, China). Dulbecco's Modified Eagle Medium/Ham's

29

F-12 (DMEM/F12) and fetal bovine serum (FBS) were obtained from Invitrogen

30

(Carlsbad, CA, USA). Other chemicals were all of the highest purity available from

IDA,

acid,

hLys,

1,4-butanediol

diglycidyl

(HFIP),

dimethyl

ether,

thioflavin

sulfoxide

4


zinc T

(DMSO),

chloride, (ThT),

and

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local sources.

2

Synthesis and Characterization of IDA-hLys. IDA was modified onto the surface

3

of hLys with 1,4-butanediol diglycidyl ether as linker, as illustrated in Scheme 1. The

4

modification used the reaction between epoxy group and amine or imino group to

5

realize. Firstly, hLys solution (100 mL) was prepared by dissolving protein power in

6

0.1 M pH 9.0 borate buffer at 0.5 mg/mL. Then, 1.875 mL 1,4-butanediol diglycidyl

7

ether was slowly added to the protein solution. The reaction mixture was kept at 37 °C

8

in a water bath. After reaction for 12 h, 0.1 M pH 9.0 borate buffer was used to

9

dialyze the uncombined ether. At the same time, a small part of the reaction solution

10

was dialyzed in deionized water then analyzed by matrix-assisted laser desorption

11

ionization time-of-flight mass spectroscopy (Autoflex Tof/TofIII, Bruker Daltonics

12

Inc, Billerica, MA, USA) to determine the molecular weight of intermediate product.

13

At last, 452.73 mg IDA was added to the intermediate product solution. After reaction

14

at 37 °C for 24 h, deionized water was used to dialyse the uncombined IDA then

15

freezed-dried under vacuum for 24 h. The lyophilized IDA-hLys was stored at -20 °C.

16

IDA-hLys and hLys were analyzed by mass spectroscopy to determine its

17

modification degree in ultrapure water at 1.0 mg/mL. The zata potentials, molecular

18

sizes, far-UV circular dichroism spectra and intrinsic fluorescence spectra of hLys and

19

IDA-hLys were respectively measured on a Zetasizer Nano (Malvern Instruments,

20

Worcestershire, UK), circular dichroism spectrometer (JASCO J-815, Tokyo, Japan)

21

and fluorescence spectrometer (Perking Elmer LS-55, MA, USA) in buffer A (20 mM

22

HEPES, 100 mM NaCl, pH 7.4) in 1.0 mg/mL solution at 37 °C.

23

Aβ40 Monomer Solution Preparation. Aβ40 monomer was pre-treated by a

24

reported method.30 The lyophilized Aβ40 power was thawed at room temperature for

25

20 min before use, and then dissolved in HFIP to 1.0 mg/mL to remove pre-existing

26

amyloid fibrils. The solution was sonicated for 10 min in ice bath. After centrifugation

27

(16,000 g) for 20 min at 4 °C，the upper 75% of the supernatant was collected then

28

freezed-dried to remove HFIP under vacuum for 24 h. The lyophilized protein was

29

immediately stored at -20 °C.

30

The treated Aβ40 was dissolved by 20 mM NaOH at 275 µM, and sonicated for 10 5


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min in ice bath followed by centrifugation (16,000 g) for 20 min at 4 °C. The upper

2

75% of the supernatant was carefully collected. For hLys/IDA-hLys inhibiting Aβ40

3

aggregation experiments, Aβ40 solution was added to hLys/IDA-hLys in buffer A to

4

the final concentration of 25 µM. For hLys/IDA-hLys inhibiting Zn2+-mediated Aβ40

5

aggregation experiments, Zn2+ in buffer A was mixed with Aβ40 monomer solution,

6

and buffer A containing various concentrations of hLys/IDA-hLys were added 2

7

minutes later. The final concentrations of Aβ40 and Zn2+ were 25 µM and 12.5 µM,

8

respectively.

9

Thioflavin T Fluorescent Assay. The assay samples (200 µL) were mixed in a

10

96-well plate, containing 25 µM Aβ40 monomers, 25 µM ThT, 12.5 µM Zn2+ and/or

11

different concentrations of hLys or IDA-hLys in buffer A. The ThT fluorescent

12

kinetics was measured using a fluorescent plate reader (SpectraMax M2e, Molecular

13

Devices, USA) at 10 min reading intervals and 5 s shaking before read at 37 °C. The

14

excitation and emission wavelengths were 440 nm and 480 nm, respectively. The

15

fluorescent intensity of the samples without Aβ40 was subtracted as background from

16

each read with Aβ40.

17 18 19

As the aggregation kinetics of Aβ40 showed sigmoidal curve, the experimental data were fitted as:31 y=y0 +

ymax -y0

(1)

1+e-(t-t1/2 )k

20

where y is the ThT fluorescence intensity at time t, y0 and ymax are the minimum and

21

maximum fluorescence intensities, respectively, t1/2 is the time when the intensity

22

reaches half the maximum intensity, and k is the elongation rate constant. And the lag

23

time (Tlag) were calculated from the fitted parameters as:31 2

24

Tlag =t1/2- k

(2)

25

Atomic Force Microscope. Aβ40 (25 µM) in the absence and presence 12.5 µM

26

Zn2+ and/or different concentrations of hLys or IDA-hLys were incubated at 37 °C in

27

a fluorescent plate reader for 72 h. In addition, 5 µM Aβ40 monomer solution mixed

28

with the mixture of 20 µM Zn2+ and 10 µM hLys or IDA-hLys. 10 µL Aβ40 samples

29

were deposited on freshly cleaved mica for 2 min, then ultrapure water was used to 6


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wash salt ions of the solution. The samples air-dried or dried in a steam of dry

2

nitrogen. Multi-mode atomic force microscope (CSPM5500, Benyuan, China) was

3

employed to image Aβ40 specimens. The images were collected in tapping mode.

