Modification of Serum Albumin by High Conversion of Carboxyl to

Apr 9, 2019 - Cell viability assays also confirmed that the detoxification of 5 μM BSA-B4 was superior over 25 μM native BSA by increasing cell viab...
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Modification of Serum Albumin by High Conversion of Carboxyl to Amino Groups Creates a Potent Inhibitor of Amyloid β‑Protein Fibrillogenesis Wenjuan Wang, Xiaoyan Dong, and Yan Sun* Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

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S Supporting Information *

ABSTRACT: Fibrillogenesis of amyloid β-protein (Aβ) has been thought to be implicated in the progression of Alzheimer’s disease (AD). Therefore, development of highefficiency inhibitors is one of the strategies for the prevention and treatment of AD. Serum albumin has been found to capture Aβ monomers through its hydrophobic groove and suppress amyloid formation, but the inhibition efficiency is limited. Inspired by the strong inhibition potency of a basic protein, human lysozyme, we have herein proposed to develop a basified serum albumin by converting carboxyl groups into amino groups with ethylenediamine conjugated on the protein surface. The idea was verified with both bovine and human serum albumins (BSA/HSA). Four basified BSA (BSA-B) preparations with amino modification degrees (MDs) from 8.0 to 41.5 were first synthesized. Extensive biophysical and biological analyses revealed that the inhibition potency significantly increased with increasing amino MD. BSA-B of the highest MD (41.5), BSA-B4, which had an isoelectric point of 9.7, presented strong inhibition on Aβ42 fibrillation at a concentration as low as 0.5 μM, at which it functioned similarly with 25 μM native BSA to impede 25 μM Aβ fibrillation. Cell viability assays also confirmed that the detoxification of 5 μM BSA-B4 was superior over 25 μM native BSA by increasing cell viability from 60.6% to 96.0%. Fluorescence quenching study unveiled the decrease of the binding affinity between Aβ42 and the hydrophobic pocket region of BSA-B4, while quartz crystal microbalance experiments demonstrated that the binding constant of BSA-B4 to Aβ42 increased nearly 5 times. Therefore, the increase of electrostatic interactions between BSA-B4 and Aβ42 was the main reason for its high potency. Hence, aminated BSA achieved a conversion of binding way to Aβ from a mainly single-site hydrophobic binding to multiregional electrostatic interactions. Similar results were obtained with basified HSA preparations on inhibiting the amyloid formation and cytotoxicity. This work has thus provided new insights into the development of more efficient protein-based inhibitors against Aβ fibrillogenesis.

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INTRODUCTION

is one of pivotal ways to reduce the risk of AD onset and progression. Considerable efforts and progress have been made to design and develop various kinds of inhibitors against on-pathway aggregation of Aβ.8−12 Among these inhibitors, peptide-/ protein-based agents have been given close attention for their high biocompatibility. Human serum albumin (HSA) is the most abundant plasma protein in human blood (400−600 μM)13 and has been found to directly bind Aβ monomers or small oligomers at a 1:1 stoichiometry mainly through its hydrophobic groove.14,15 The dissociation constant of HSA-Aβ is within the range of 5−10 μM.16,17 More than 90% of Aβ species in blood plasma is found to be bound to HSA, and serum albumin levels are directly associated with cognitive impairment, as AD patients exhibit decreased serum albumin levels.18 Thus, serum albumin is expected to function as a promising agent for inhibiting Aβ aggregation and providing

