Appl Microbiol Biotechnol (2016) 100:825–835 DOI 10.1007/s00253-015-7001-7
APPLIED MICROBIAL AND CELL PHYSIOLOGY
A specific RAGE-binding peptide biopanning from phage display random peptide library that ameliorates symptoms in amyloid β peptide-mediated neuronal disorder Cuizan Cai 1 & Xiaoyong Dai 2 & Yujie Zhu 1 & Mengyang Lian 1 & Fei Xiao 3 & Fangyuan Dong 1 & Qihao Zhang 2 & Yadong Huang 2 & Qing Zheng 1
Received: 9 June 2015 / Revised: 1 September 2015 / Accepted: 10 September 2015 / Published online: 24 October 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Alzheimer’s disease (AD) is an age-related neurodegenerative disorder in which amyloid β (Aβ) peptide accumulates in the brain. The receptor for advanced glycation end product (RAGE) is a cellular binding site for Aβ peptide and mediates amyloid β-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a specific high-affinity RAGE inhibitor (APDTKTQ named RP-1) from a phage display library. RP-1 bound to RAGE and inhibited Aβ peptide-induced cellular stress in human neuroblastoma SH-SYSY cells in vitro. Three amino acids in RP-1 are identical to those in the Aβ peptide. RP-1 shows high homology to the 16–23 (KLVFFAED) regions in Aβ peptide and highaffinity RAGE. Functional analyses indicated that RP-1 significantly reduced the level of reactive oxygen species (ROS) and ROS products and that it enhanced catalase and glutathione peroxidase (GPx) activity. Furthermore, it inactivated caspase3 and caspase9 and inhibited the upregulation of RAGE, nuclear factor-κB (NF-κB), and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) protein expression. In addition, RP-1 activated the PI3K/AKT signaling pathway, inhibiting the interaction between Bax and Bcl-2. Our data suggest that RP-1 is a potent RAGE blocker that effectively controls the progression of Aβ peptide-mediated brain Cuizan Cai and Xiaoyong Dai contributed equally to this work. * Qing Zheng [email protected] 1
College of Pharmacy, Jinan University, 510632 Guangzhou, Guangdong, People’s Republic of China
2
Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, 510632 Guangzhou, People’s Republic of China
3
Department of Pharmacology, School of Medicine, Jinan University, 510632 Guangzhou, Guangdong, People’s Republic of China
disorders and that it may have potential as a diseasemodifying agent for AD. Keywords Alzheimer’s disease . RAGE . Phage display . Aβ
Introduction Alzheimer’s disease (AD) is the result of neuronal degeneration associated with senile plaques in the brain (Vickers et al. 2000). Amyloid β (Aβ) is found in extracellular senile plaque cores and is recognized as one of the vital neuropathological characteristic of AD (Rohn et al. 2001). Aβ peptides are the result of proteolysis of the amyloid precursor protein by sequential enzymatic actions of the beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and γ-secretase (Haass and Selkoe 2007). There are many mechanisms involved in Aβ-mediated neurotoxicity, but there is evidence showing that oxidative stress plays a key role in Aβ-mediated neurotoxicity (Miranda et al. 2000). Studies have shown that Aβ increases intracellular levels of reactive oxygen species and induces oxidative damage in cellular macromolecules, such as DNA proteins and lipids (Chonpathompikunlert et al. 2011; Pratico 2008; Behl and Moosmann 2002). Previous studies also demonstrated that overexpression of antioxidant enzymes and pretreatment with an antioxidant compound attenuated Aβinduced apoptotic cell death (Aruoma et al. 2003; Anderton 1994). The PI3K signaling pathway plays a central role in neuronal survival (Koh et al. 2003; Franke et al. 2003), with studies showing that activation of PI3K prevents Aβ-induced neuronal death (Liu et al. 2010; Zhao et al. 2011; Zeng et al. 2011; Lou et al. 2011). Previous data also demonstrated that luteolin blocks the PI3K-AKT-NF-κB-ERK1/2 pathway and attenuates the increase in ROS and elevates the Bcl-2/Bax
826
ratio, leading to decreased apoptotic cell death (Zhou et al. 2011). As reported in the literature, the receptor for advanced glycation end products (RAGE) plays an important role as a cell-surface receptor for Aβ at the blood-brain barrier and in neurons and microglia (Yan et al. 1996, 2010; Zlokovic 2011; Origlia et al. 2010). The extracellular V domain of RAGE is the key domain in Aβ binding to RAGE (Sturchler et al. 2008). In the Aβ peptide, the major binding site is localized to an eight amino acid stretch of residues in positions 16–23 of the KLVFFAED sequence, which consists of a series of hydrophobic residues flanked by two negatively charged residues at the peptide c-terminus (Gospodarska et al. 2011; Jargilo et al. 2013). Research had shown that RAGE mediates Aβ-induced oxidant stress in neurons, causing mitochondrial dysfunction and Aβ intraneuronal transport (Deane et al. 2012; Liu et al. 2012). In microglia, it amplified the Aβmediated inflammatory response and activated the nuclear factor-κB (NF-κB) pathway. In APP transgenic mice, targeted expression of RAGE in neurons accelerated cognitive decline and Aβ-induced neuronal perturbation (Zlokovic 2005). In an Aβ-rich environment, RAGE expression increased (Rong et al. 2005), and RAGE regulated the generation of the betasite amyloid precursor protein-cleaving enzyme 1 (BACE1) protein and Aβ (Cho et al. 2009). It was also shown to contribute to stroke pathology through dependent upregulation of inflammatory cytokines and NF-κB (Rong et al. 2005). Therefore, there is a need to develop new efficacious highaffinity Aβ/RAGE blockers that are safe and nontoxic. To develop such an agent, we first used a phage display library to obtain a novel peptide that bound to RAGE with high affinity. RAGE is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules, which include advanced glycation end product proteins S100/calgranulins, Aβ, and amphotericin (Koch et al. 2010). Second, we used reverse screening to obtain a high-affinity peptide, which specifically blocked the Aβ/RAGE interaction, inhibited Aβinduced cellular stress in SH-SY5Y cells, and was not toxic to cells.