4

Cell Viability Assay. The MTT assays with SH-SY5Y cells were employed to

5

examine the cytotoxicity of Aβ40 aggregates. The SH-SY5Y cells were cultured in

6

DMEM/F12 medium supplemented with 20% FBS, 2 mM L-glutamine, 100 U/mL

7

penicillin and 100 U/mL streptomycin at 37 °C under 5% CO2. A total of 5×103 cells

8

(90 µL) were cultured in a 96-well plate for 24 h. Aβ40 stock solution (25 µM) were

9

incubated with or without Zn2+ and/or hLys/IDA-hLys at 37 °C and 150 rpm for 18 h.

10

Then, the obtained samples (10 µL) were added to cells. After cultured additional 24 h,

11

10 µL 5.5 mg/mL MTT in buffer A was added into each well and incubated for

12

another 4 h. The suspension was centrifuged at 1,500 rpm for 10 min to remove the

13

supernatant. 100 µL DMSO was used to dissolve the cell, then shook at 150 rpm for

14

10 min. The absorbance at 570 nm was measured in a Plate Reader (TECAN GmbH,

15

Salzburg, Austria) and six replicates were performed for each sample. Background

16

signals from the sample treated without cells were subtracted and the cell survival

17

treated with buffer A only was set as control to normalize other data for comparison.

18

In addition, lactate dehydrogenase (LDH) release assays were conducted to

19

quantitatively assess the cell death, as described previously.23 In brief, after SH-SY5Y

20

cells were incubated for 24 h, the culture medium was replaced with an FBS-free

21

medium. Then, aged-Aβ40 aggregates as prepared above with or without Zn2+ and

22

inhibitors were added into the cells and incubated for 48 h. Extracellular LDH leakage

23

was evaluated using the LDH cytotoxicity assay kit (Beyotime, China) according to

24

the manufacturer’s instructions. Brieﬂy, the cultured cells were centrifuged at 400 g

25

for 5 min, then 80 µL supernatant was collected from each well and added to a new

26

96-well plate. To each of the wells containing 80 µL of the above supernatant, 40 µL

27

reaction buffer was added. After mixing for 30 min, the LDH release level was

28

assessed at a test wavelength of 490 nm with 630 nm as the reference wavelength. As

29

a control, the cells were incubated with 1% (v/v) Triton X-100 to get a representative

30

maximal LDH release level as 100% cytotoxicity. 7


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In the above ThT and cell viability assays, each sample was measured at least three

2

times, and the mean values and standard deviations were calculated. Student’s t-test

3

was conducted to analyze the variance and p < 0.05 or less was considered to be

4

statistically significant.

5

Isothermal Titration Calorimetry. To detect the binding affinities of

6

hLys/IDA-hLys with Zn2+ and Aβ40, ITC assays were employed using a VP-ITC

7

(MicroCal, Northampton, MA, USA) as described in literature.32 In brief, 10 µL of

8

Zn2+ solution (500 µM) in buffer A was titrated into 1.425 mL of hLys/IDA-hLys

9

solution (30 µM) 25 times at 37 °C. Moreover, hLys solution (50, 200 and 1000 µM)

10

or IDA-hLys solution (200 µM) were titrated into Aβ40 solution (20 µM) at the same

11

condition. The results were analyzed with one-site model by MicroCal Origin

12

software.

13

Stopped-Flow Fluorescent Spectroscopy Measurement. SX 20 stopped-flow

14

fluorescent instrument (Applied Photophysics, Leatherhead, UK ) was used to study

15

the interaction kinetics of Aβ40, Zn2+ and hLys/IDA-hLys on a time scale of seconds.

16

The change of Rayleigh light scattering intensity over time was detected with

17

excitation at 435 nm to reflect Aβ40 aggregation state. Any two of Aβ40, Zn2+ and

18

hLys/IDA-hLys were mixed or any two of these mixed 2 min before mixing with the

19

rest one. The time course of fluorescent intensity was recorded. The final

20

concentrations of Aβ40 and hLys/IDA-hLys were 5 µM and 10 µM, respectively. And

21

Zn2+ concentrations range from 5 µM to 50 µM. The dead time of the experiment was

22

2 ms. Pro-Date Software was used to collect and analyze the data.

23 24

RESULT AND DISCUSSION

25

Characteristics of IDA-hLys. Scheme 1 shows the two-step reaction for the

26

modification of hLys with IDA. Mass spectroscopy was used to analyze the

27

modification degree of IDA-hLys. As shown in Figure S1, the two reactions

28

successively increased the molecular weight of modified hLys to 15603.9 Da

29

(intermediate product) and 15876.2 Da (IDA-hLys), suggesting that the modification

30

of IDA increased the molecular weight by 272.3 Da. As a result, the modification 8


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degree was calculated to be 2.1 on average for coupling one IDA molecule increased

2

molecular weight by 133.1 Da (Scheme 1).

3

The zeta potential values of hLys and IDA-hLys were measured (Table S1). It is

4

seen from the table that native hLys showed positive charges (7.6 ± 0.4 mv) under the

5

physiological condition (pH 7.4) because of its basic nature (isoelectric point, 10.2).33

6

However, IDA-hLys displayed lower zeta potential value (2.6 ± 0.5 mv) than hLys.

7

This means that the modifications led to the reduction of amino groups on the protein

8

surface. The two carboxyl groups in IDA also contributed to the zeta potential

9

decrease of IDA-hLys.

10

Figure S2 shows that hLys and IDA-hLys were both about 5 nm in size, the same as

11

that reported in literature.34 In addition, IDA-hLys kept the same circular dichroism

12

spectra and fluorescence spectra as hLys did (Figures S3 and S4). The results

13

confirmed that IDA-hLys had similar stability with native hLys. This would ensure

14

IDA-hLys to work as a stable and biocompatible agent.