Protein misfolding and deposition in human tissues are pathologically associated with various lethal diseases,1,2 including Alzheimer’s disease (AD), which is recognized as the most common neurodegenerative disease.3 Pathologically, one of the hallmarks of AD is the accumulation of misfolded amyloid β-protein (Aβ) aggregates in the brain. Aβ with 39− 43 amino acid residues is found in human cerebrospinal fluid (CSF) and normally produced as a cleavage product of amyloid β precursor protein.4 The 40 amino acid long amyloidogenic peptide (Aβ40) is the most abundant form, while 42 amino acid long analogue (Aβ42) is more aggregationprone and neurotoxic.5 Aβ species are hydrophobic, net negative, and intrinsically soluble disordered peptides that can form diverse fibrillary aggregates rich in β-sheet structures including oligomers, protofibrils, and mature fibrils.6 Aβ oligomers and protofibrils are generally considered to be the key contributors to neuron dysfunction and death.7 Hence, the development of antiaggregation agents to regulate Aβ toxicity © XXXX American Chemical Society

Received: March 20, 2019 Published: April 9, 2019 A

DOI: 10.1021/acs.bioconjchem.9b00209 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry protection from the progression of AD.19,20 Therefore, plasma exchange with HSA has been clinically applied to AD patients to modify Aβ concentrations in plasma and CSF.21 However, HSA concentration in the CSF (1−5 μM) is much lower than in plasma and the ability of HSA in CSF to sequestrate free Aβ is quite limited, which might explain why Aβ plaques are deposited in brain rather than in peripheral tissues.22 Recently, Xie et al. designed acidulated bovine serum albumin (A-BSA) as well as acidulated HSA (A-HSA).23,24 The acidification of serum albumin elevated the inhibitory effects of albumin on Aβ aggregation; it reduced the thioflavin T (ThT) intensity of Aβ that reflects the content of β-sheet by about 47% at equimolar A-BSA/A-HSA: Aβ peptides ratios, while native albumin, only reduced by about 25%. The research revealed that the enhanced inhibition effect of A-HSA/A-BSA was due to the hydrophobic binding−electrostatic repulsion (HyBER) effect of the acidulated albumin on the bound Aβ42. Though the inhibitory effect of A-HSA/BSA has been increased by nearly twice, they were still weaker than native human lysozyme (hLys), which has been found to colocalize with Aβ plaques in AD patients25 and could completely inhibit the aggregation of Aβ peptides at a 1:1 ratio.26,27 The possible explanation could be that hLys is a native basic protein with isoelectric point (pI) of about 10.2,28 while Aβ is an acidic peptide (pI, 5.5) .29 So hLys could not only interact with the C-terminus of Aβ peptide via the hydrophobic pocket but also stabilize the Aβ peptide by the widespread attractive electrostatic interactions due to the locally concentrated arginine and lysine residues on its surface.26,30 Lysozyme presents strong inhibition on Aβ peptide, emphasizing the importance of the electrostatics of a protein inhibitor, but its concentration in plasma and CSF is much lower than serum albumin. Inspired by the above properties of albumin and lysozyme, we have herein proposed to develop a new improved proteinbased Aβ inhibitor by coupling ethylenediamine onto albumin surface to convert carboxyl groups into amino groups, leading to the production of amino-modified serum albumin. Because BSA and HSA have similar structures and high sequence identities (76%)31 and FDA has approved BSA for drug formulation and delivery due to its nonimmunogenicity, richness, low cost, and its wide acceptance in the biopharmaceutical applications,32−34 we first prepared basified BSA (BSA-B) to start the research and then the results were demonstrated with basified HSA (HSA-B). The aminated or basified albumin has albumin-like structures but contains more positive charges on the surface. Extensive biophysical and biological assays confirmed the inhibitory effect of BSA-B/ HSA-B on Aβ42 aggregation and cytotoxicity. The inhibition mechanism was explored with BSA-B to advance the design of inhibitors against Aβ fibrillogenesis.

determined and listed in Table 1. The highest MD value achieved from the modifications was 41.5. From the structure Table 1. Comparison of Physicochemical Properties of BSA and BSA-Bs sample

MD

number of amino groups

BSA BSA-B1 BSA-B2 BSA-B3 BSA-B4

8.0 11.7 17.0 41.5

60 68.0 71.7 77.7 101.5

a

number of carboxyl groups 100 92 88.3 83 58.5

Dhb (nm)

pIc

± ± ± ± ±

4.6 5.2 5.9 8.3 9.7

9.15 9.91 9.79 9.47 9.18

0.19 0.66 0.19 1.36 0.35

a

Modification degree (MD) was determined by mass spectrometry. Hydrodynamic diameter (Dh) was determined in the PBS (pH 7.4) by dynamic light scattering method. cIsoelectric point (pI) was determined in deionized water by measuring the ζ potential change as a function of pH. b

of BSA,31 the amino and carboxyl groups of the BSA-B preparations were estimated and are also listed in Table 1. The structural properties of BSA-Bs were then investigated and compared with native BSA. The far-UV circular dichroism (CD) and fluorescence spectroscopic analyses (Figure S3) confirmed the consistency of the basified BSA preparations with native BSA in the secondary and tertiary conformations. In addition, the hydrodynamic sizes of BSA and BSA-Bs were quite comparable (Table 1), indicating that the BSA-Bs remained monodispersed without chemical cross-linking. Then, the ζ potential values of BSA and BSA-Bs as a function of pH in water were measured (Figure S4). It can be seen that the pI values of BSA-Bs increased from 4.6 of native BSA, and a pI of 9.7 was reached with BSA-B4 (Table 1). This indicates that the high conversion of carboxyl to amino groups has created a basic protein. Inhibition on Aβ42 Fibrillation. ThT could bind to βsheet structures existing in amyloid fibrils and profibrils and then exhibit enhanced fluorescence upon binding to amyloid fibril.35 Thus, a ThT-based fluorescence assay was employed to investigate the inhibitory effect of BSA-Bs on Aβ42 fibrillation. As shown in Figure 1, BSA and the four BSA-Bs impeded Aβ42

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RESULTS AND DISCUSSION Characteristics of BSA-Bs. BSA-B was prepared by coupling ethylenediamine onto the BSA surface, as illustrated in Figure S1 in Supporting Information. The modification degrees (MDs) of the four BSA-Bs were determined from their molecular weights measured by mass spectrometry. As shown in Figure S2, the molecular weights of the four BSA-B preparations increased by 336, 494, 717, and 1747, respectively, as compared with native BSA. Because coupling one ethylenediamine molecule increases the molecular weight by 42.1 Da, the average MDs of the BSA-B preparations were

Figure 1. Relative ThT fluorescence intensity of Aβ42 aggregated with varying BSA or BSA-Bs concentrations (0−25 μM) after 48 h coincubation at 37 °C. Aβ42 concentration was 25 μM. The fluorescence of Aβ42 after incubation for 48 h was set to 100%. Error bars correspond to standard deviations (n = 3): (###) p < 0.001 as compared to the Aβ42-treated group. Values of p < 0.001 for the pairs of data sets are marked with ∗∗∗. B

DOI: 10.1021/acs.bioconjchem.9b00209 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 2. AFM images of 25 μM Aβ42 incubated without or with different concentrations (0.1−25 μM) of BSA or BSA-B3/4 for 72 h. Incubations were performed in PBS (pH 7.4) at 37 °C.

Figure 3. Detoxification effects of BSA/BSA-Bs on Aβ42 toxicity to SH-SY5Y cells determined by (a) MTT assay and (b) LDH leakage assay. The cell viability treated with PBS buffer only was set to 100% in the MTT assay. In the LDH leakage assay, the cells treated with 1% (v/v) Triton X100 in FBS-free medium were set to be 100% cytotoxicity. Aβ42 concentration was 25 μM. Error bars correspond to standard deviations (n = 6): (#) p < 0.05, (##) p < 0.01, and (###) p < 0.001 as compared to the Aβ42-treated group. Values of p < 0.01 and p < 0.001 for the pairs of data sets are marked with ∗∗ and ∗∗∗, respectively.