15

IDA-hLys Inhibits Aβ40 and Zn2+-Mediated Aβ40 Aggregation. Once binding to

16

amyloid fibrils and profibrils, ThT, which is a benzothiazole dye, exhibits enhanced

17

fluorescence with excitation and emission at 440 nm and 480 nm, respectively.35,36

18

Thus, ThT fluorescence assays were used to monitor the inhibitory effects of hLys and

19

IDA-hLys on Aβ40 amyloid formation and Zn2+-mediated Aβ40 aggregation. Figure 1A

20

shows that the ThT fluorescence intensity (FI) of Aβ40 (25 µM) exhibited a sigmoidal

21

appearance, as reported previously.25,37-39 However, the ThT intensity decreased

22

obviously in the presence of hLys or IDA-hLys at 2.5-12.5 µM (Figure 1B). This is

23

consistent with previous reports that hLys inhibited the aggregation of Aβ40 and Aβ42

24

effectively.25,27 The inhibitory effect of hLys on Aβ40 fibrillization showed a

25

dose-dependent manner, and so did IDA-hLys. Compared with native hLys, however,

26

IDA-hLys showed somewhat weaker inhibitory effect on Aβ40 aggregation (Figure

27

1B). It is considered due to the decrease of positive charges on IDA-hLys surface,

28

which weakened the electrostatic interaction with the acidic protein Aβ40 that has an

29

isoelectric point of 5.5 and carries negative charges at pH 7.4.40 Moreover, the lag

30

phase of Aβ40 aggregation with 2.5 µM hLys/IDA-hLys was shorter than that of Aβ40 9


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1

alone (Table 1). This might imply that low hLys/IDA-hLys to Aβ40 concentration ratio

2

resulted in a high local bound Aβ40 concentration on hLys/IDA-hLys surface (i.e., the

3

molecular number of bound Aβ40 per hLys/IDA-hLys molecule), which favored Aβ40

4

nucleation on the surfaces.41 However, with increasing the molar ratio of

5

hLys/IDA-hLys to Aβ40, the lag phase time prolonged and the final plateau ThT FI

6

value decreased noticeably (Table 1 and Figure 1). This indicates that increasing

7

hLys/IDA-hLys concentration gave rise to both the reduction of free monomers in

8

solution and the local bound Aβ40 concentration on the protein surfaces (i.e., the

9

molecular number of bound Aβ40 per hLys/IDA-hLys molecule), resulting in the

10

decreases in Aβ40 oligomerization, nucleation and fibrillation.41 At 12.5 µM hLys or

11

IDA-hLys, amyloid formation was fully inhibited till 72 h (Figure 1A, Lines 4 and 7).

12

The results demonstrated the potency of IDA-hLys on inhibiting Aβ40 self-assembly.

13

Next, the inhibitory effects of hLys and IDA-hLys on Zn2+-mediated Aβ40

14

aggregation were analyzed by ThT assays (Figure 2). A fast increase of FI was

15

observed for Zn2+-Aβ40 aggregation during the first 2 h (Figure 2, Line 1), suggesting

16

that Zn2+ addition caused the rapid Aβ40 aggregation, consistent with previous

17

reports.42-45 Then, the ThT FI remained unchanged because Zn2+ stabilized the

18

non-fibrillar Aβ40 oligomers and formed Zn2+-Aβ40 aggregates.42,46 The inhibitory

19

effect of hLys on Zn2+-mediated Aβ40 aggregates was inappreciable (Figure 2, Lines

20

2-4); with increasing hLys concentration, the ThT fluorescence curves changed little

21

and almost overlapped with the curve of Zn2+-Aβ40 system (Figure 2, Line 1).

22

However, the addition of IDA-hLys changed Zn2+-mediated Aβ40 aggregation

23

remarkably (Figure 2, Lines 5-7). The ThT FI of Zn2+-Aβ40 aggregates decreased with

24

IDA-hLys concentration, confirming that the inhibitory effect of IDA-hLys increased

25

with its concentration. When its concentration reached 12.5 µM, the ThT FI was

26

reduced by 66%. The results indicate that IDA-hLys not only chelated Zn2+ but also

27

effectively inhibited Aβ40 aggregation. Therefore, it is considered that IDA coupling

28

onto hLys surface contributed to the happening of the strong inhibition effect on

29

Zn2+-mediated Aβ40 aggregation.

30

Furthermore, the morphology of Aβ40 aggregates was observed by atomic force 10


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microscope (AFM) to confirm the inhibitory effect of IDA-hLys (Figure 3). After

2

incubation for 72 h, Aβ40 fibril presented long, serried and entangled structure as

3

observed in previously published experiments (Figure 3A).25,47 By contrast, Aβ40

4

fibrils appeared in shorter and amorphous structures in the presence of the inhibitors

5

(Figure 3B,C, Figure S5A-D). Figure 3D shows that Zn2+ facilitated the generation of

6

non-fibrillar aggregates, in agreement with the literature data.44,46 The morphology of

7

Zn2+-Aβ40 aggregates with hLys also displayed similar non-fibrillar aggregates

8

(Figure 3E, Figure S5E,F, and Table 2) due to the weak interaction of hLys with Zn2+.

9

Differently, in the presence of IDA-hLys, Zn2+-mediated Aβ40 aggregates presented a

10

dramatic change. That is, bigger aggregates disappeared and smaller amorphous

11

aggregates were observed (Figure 3F, Figure S5G,H, and Table 2). The results proved

12

that IDA-hLys altered the morphology of Zn2+-Aβ40 aggregates by remarkably

13

changing the pathway of Zn2+ mediated Aβ40 aggregation.

14

From the results described in Figures 1 to 3, it can be concluded that IDA-hLys

15

efficiently inhibited both Aβ40 and Zn2+-mediated Aβ40 aggregation and greatly

16

disturbed the amyloidosis pathway.