fibrillation in a dose-dependent manner and exhibited roughly the same inhibition ability at equimolar ratio. Within the experimental concentration range, BSA-B1, BSA-B2, and BSA presented comparable inhibitory effects on Aβ42 fibrillation, indicating the binding force between Aβ42 and two low-MD BSA-Bs of which the pI values were less than 7 was still dominated by hydrophobic interactions like native BSA.15 By contrast, the two BSA-Bs of higher MDs, BSA-B3 and -B4, showed stronger inhibitory effects on Aβ42 fibrillation. Particularly, BSA-B4 presented high inhibitory potency at a low concentration, as evidenced by 55% decrease in ThT fluorescence intensity of Aβ42 at 0.5 μM, while native BSA just showed inappreciable effect at the same concentration. Four HSA-Bs in the similar amino MD range were prepared by the same procedure, and the properties of the HSA-Bs are listed in Table S1. Because HSA and BSA have a high degree of

structure and property similarities (Table S2), the four HSA-Bs showed similar inhibition potencies with the BSA-Bs (Figure S5). The results indicate that a potent inhibitor of Aβ fibrillation could be prepared by conjugating as many as possible positively charged groups on the surface of BSA/HSA. It might be due to the enhanced electrostatic attractions between the high-MD BSA-B/HSA-B and Aβ that carries negative charges at pH 7.4.29 Atomic force microscope (AFM) observation was then performed to investigate the inhibition effect of BSA-Bs on Aβ42 fibrillation (Figure 2). Pure Aβ42 (25 μM) formed typical elongated and tangled fibrils after 72 h incubation, consistent with literature results.36 Co-incubations of Aβ42 with BSA, BSA-B1, and BSA-B2 resulted in similar changes of the fibrils within inhibitor concentrations (Figure S6), indicating that BSA-B1, BSA-B2, and BSA exhibited similar inhibition effects, C

DOI: 10.1021/acs.bioconjchem.9b00209 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 4. Far-UV circular dichroism spectra of 25 μM Aβ42 incubated in the absence and presence of 0.5 μM BSA/BSA-Bs at (a) 0 h and (b) 48 h.

release triggered by 25 μM BSA-B4 alone (∼45% in Figure S7b) was higher than that triggered by the mixture of 25 μM BSA-B4 and Aβ42. This suggested that the Aβ42-BSA-B4 complexes displayed weaker toxicity than Aβ42 or BSA-B4 alone. Namely, Aβ42 bound on BSA-B4 actually protected the cells from BSA-B4 to a certain extent, as Aβ42 species might bind onto the BSA-B4 surface and cover the excessive positive charges of BSA-B4. Although BSA-B4 at high concentrations showed cytotoxicity as well, BSA-B4 at low concentrations (0.5−5 μM) showed effectively high antifibrillogenesis and detoxification without any cytotoxicity. In the present case, considering that the in vivo administration of an inhibitor is generally low, the cytotoxicity of BSA-B4 presented at high concentrations would not hamper its potential applications. MTT assays were also performed with HSA-Bs, as shown in Figure S8. Similar detoxification effects of HSA-Bs were obtained with BSA-Bs. HSA-B3/4 could significantly reduce cytotoxicity of Aβ42 at 0.5 μM, and 5 μM BSA-B4 increased the cell viability to ∼92%, greatly superior over native HSA. This further verified the applicability of this design strategy on HSA. From the results discussed above, we can conclude that BSA-Bs/HSA-Bs of high conversion of carboxyl groups to amino groups (BSA-B3/4 or HSA-B3/4) could deflect from the amyloid aggregation pathway and significantly inhibit Aβ42 fibrillation, leading to nontoxic or low-toxicity aggregates. In particular, BSA-B4/HSA-B4 had a pronounced performance at very low concentrations (inhibitor/Aβ42 = 0.02−0.2), presenting great advantages over other protein-based inhibitors reported previously, such as A-BSA/A-HSA and hLys.23,26,42 Effects on the Secondary Structures of Aβ 42 Aggregates. To gain an insight into the effect of BSA-Bs on the conformational transition of Aβ42 aggregation, CD measurements were applied to monitor the secondary structures of Aβ42 over a span of time. Due to BSA itself at high concentrations having a strong far-UV CD signal (Figure S3a), which greatly disturbs the detection and analysis of Aβ42, only a set of BSA and BSA-Bs at a low concentration (0.5 μM) was tested. As shown in Figure 4a, the initial secondary structures of Aβ42 with or without BSA/BSA-Bs all presented a random coil with a representative negative minimum around 200 nm. After 48 h, the CD spectrum of Aβ42 alone showed one positive peak at 200 nm and one negative valley around 215 nm (Figure 4b), confirming the formation of a typical βsheet structure.43 In the presence of 0.5 μM BSA or BSA-Bs, the negative ellipticity of Aβ42 around 215 nm decreased with the increase of MD (Figure 4b). Interestingly, it can be seen that the CD spectrum of Aβ42 mixed with 0.5 μM BSA-B4 did

consistent with that observed above in the ThT assays (Figure 1). This further proves that low modification of positive charges has little improvement on inhibition effects. In the presence of BSA-B3 or BSA-B4, particularly BSA-B4, some shorter, thinner, and broken Aβ42 fibrils or amorphous aggregates were observed at low concentrations (95%, lyophilized powder), synthesized by routine solid-phase peptide synthesis and Fmoc chemistry, were purchased from GL Biochem (Shanghai, China). Human neuroblastoma SH-SY5Y cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium/Ham’s F-12 (DMEM/F12) were obtained from Invitrogen (Carlsbad, CA, USA). All other reagents were of analytical grade and purchased from local sources. Deionized water filtered through 0.22 μm filters was used for all solution preparations. Synthesis and Characterization of Basified BSA (BSAB). At first, 1 mL of ethylenediamine was added into 0.1 M MES buffer (40 mL), and the solution was maintained at pH 6.0 by titrating 12 M HCl. BSA powder (400 mg) was dissolved into the above solution. The solution was divided into four equal volumes of solutions into four flasks, and to each were added 2, 5, 10, and 40 mg of EDC to achieve four different MDs of amino groups. The reaction mixture was kept at 25 °C for 24 h, and the resulting reactant was purified by dialysis with a membrane dialyzer (MWCO, 10−14 kDa) against deionized water for 4 days. At last, the modified protein (BSA-B) preparation was collected, lyophilized, and stored at −20 °C before use. HSA-Bs were prepared by the same method. BSA-Bs and BSA were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (Autoflex Tof/TofIII, Bruker Daltonics Inc., Billerica, MA, USA) to determine the MD. The ζ potentials of BSA and BSA-B at different pH values were determined by Zetasizer Nano (Malvern Instruments, Worcestershire, U.K.) in deionized water. The molecular sizes and the intrinsic fluorescence

Figure 7. Adsorption isotherms and respective model fits for determinations of affinity and kinetic rate constants by the QCM experiments. Adsorption curves of Aβ42 on (a) BSA- or (b) BSA-B4modified sensors and the corresponding fitting curves to eq 6 (black dashed lines). (c) The fitted values from part a and part b were substituted into eq 7, resulting in ka = 2.0 × 10−3 μM−1 min−1, kd = 5.0 × 10−3 min−1, KA = 4.0 × 105 M−1 for Aβ-BSA and ka = 6.7 × 10−3 μM−1 min−1, kd = 3.5 × 10−3 min−1, KA = 1.9 × 106 M−1 for Aβ-BSAB4.