17

IDA-hLys Alleviates the Cytotoxicities of Aβ40 and Zn2+-Aβ40 Aggregates. To

18

assess the effect of IDA-hLys on the cytotoxicities of Aβ40 and Zn2+-Aβ40 aggregates,

19

MTT and LDH release assays were performed with SH-SY5Y cells. As shown in

20

Figure S6, hLys and IDA-hLys presented no cytotoxicity to SH-SY5Y cells,

21

indicating that IDA-hLys was biocompatible. The addition of 25 µM Aβ40 aggregates

22

alone led to about 40% cell death (Figure 4A, Lane 2). However, the cell viability

23

with Aβ40 species gradually increased with hLys/IDA-hLys concentrations, implying

24

that both hLys and IDA-hLys relieved the cytotoxicity of Aβ40 fibrils effectively

25

(Figure 4A, Lanes 3-7). Compared to native hLys, IDA-hLys showed somewhat

26

weaker inhibitory effect on cytotoxicity of Aβ40 aggregates, similar with that in the

27

ThT assay (Figure 2). Moreover, in the LDH release assays, similar results were

28

obtained, as shown in Figure 4B.

29

Figures 4C and 4D show the influence of hLys and IDA-hLys on the cytotoxicity of

30

Zn2+-Aβ40 species. In the MTT assays, 12.5 µM Zn2+ presented little toxicity to the 11


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

cultured cells (Figure 4C, Lane 3). However, Aβ40 aggregates showed about 40% cell

2

death and Zn2+-Aβ40 aggregates led to about 53% cell death (Figure 4C, Lanes 2 and

3

4). This implies that Zn2+ greatly enhanced the toxicity of Aβ40 species, which was in

4

agreement with previous studies.45,46 When different hLys was co-incubated with

5

Zn2+-Aβ40 system, the cytotoxicities of Zn2+-Aβ40 species were not pronouncedly

6

mitigated (Figure 4C), indicating that hLys was ineffective on diminishing the effect

7

of Zn2+ on Aβ40 aggregation. However, in the presence of IDA-hLys, the cell viability

8

enhanced significantly. As compared with the Zn2+-Aβ40 system, 25 µM IDA-hLys

9

increased the cell viability by about 64% (Figure 4C, Lane 9, red). Similar inhibitory

10

effects of hLys and IDA-hLys on Zn2+-Aβ40 induced cytotoxicity were observed using

11

the LDH leakage assay (Figure 4D), indicating that IDA-hLys effectively attenuated

12

the cytotoxicity of Zn2+-Aβ40 species.

13

Compared with literature data, IDA-hLys had pronounced advantage over other

14

bifunctional agents on detoxification.48,49 For example, equimolar EGCG diminished

15

the cytotoxicity of metal-Aβ species towards murine Neuro-2a neuroblastoma cells

16

only about 15%;48 as compared to Zn2+-Aβ40 aggregates-treated group, naturally

17

occurring polyphenolic glycosides verbascoside and its esterified derivative increased

18

the cell viability only by 16% and 10%, respectively.49

19

Mechanistic Analysis. The above results showed that IDA-hLys could suppress the

20

aggregation and cytotoxicities of Aβ40 and Zn2+-Aβ40 species. In order to further

21

analyze the working mechanism of IDA-hLys, we used isothermal titration

22

calorimetry (ITC) to investigate the binding properties of hLys/IDA-hLys with Zn2+

23

and Aβ40. The titration results of Zn2+ to hLys and IDA-hLys are shown in Figure S7

24

and Table 3. As shown in Figure S7A, calorimetric titration of Zn2+ to hLys revealed

25

that the enthalpy variable values were randomly distributed and the data could not be

26

fitted to a model. The upper limit of dissociation constant (Kd) value that can be

27

determined by the VP-ITC is in the magnitude of mmol/L.50 Thus, the Kd value of

28

hLys for Zn2+ should be about or greater than a few mmol/L, indicating the

29

inappreciable affinity of hLys for Zn2+. By contrast, as shown in Figure S7B and Table

30

3, the Kd of Zn2+ with IDA-hLys was determined to be 3.6 µM. It has been previously 12


Page 12 of 34

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Langmuir

1

determined that the Kd between Aβ40 and Zn2+ was 65 µM.51 Therefore, IDA-hLys has

2

stronger affinity for Zn2+ than Aβ40 does. It means that IDA-hLys could remarkably

3

inhibit Zn2+ binding to Aβ40 and remove Zn2+ from Aβ40 aggregates, leading to the

4

inhibitory effect on Zn2+-Aβ40 aggregation and cytotoxicity.

5

Furthermore, the titration of hLys/IDA-hLys to Aβ40 were performed (Figure S8).

6

Several concentrations of hLys were used to titrated 20 µM Aβ40, and the results could

7

not be fitted to a model (Figure S8A-C), indicating that the binding affinity between

8

hLys and Aβ40 could not be detected by the VP-ITC. IDA-hLys displayed a similar

9

result with hLys (Figure S8D), indicating that IDA modification had inappreciable

10

effect on the binding affinity between hLys and Aβ40. Although the binding affinity

11

could not be detected, it does not mean that there are no interactions between

12

hLys/IDA-hLys and Aβ40. Some complications in the titration process, such as the

13

self-aggregation of Aβ40, and the influence of hLys/IDA-hLys at low concentrations in

14

the beginning of the titration, which favored Aβ40 nucleation (Figure 1 and Table 1),

15

might influence the titration results. The interaction will be discussed more

16

specifically below in the mechanistic model.

17

Moreover, stopped-flow fluorescent measurements were used to study the rapid

18

kinetics of Zn2+-mediated Aβ40 aggregation. We monitored the Rayleigh light

19

scattering intensity as direct internal probes to measure the changes in Aβ40

20

aggregation states on second timescales.42 As demonstrated in Figure S9A, Aβ40 alone

21

remained the unchanged over the time range. By contrast, Zn2+ induced noticeable

22

Aβ40 aggregation during the first milliseconds to seconds, and the aggregation rate

23

increased with Zn2+ concentrations, which is in agreement with literatures.43,52 As

24

shown in Figure S9B, the scattering signals of Aβ40 mixed with hLys/IDA-hLys

25

increased slowly in a similar pattern. It indicates that Aβ40 binds to hLys/IDA-hLys

26

rapidly. The binding would be the cause of the remarkable inhibitory effect of

27

hLys/IDA-hLys on Aβ40 self-aggregation discussed above (Figure 1), although the

28

binding affinity could not be detected by the VP-ITC (Figure S8). For the Zn2+ plus

29

hLys/IDA-hLys systems, it is seen that the samples comprising different

30

concentrations of Zn2+ mixed with hLys/IDA-hLys (10 µM) showed inappreciable 13


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

change in the scattering signal (Figure S9C). This suggests that the interaction

2

between Zn2+ and hLys/IDA-hLys induced negligible changes in size. However, when

3

5 µM Aβ40 monomer solution was added to the mixture of hLys/IDA-hLys (10 µM)

4

and Zn2+ (20 µM), pronounced increases in signals were observed (Figure 5A), and

5

the changes in aggregate size were much more remarkable than those of the samples

6

of Aβ40 mixed with either Zn2+ or hLys/IDA-hLys (Figure S9A,B). This indicates that

7

ternary complexes of large aggregates were formed in seconds. Compared with native

8

hLys, IDA-hLys effectively decreased the aggregate size of the ternary complexes

9

(Figure 5A and Figure S10). The higher affinity of IDA-hLys for Zn2+ would be the

10

cause of this phenomenon.

11

Besides, the effect of Zn2+ on the fast kinetics of hLys/IDA-hLys-mediated Aβ40

12

aggregation was investigated to provide further insight into the inhibition effect of

13

IDA-hLys (Figure 5B). It is seen that 20 µM Zn2+ did not give rise to significant

14

change in the scatting signals of 10 µM hLys/IDA-hLys-mediated 5 µM Aβ40

15

aggregates within 100 s, indicating that 20 µM Zn2+ had inappreciable effect on the

16

hLys/IDA-hLys-Aβ40 species. It suggests that the binding of hLys/IDA-hLys with

17

Aβ40 blocked the binding sites of Zn2+ and Aβ40 and inhibited the effect of Zn2+ on

18

Aβ40 aggregation. However, this effect of hLys was limited. As demonstrated in

19

Figure 5B, when Zn2+ concentration was increased to 50 µM, the scattering signal of

20

hLys-mediated Aβ40 aggregates increased obviously. By contrast, 50 µM Zn2+ did not

21

affect the scattering signal of IDA-hLys-mediated Aβ40 aggregates. This indicates that

22

IDA-hLys effectively protected Aβ40 from Zn2+-mediated aggregation for its stronger

23

affinity for Zn2+.

24

Furthermore, the effect of hLys/IDA-hLys on Zn2+-Aβ40 aggregates was

25

investigated by fast kinetics (Figure 5C). Firstly, we incubated 5 µM Aβ40 with

26

different concentrations of Zn2+, and 2 min later mixed with 10 µM hLys/IDA-hLys in

27

the stopped-flow apparatus. As demonstrated in Figure 5C, the starting points of Lines

28

1, 3 and 5 were different from those of Lines 2, 4 and 6 in Figure 5C. It is considered

29

due to the difference in aggregate sizes caused by different Zn2+ concentrations (20

30

and 50 µM). Namely, the size of 50 µM Zn2+-Aβ40 aggregates was larger than that of 14


Page 14 of 34

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Langmuir

1

20 µM Zn2+-Aβ40 aggregates. By addition of 10 µM hLys, the signals of Zn2+-Aβ40

2

system kept the same as that of control (addition of buffer A) due to the weak

3

interactions between hLys and Zn2+. However, addition of 10 µM IDA-hLys gave rise

4

to a dramatic decrease of the signal of Zn2+-Aβ40 aggregates with either 20 or 50 µM

5

Zn2+ (Figure 5C). This indicates that IDA-hLys could sequester Zn2+ from Zn2+-Aβ40

6

aggregates and effectively depolymerize the aggregates.

7

To sum up, we proposed a mechanistic model to interpret the effects of IDA-hLys

8

on Zn2+-mediated Aβ40 aggregation (Figure 6). Aβ40 monomers self-associate into

9

oligomers and fibrils through β-sheet-rich structures (Figure 6A), while Zn2+ rapidly

10

causes the generation of Zn2+-Aβ40 aggregates of higher neurotoxicity (Figure 6B). It

11

has been shown that hLys inhibited the fibrillation and cytotoxicity of Aβ40 through

12

binding Aβ40 in seconds (Figure 6C). For better understanding of the effect of hLys on

13

Aβ40 aggregation, the structures of Aβ40 and hLys are analyzed. As shown in Figure

14

S11A, hLys surface is distributed by positive-potential areas and hydrophobic patches

15

at pH 7.4.53 Under the same condition, the N-terminus of Aβ40 carries negative

16

charges, and its hydrophobic parts include the central hydrophobic core and the

17

C-terminus hydrophobic residues (Figure S11B).54 Molecular dynamics simulations

18

have indicated that hLys stabilizes the N-terminus of Aβ40 by electrostatic interactions

19

and interacts with the C-terminus of Aβ40 via hydrophobic surface.25 Moreover, the

20

binding of Aβ40 blocks the substrate binding site of lysozyme, which locates at

21

Arg62-Trp64 and Asp102-Arg107 (plus Ser24).25 So, electrostatic and hydrophobic

22

interactions may be the key for hLys to prevent Aβ40 aggregation. However, hLys has

23

low binding affinity for Zn2+. Therefore, hLys has inappreciable inhibitory effect on

24

Zn2+-mediated Aβ40 aggregation and cytotoxicity (Figure 6D). In the absence of Zn2+,

25

IDA-hLys had a similar inhibitory effect on Aβ40 aggregation by altering the pathway

26

of fibrillation (Figure 6E). In addition, in the presence of Zn2+, IDA-hLys could

27

sequester Zn2+ from Zn2+-Aβ40 aggregates and effectively alter the pathway of

28

Zn2+-mediated Aβ40 aggregation for its strong affinity for Zn2+. Moreover, the binding

29

of Aβ40 with IDA-hLys could block the binding sites of Zn2+ and Aβ40 and attenuated

30

the effect of Zn2+ on Aβ40 aggregation and cytotoxicity (Figure 6F). Thus, IDA-hLys 15


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1

works as a bifunctional agent in inhibiting Zn2+-mediated Aβ40 aggregation and

2

cytotoxicity. The finding of the bifunctional feature of IDA-hLys would benefit in the

3

design of more potent bifunctional or even multifunctional agents via conjugation of

4

more functional groups (e.g., metal chelators and inhibitors) onto a protein surface.