steric effect, BSA selectively binds Aβ monomers or oligomers mainly through the hydrophobic pocket.15 By contrast, BSABs, represented by BSA-B4, could interact with more Aβ monomers, oligomers, and protofibrils by the widely distributed positive charges on the surface (Figure 6), as presented in Figure 8c and Figure 8d. The enhanced electrostatic attractions benefit in the generation of various coclusters of Aβ-BSA-B4, which are amorphous in morphology and have significantly less seeding ability for nucleation and amyloid formation. Thus, BSA-B4 at low concentrations shows a potent inhibiting capability on the on-pathway aggregation and cytotoxicity of Aβ42. Because of the similar structures and properties of BSA and HSA (Table S2) and the similar effects of BSA-B4 and HSA-B4 (Figures S5 and S8), it is considered that HSA-B4 worked by a similar mechanism on inhibiting amyloid fibrillogenesis with BSA-B4. H

DOI: 10.1021/acs.bioconjchem.9b00209 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 8. Schematic representations of Aβ aggregation pathways influenced by BSA and BSA-B4. (a) On-pathway fibrillation: Aβ monomers selfaggregate into oligomers, protofibrils, and fibrils with high neurotoxicity. (b) Native BSA binds Aβ monomers mainly through the hydrophobic groove at a 1:1 stoichiometry, reducing fibril production. (c) Aβ aggregation in the presence of BSA-B4: the widely distributed positive charges promote BSA-B4 to bind more Aβ monomers, oligomers, and protofibrils to form various coclusters of Aβ-BSA-B4, thus powerfully suppressing the conformational transition to β-sheet structure and the following Aβ fibrillation. (d) Schematic representation of the widely distributed electrostatic interactions between Aβ species and BSA-B4. The enhanced electrostatic interactions disrupt the formation of highly ordered β-sheet structures.

the single time-point ThT-binding assay) were performed in triplicate. Atomic Force Microscope (AFM). Aβ (25 μM) in the absence and presence of different concentrations of BSA or BSA-Bs was incubated at 37 °C in a fluorescence plate reader. The sample (50 μL) of the incubation was deposited on freshly cleaved mica for 2 min, and then salt ions of the solution were washed out with ultrapure water. The AFM sample was dried in a steam of dry nitrogen. Micrographs of the Aβ specimen were examined on a multimode atomic force microscope (CSPM5500, Benyuan, China) and collected in tapping mode. Circular Dichroism (CD) Spectroscopy. A freshly prepared stock solution of Aβ42 (275 μM) was diluted to 100 mM PBS (10 mM NaCl, pH 7.4) to a final concentration of 25 μM, with or without 0.5 μM BSA or BSA-B, then incubated in a shaking incubator at 150 rpm and 37 °C. The CD spectra of samples immediately taken after preparation (0 h) and taken after incubation for 48 h were measured by a J810 circular dichroism spectropolarimeter (JASCO, Japan). Far-UV (190−260 nm) measurements were carried out with a 1 mm path length quartz cell at a scanning speed of 100 nm/ min and a bandwidth of 1 nm. The signals of solution without Aβ42 were set as the background and subtracted from the sample spectra. Cell Viability Assay. The cytotoxicity of Aβ42 aggregates was determined by MTT assays and LDH release assays with SHSY-5Y cells. MTT assays and LDH release assays were all carried out in sextuplicate. The SHSY-5Y cells were cultured in a medium of DMEM/F12 supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in an atmosphere of 5% CO2. A total of 8 × 103 cells (80 μL) were cultured in a 96-well plate for 24 h. Then 20 μL Aβ42 solutions (25 μM) which were incubated with or without inhibitors at