5 6

CONCLUSIONS

7

In this work, we have developed IDA-modified human lysozyme as a bifunctional

8

agent to inhibit Aβ40 fibrillization and modulate Zn2+-associated Aβ40 aggregation.

9

Although slightly weaker than hLys, IDA-hLys still has noticeable inhibitory effect on

10

Aβ40 self-aggregation by altering the pathway of Aβ40 fibrillation. Moreover,

11

IDA-hLys shows prominent inhibitory effect on Zn2+-mediated Aβ40 aggregation and

12

cytotoxicity due to its strong binding affinity for Zn2+. In addition, it can protect Aβ40

13

against Zn2+-induced aggregation and depolymerize Zn2+-Aβ40 aggregates in seconds.

14

Therefore, IDA-hLys works as a bifunctional agent for inhibiting Aβ40 fibrillization

15

and mitigating Zn2+-mediated Aβ40 aggregation and cytotoxicity. The findings

16

provided new insights into the design of bifunctional agents against metal-mediated

17

Aβ aggregation.

18 19

ASSOCIATED CONTENT

20

Supporting Information

21

Mass spectra, zeta potentials, size distributions, far-UV circular dichroism spectra and

22

fluorescence intensities of hLys and IDA-hLys, AFM measurements of the Aβ40

23

species, cytotoxicity assays of hLys and IDA-hLys, calorimetric titration assays,

24

kinetic traces by stopped-flow fluorescence experiments and surface models of hLys

25

and Aβ40. This material is available free of charge via the Internet at

26

http://pubs.acs.org.

27 28

AUTHOR IMFORMATION

29

Corresponding Author

30

*Tel: +86 22 27403389; Fax: +86 22 27403389; E-mail address: [email protected] (Y. 16


Page 16 of 34

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Langmuir

1

Sun).

2

ORCID

3

Xiaoyan Dong: 0000-0002-8040-5897

4

Yan Sun: 0000-0001-5256-9571

5

Author Contributions

6

Y.S designed the research; X.L. performed the experiments; X.L. and B.X. analyzed

7

the data; X.L., B.X., X.D., and Y.S wrote or contributed to the writing of the

8

manuscript.

9

Notes

10

The authors declare no competing financial interest.

11 12

ACKNOWLEDGMENTS

13

This work was funded by the National Natural Science Foundation of China (Nos.

14

91634119 and 21621004) and the Natural Science Foundation of Tianjin from Tianjin

15

Municipal Science and Technology Commission (Contract No.16JCZDJC32300).

16 17 18 19 20 21 22 23

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(40) Wood, S. J.; Maleeff, B.; Hart, T.; Wetzel, R. Physical, morphological and

23

functional differences between pH 5.8 and 7.4 aggregates of the Alzheimer's amyloid

24

peptide A β. J. Mol. Biol. 1996, 256, 870-877.

25

(41) Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Dawson, K. A.; Linse, S.

26

Dual effect of amino modified polystyrene nanoparticles on amyloid β protein

27

fibrillation. ACS Chem. Neurosci. 2010, 1, 279-287.

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(42) Noy, D.; Solomonov, I.; Sinkevich, O.; Arad, T.; Kjaer, K.; Sagi, I.

29

Zinc-amyloid β interactions on a millisecond time-scale stabilize non-fibrillar

30

Alzheimer-related species. J. Am. Chem. Soc. 2008, 130, 1376-1383. 21


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(43) Guo, J.; Yu, L.; Sun, Y.; Dong, X. Kinetic Insights into Zn2+-Induced Amyloid

2

β-Protein Aggregation Revealed by Stopped-Flow Fluorescence Spectroscopy. J. Phys.

3

Chem. B 2017, 121, 3909-3917.

4

(44) Chen, W. T.; Liao, Y. H.; Yu, H. M.; Cheng, I. H.; Chen, Y. R. Distinct Effects

5

of Zn2+, Cu2+, Fe3+, and Al3+ on Amyloid-β Stability, Oligomerization, and

6

Aggregation

7

PROTOFIBRIL FORMATION. J. Biol. Chem. 2011, 286, 9646-9656.

AMYLOID-β

DESTABILIZATION

PROMOTES

ANNULAR

8

(45) Du, X.; Li, H.; Wang, Z.; Qiu, S.; Liu, Q.; Ni, J. Selenoprotein P and

9

selenoprotein M block Zn2+-mediated Aβ42 aggregation and toxicity. Metallomics

10

2013, 5, 861-870.

11

(46) Solomonov, I.; Korkotian, E.; Born, B.; Feldman, Y.; Bitler, A.; Rahimi, F.; Li,

12

H.; Bitan, G.; Sagi, I. Zn2+-Aβ40 complexes form metastable quasi-spherical

13

oligomers that are cytotoxic to cultured hippocampal neurons. J. Biol. Chem. 2012,

14

287, 20555-20564.

15

(47) Liu, R.; Barkhordarian, H.; Emadi, S.; Park, C. B.; Sierks, M. R. Trehalose

16

differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42.

17

Neurobiol. Dis. 2005, 20, 74-81.

18

(48) Hyung, S. J.; DeToma, A. S.; Brender, J. R.; Lee, S.; Vivekanandan, S.; Kochi,

19

A.; Choi, J. S.; Ramamoorthy, A.; Ruotolo, B. T.; Lim, M. H. Insights into

20

antiamyloidogenic properties of the green tea extract (-)-epigallocatechin-3-gallate

21

toward metal-associated amyloid-β species. Proc. Natl. Acad. Sci. U. S. A. 2013, 110,

22

3743-3748.

23

(49) Korshavn, K. J.; Jang, M.; Kwak, Y. J.; Kochi, A.; Vertuani, S.; Bhunia, A.;

24

Manfredini, S.; Ramamoorthy, A.; Lim, M. H. Reactivity of metal-free and

25

metal-associated amyloid-β with glycosylated polyphenols and their esterified

26

derivatives. Sci. Rep. 2015, 5, 17842.

27

(50) Li, M.; Dong, X.; Liu, Y.; Sun, Y. Brazilin Inhibits Prostatic Acidic

28

Phosphatase Fibrillogenesis and Decreases its Cytotoxicity. Chem.-Asian. J. 2017, 12,

29

1062-1068.

30

(51) Tougu, V.; Karafin, A.; Palumaa, P. Binding of zinc (II) and copper (II) to the 22


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1

full‐length Alzheimer’s amyloid-β peptide. J. Neurochem. 2008, 104, 1249-1259.

2

(52) Xie, B.; Dong, X.; Wang, Y.; Sun, Y. Multifunctionality of Acidulated Serum

3

Albumin on Inhibiting Zn2+-Mediated Amyloid β-Protein Fibrillogenesis and

4

Cytotoxicity. Langmuir 2015, 31, 7374-7380.

5

(53) Durek, T.; Torbeev, V. Y.; Kent, S. B. Convergent chemical synthesis and

6

high-resolution X-ray structure of human lysozyme. Proc. Natl. Acad. Sci. U. S. A.

7

2007, 104, 4846-4851.

8 9

(54) Serpell, L. C. Alzheimer’s amyloid fibrils: structure and assembly. BBA-Mol. Basis Dis. 2000, 1502, 16-30.

10

23


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Page 24 of 34

1

Table 1. Lag Times for Amyloid Formation Kinetics of Aβ40 (25 µM) at Different

2

Conditions. Inhibitor

Lag time (h) Aβ

Aβ:Inhibitor=1:0.1

Aβ:Inhibitor=1:0.25

None

16.3 ± 0.7

-

-

hLys

-

14.4 ± 1.1

44.9 ± 0.7

IDA-hLys

-

8.8 ± 0.8

14.8 ± 0.6

3 4

Table 2. The Average Sizes of Zn2+-Aβ40 Aggregates in the Absence and Presence

5

of Inhibitors. Inhibitor

Size (nm) Zn2+-Aβ

1.25 µM Inhibitor

6.25 µM Inhibitor

12.5 µM Inhibitor

None

294 ± 35

-

-

-

hLys

-

290 ± 24

286 ± 31

287 ± 20

IDA-hLys

-

242 ± 22

218 ± 15

213 ±26

6 7

Table 3. Thermodynamic Parameters and Dissociation Constant for Zn2+

8

Binding to IDA-hLys. Thermodynamic

N

constants Value

1.0 ± 0.1

Kd

∆G

∆H

T∆S

(µM)

(kcal/mol)

(kcal/mol)

(kcal/mol)

3.6 ± 0.2

-7.7 ± 0.3

-3.2 ± 0.2

4.5 ± 0.1

9

24


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1

Scheme 1. Reaction Scheme for the Modification of hLys with IDA.

2 3

25


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1 2

Figure 1. (A) Kinetics of the inhibition of Aβ40 self-aggregation by ThT fluorescence

3

assays. (B) The final ThT FI of samples in (A) after incubated for 72 h when the FI

4

arrive the plateau. Lines or Lanes denote the following conditions: Line/Lane 1, Aβ40

5

alone; Line/Lane 2, Aβ40 + 2.5 µM hLys; Line/Lane 3, Aβ40 + 6.25 µM hLys;

6

Line/Lane 4, Aβ40 + 12.5 µM hLys; Line/Lane 5, Aβ40 + 2.5 µM IDA-hLys; Line/Lane

7

6, Aβ40 + 6.25 µM IDA-hLys; Line/Lane 7, Aβ40 + 12.5 µM IDA-hLys. The final

8

concentration of Aβ40 was 25 µM. All measurements were conducted in buffer A at

9

37 °C. ***, p < 0.001, **, p < 0.01 as compared to lane 1. The values of p < 0.05 for

10

the pairs of data sets are marked with #.

11

26


Page 26 of 34

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Figure 2. ThT fluorescence kinetic assays for the inhibition of Zn2+-Aβ40 aggregation.

3

Lines denote the following conditions: Line 1, Aβ40 + Zn2+; Line 2, Aβ40 + Zn2+ + 2.5

4

µM hLys; Line 3, Aβ40 + Zn2+ + 6.25 µM hLys; Line 4, Aβ40 + Zn2+ + 12.5 µM hLys;

5

Line 5, Aβ40 + Zn2+ + 2.5 µM IDA-hLys; Line 6, Aβ40 + Zn2+ + 6.25 µM IDA-hLys;

6

Line 7, Aβ40 + Zn2+ + 12.5 µM IDA-hLys. The final concentrations of Aβ40 and Zn2+

7

were 25 µM and 12.5 µM, respectively. All experiments were in buffer A at 37 °C.

8

27


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1 2

Figure 3. AFM measurements of the (A) Aβ40, (B) Aβ40 + hLys, (C) Aβ40 + IDA-hLys,

3

(D) Aβ40 + Zn2+, (E) Aβ40 + Zn2+ + hLys, and (F) Aβ40 + Zn2+ + IDA-hLys after 72 h

4

of incubation at 37 °C. The final concentration of Aβ40 was 25 µM, and that of Zn2+,

5

hLys and IDA-hLys were all 12.5 µM.