spectra of BSA and BSA-Bs were respectively measured on a Zetasizer Nano and a fluorescence spectrometer (PerkinElmer LS-55, MA, USA) in PBS buffer (100 mM sodium phosphate, 10 mM NaCl, pH 7.4) in 0.5 mg/mL solution at 37 °C. Preparation of Aβ Monomer Solution. Aβ42 and Aβ40 monomer solutions were pretreated as described in the literature.57 The lyophilized Aβ powder was dissolved in HFIP to 1.0 mg/mL, set quietly for 2 h, and sonicated for 30 min in ice bath to destroy pre-existing amyloid fibrils. HFIP was then removed using a vacuum freeze drier (Labconco, MO, USA). The lyophilized Aβ was immediately stored at −20 °C. Before use, the treated Aβ was dissolved in 20 mM NaOH at 275 μM and sonicated for 10 min in ice bath. After centrifugation at 16 000g for 20 min at 4 °C, the upper 3/4 of the supernatant was carefully collected as a stock solution of Aβ. Aggregation experiments of Aβ40 or Aβ42 were initiated by addition of the stock solution to PBS buffer (100 mM sodium phosphate, 10 mM NaCl, pH 7.4), leading to a final Aβ concentration of 25 μM. This solution with or without different concentrations of BSA/BSA-B was used for the following studies of Aβ aggregation and cytotoxicity. Thioflavin T Fluorescent Assay. The fluorescent assays were performed at 37 °C by a fluorescence plate reader (Infinite M200 Pro, TECAN, Salzburg, Austria) with excitation at 440 nm, emission at 480 nm, and 10 s shaking before fluorescent intensities were detected. The assay samples were prepared as described above, containing 25 μM Aβ monomers, 25 μM ThT, and different concentrations of inhibitors. The blank sample contained everything but Aβ. The fluorescence data were processed by subtracting the blank (control). As Aβ40 presents much slower aggregation rate with a sigmoidal curve and an obvious lag phase, Aβ40 was used for kinetic studies. All experiments (the real-time fluorescent assay and I

DOI: 10.1021/acs.bioconjchem.9b00209 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry 37 °C and 150 rpm for 18 h were added to the cells. To investigate the effect of the inhibitors on cell viability, different concentrations of inhibitors solution containing everything except Aβ were added into the cells. After additional 24 h incubation, 10 μL of 5.5 mg/mL MTT in 100 mM PBS (10 mM NaCl, pH 7.4) was added into each well and incubated for another 4 h. The culture medium was removed after centrifuging the suspension at 1500 rpm for 10 min, and the precipitated cells were lysed using 100 μL of DMSO. When the purple crystals were dissolved completely, the absorbance signals were recorded in a plate reader (TECAN GmbH, Salzburg, Austria) at 570 nm. All the experiments were carried out with six replicates. Background signals were recorded from the samples treated without cells. The cell viability was processed by subtracting the blank, and the cell survival treated with PBS buffer only was set as control (100%) to normalize other data for comparison. LDH release assay was also conducted to quantitatively assess the cell death. Briefly, a density of 8 × 103 SH-SY5Y cells was placed per well in 96-well plates and replaced with FBS-free medium (80 μL) after 24 h incubation. Then an amount of 20 μL of the end-point Aβ42 products was added into the SH-SY5Y cells and incubated for 48 h. Finally, the extracellular LDH leakage was evaluated using the LDH assay kit (Beyotime, Shanghai, China). For the assay, the culture medium was centrifuged at 400g for 5 min, and 80 μL of supernatant was collected and mixed with 40 μL of reaction buffer prepared according to the manufacturer’s instructions. The LDH release level was assessed at 490 nm with a reference wavelength of 630 nm after shaking for 30 min in 37 °C. As a control, the cells incubated with 1% (v/v) Triton X-100 were set as the maximal LDH release as 100% cytotoxicity. Fluorescence Titration Experiment. Fluorescence quenching experiments of BSA/BSA-B4 were performed at a fixed excitation wavelength of 280 nm, and the emission spectra were recorded in the range of 300−400 nm. The maximum emission intensity at 332 nm was used for analysis. Upon titration of 0.5 mM Aβ42 solution (15 μL) into 2 μM BSA solution (2.1 mL) in a cuvette, the mixture was gently shaken and kept for 1 min for equilibrium. The background signal of Aβ42 solution without BSA/BSA-B4 was subtracted from the spectra with BSA/BSA-B4. Isothermal Titration Calorimetry (ITC). ITC experiment was conducted using a VP-ITC (MicroCal, Northampton, MA, USA). Briefly, 10 μL of BSA solution (300 μM) or BSA-B4 solution (100 μM) in PBS (pH 7.4) was titrated into 1.425 mL of Aβ42 solution (30 μM) 25 times at 37 °C. The heat of dilution was acquired under the same condition by injecting BSA/BSA-B4 into the cell containing the sample buffer only and subtracted from the corresponding experimental group. The ITC results were analyzed using the one-site model with MicroCal Origin 7.0 software.27 Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). A Q-Sense E1 system (Q-sense, Gothenburg, Sweden) was used to monitor the adsorption of Aβ42 monomers. Gold-coated quartz crystal chip was used as the sensor. The BSA-/BSA-B4-modified sensors were prepared by the following method. Briefly, the clean chips were immersed in ethanol solution of 5 mM MUA for 16 h at 25 °C. After washing with ethanol and deionized water, the chips were activated for 1 h using 0.4 M EDC and 0.1 M NHS in MES buffer (pH 5.0). Then the chips were immersed in 2 mg/mL BSA/BSA-B4 in PBS (pH 7.4) for 12 h. After washing with

PBS and deionized water, the remaining activated sites were blocked by reaction with 0.5 M ethanolamine for 1 h. Finally, the chips were rinsed with deionized water, dried with N2, and stored at 4 °C. The BSA-/BSA-B4-modified sensor was put into the flow module and equilibrated with degassed PBS (100 mM, pH 7.4) at 37 °C. Then, a set of fresh Aβ42 at different concentrations (1−25 μM) was separately pumped into the system. At the end of the experiment, the running solution of Aβ42 was replaced with the injection of PBS. A flow rate of 30 μL/min was maintained for all experiments. Time-resolved changes in the shift of dissipation (ΔD) and resonant frequency (Δf) were recorded. All data presented here were recorded at the third overtone. Statistical Analysis. The results were expressed as the mean ± standard deviation. One-way ANOVA with Tukey test was carried out for statistical comparisons to analyze the variance, and p < 0.05 or less was considered to be statistically significant.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.9b00209. Mass spectra, far-UV circular dichroism spectra, fluorescence spectra and ζ potentials of BSA and BSABs, AFM images of Aβ42 species, cytotoxicity assays, Aβ42 aggregation kinetics, fluorescence quenching spectra and fitting curves, the structures of Aβ40/42 and BSA, Aβ42 aggregation kinetics incubated in different NaCl concentrations, and calorimetric titration assays, ITC, time-resolved QCM isotherms (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86 22 27404981. Fax: +86 22 27403389. ORCID

Xiaoyan Dong: 0000-0002-8040-5897 Yan Sun: 0000-0001-5256-9571 Author Contributions

X.D. and Y.S. designed the research. W.W. performed the experiments. W.W. and X.D. analyzed the data. W.W., X.D., and Y.S wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Natural Science Foundation of China (Grants 21621004 and 91634119). REFERENCES

(1) Selkoe, D. J. (2003) Folding proteins in fatal ways. Nature 426, 900−904. (2) Debnath, K., Pradhan, N., Singh, B. K., Jana, N. R., and Jana, N. R. (2017) Poly(trehalose) nanoparticles and prevent amyloid aggregation and suppress polyglutamine aggregation in a Huntington’s disease model mouse. ACS Appl. Mater. Interfaces 9, 24126− 24139. (3) Clark, L. F., and Kodadek, T. (2016) The immune system and neuroinflammation as potential sources of blood-based biomarkers for J

DOI: 10.1021/acs.bioconjchem.9b00209 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

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DOI: 10.1021/acs.bioconjchem.9b00209 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.bioconjchem.9b00209 Bioconjugate Chem. XXXX, XXX, XXX−XXX