6

28


Page 28 of 34

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Figure 4. Cytotoxicity of SH-SY5Y cells incubated with Aβ40 aggregates using MTT

3

assay and LDH leakage assay. The samples obtained by pre-incubation at different

4

conditions: (A) Lane 1, buffer A; Lane 2, Aβ40 aggregates; Lane 3, 2.5 µM

5

hLys/IDA-hLys + Aβ40; Lane 4, 6.25 µM hLys/IDA-hLys + Aβ40; Lane 5, 12.5 µM

6

hLys/IDA-hLys + Aβ40; Lane 6, 17.5 µM hLys/IDA-hLys + Aβ40; Lane 7, 25 µM

7

hLys/IDA-hLys + Aβ40. (B) Lane 1, 1% (v/v) Triton X-100 in FBS-free medium; Lane

8

2, buffer A; Lane 3, Aβ40 aggregates; Lane 4, 2.5 µM hLys/IDA-hLys + Aβ40; Lane 5,

9

6.25 µM hLys/IDA-hLys + Aβ40; Lane 6, 12.5 µM hLys/IDA-hLys + Aβ40; Lane 7,

10

17.5 µM hLys/IDA-hLys + Aβ40; Lane 8, 25 µM hLys/IDA-hLys + Aβ40. (C) Lane 1,

11

buffer A; Lane 2, Aβ40 aggregates; Lane 3, Zn2+; Lane 4, Zn2+-Aβ40 aggregates; Lane

12

5, 2.5 µM hLys/IDA-hLys-mediated Zn2+-Aβ40 aggregates; Lane 6, 6.25 µM

13

hLys/IDA-hLys-mediated

Zn2+-Aβ40

aggregates;

Lane

7,

12.5

µM

14


Zn2+-Aβ40

aggregates;

Lane

8,

17.5

µM

15


Zn2+-Aβ40

aggregates;

25

µM

16

hLys/IDA-hLys-mediated Zn2+-Aβ40 aggregates. (D) Lane 1, 1% (v/v) Triton X-100 in

17

FBS-free medium; Lane 2, buffer A; Lane 3, Aβ40 aggregates; Lane 4, Zn2+; Lane 5,

18

Zn2+-Aβ40 aggregates; Lane 6, 2.5 µM hLys/IDA-hLys-mediated Zn2+-Aβ40 29


Lane

9,

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Page 30 of 34

1

aggregates; Lane 7, 6.25 µM hLys/IDA-hLys-mediated Zn2+-Aβ40 aggregates; Lane 8,

2

12.5 µM hLys/IDA-hLys-mediated Zn2+-Aβ40 aggregates; Lane 9, 17.5 µM

3


4

hLys/IDA-hLys-mediated Zn2+-Aβ40 aggregates. The concentrations of aged-Aβ40 and

5

Zn2+ were 25 µM and 12.5 µM, respectively. ###, p < 0.001 as compared to lane 1 (A,

6

C) or lane 2 (B, D). ***, p < 0.001, **, p < 0.01, and *, p < 0.05 as compared to lane

7

2 (A) or lane 3 (B) or lane 4 (C) or lane 5 (D). The values of p < 0.001 and p < 0.01

8

for the pairs of data sets are marked with +++ and ++, respectively.

Zn2+-Aβ40

aggregates;

9

30


Lane

10,

25

µM

Page 31 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2

Figure 5. Kinetic traces followed by stopped-flow fluorescence of (A) Aβ40 monomer

3

solution mixed with (Line 1) the mixture of 20 µM Zn2+ and hLys or (Line 2) the

4

mixture of 20 µM Zn2+ and IDA-hLys, (B) buffer A mixed with (Line 1) the mixture

5

of Aβ40 and hLys or (Line 2) the mixture of Aβ40 and IDA-hLys, 20 µM Zn2+ solution

6

mixed with (Line 3) the mixture of Aβ40 and hLys or (Line 4) the mixture of Aβ40 and

7

IDA-hLys, or 50 µM Zn2+ solution mixed with (Line 5) the mixture of Aβ40 and hLys

8

or (Line 6) the mixture of Aβ40 and IDA-hLys, (C) buffer A mixed with (Line 1) the 31


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1

mixture of Aβ40 and 20 µM Zn2+ or (Line 2) the mixture of Aβ40 and 50 µM Zn2+,

2

hLys solution mixed with (Line 3) the mixture of Aβ40 and 20 µM Zn2+ or (Line 4) the

3

mixture of Aβ40 and 50 µM Zn2+, or IDA-hLys solution mixed with (Line 5) the

4

mixture of Aβ40 and 20 µM Zn2+ or (Line 6) the mixture of Aβ40 and 50 µM Zn2+. The

5

final concentrations of Aβ40 and IDA-hLys were 5 µM and 10 µM, respectively.

6

32


Page 32 of 34

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1 2

Figure 6. Schematic representations of the bifunctional effects of IDA-hLys on

3

Zn2+-mediated Aβ40 aggregation and cytotoxicity. (A) On-pathway aggregation: Aβ40

4

monomers convert into β-sheet conformations, then assemble into oligomers and

5

fibrils of high neurotoxicity. (B) Zn2+-mediated Aβ40 aggregation: Zn2+ rapidly

6

induces the formation of Zn2+-Aβ40 aggregates with even higher neurotoxicity. (C)

7

Aβ40 aggregation in the presence of hLys: hLys inhibits the fibrillation and

8

cytotoxicity of Aβ40 through binding Aβ40 in seconds. (D) Zn2+-mediated Aβ40

9

aggregation in the presence of hLys: hLys has no influence on Zn2+-mediated Aβ40

10

aggregation. (E) Aβ40 aggregation in the presence of IDA-hLys: IDA-hLys has

11

slightly weaker inhibitory effect on Aβ40 aggregation than hLys due to decreased

12

positive surface charges. (F) Zn2+-mediated Aβ40 aggregation in the presence of

13

IDA-hLys: IDA-hLys can sequester Zn2+ from Zn2+-Aβ40 aggregates and protect Aβ40

14

from Zn2+-mediated aggregation, resulting in inhibiting Zn2+-Aβ40 aggregation and

15

cytotoxicity. The number of skulls represents the toxicity level. 33


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1 2

Table of Contents Graphic

34


Page 34 of 34

Bifunctionality of Iminodiacetic Acid-Modified Lysozyme on Inhibiting

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