Materials and methods Materials A ph.D.-T™ Phage Display Peptide Library Kit and Escherichia coli ER2738 were purchased from New England Biolabs (Beverly, MA, USA). Human neuroblastoma SH-SY5Y cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA, CRL-2266). A caspase3 activity assay kit, caspase9 activity assay kit, reactive oxygen species (ROS) assay kit, cellular glutathione peroxidase (GPx) assay kit, lipid peroxidation MDA assay
Appl Microbiol Biotechnol (2016) 100:825–835
kit, and JC-1 mitochondrial membrane potential (MMP) assay kit were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Anti-RAGE antibody (Abcam, Cambridge, UK) PI3K antibody, P-PI3K antibody, AKT antibody, P-AKT antibody, NF-κB antibody, Bax antibody, β-actin antibody, and goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Bcl-2 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), sheep anti-rabbit IgG H&L Dylight 488 and Northernlights anti-rabbit IgG NL 557 were purchased from R&D (R&D, USA). Human soluble receptor for advanced glycation end products (sRAGE) were obtained from R&D (R&D, USA). Amyloid β 1-42 (Aβ1–42) were purchased from Sigma (Shanghai, China). Phage display biopanning procedures sRAGE-coated 96-well microtiterplates were incubated overnight at 4 °C and then blocked with bovine serum albumin (BSA) for 2 h at room temperature. The plate wells were then washed six times with 0.05 % Tween-20 in TBS (TBST). Then, 10 μl of original library (2×1011 plaque-forming units, PFUS) were diluted in 100 μl of TBST and added to plate wells for 2 h at room temperature, with gentle agitation. Removing the unbound phages uses 0.05 % TBST and then bound phages with 0.1 M glycine-HCl (pH 2.2) were eluted and neutralized with 1 M Tris–HCl (pH 9.1). The bound phages were amplified by infecting them with liquid E. coli ER2738 culture. After being purified and concentrated, these amplified phages were used for additional rounds of biopanning. Reverse biopanning To obtain an efficacious high-affinity Aβ/RAGE blocker, we performed inversion screening. sRAGE-coated 96-well microtiterplates were incubated overnight at 4 °C and then blocked with BSA for 2 h at room temperature. The plate wells were then washed six times with 0.05 % TBST, followed by blocking with 10 μg of Aβ1–42 for 2 h at room temperature and washing six times with TBST. The phages from the fourth round (phage display biopanning procedures) were added to microtiterplates and the unbound positive phages were collected. DNA sequencing of the selected phage clones After the inversion biopanning, 40 phage clones were randomly selected and the ssDNA was prepared as described by the standard protocol (NEB). Briefly, the phage clones were precipitated with polyethylene glycol (PEG)/NaCl for 10 min at room temperature. After centrifuged at 12,000 rpm for
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10 min, the pellet was resuspended in 100 μl iodide buffer, and DNA was precipitated with 250 μl ethanol for 10 min at room temperature. After washing with 70 % ethanol, the DNA was resuspended in 30 μl TE buffer 10 mM Tris–HCl (pH 8.0), 1 mM EDTA. DNA sequencing was performed with −96 gIII primer by Invitrogen Corporation (Shanghai, China).
was measured at 370 nm. An unrelated peptide was used as a negative control. The competitive inhibition of binding of other phage clones (clone P-22, P-31) to sRAGE by the RP-1 peptide was conducted using the same method.
ELISA assay of high-affinity sRAGE-binding phages
Western blot analysis was used to analyze the expression of RAGE in SH-SY5Y cells. Briefly, the SH-SY5Y cells were maintained in DMEM medium, supplemented with 10 % of FBS, and incubated in a humidified atmosphere of 5 % CO2 at 37 °C for 24 h. SH-SY5Y cell homogenates were separated on 10 % SDS-PAGE gels, then transferred to a PVDF membrane, and incubated with the primary anti-RAGE antibody overnight at 4 °C. Goat anti-rabbit IgG and HRP-linked antibody (1:1000) were then added and incubated for 2 h at room temperature. Expression of RAGE in SH-SY5Y cells enhanced chemiluminescence was used for visualization, and confocal microscopy was used to directly observe the binding of the synthetic RP-1 peptide to the SH-SY5Y cells. Briefly, the SHSY5Y cells were washed three times with PBS and fixed with 5 % paraformaldehyde for 15 min at room temperature, followed by washing with PBS three times and blocking with 5 % BSA for 15 min at room temperature. Next, the cells were incubated with FITC-conjugated RP-1 for 2 h at room temperature. The colocalization of the positive peptide and RAGE on the surface of the SH-SY5Y cells were observed with confocal microscopy. Briefly, the SH-SY5Y cells were first washed three times with PBS and fixed with 5 % paraformaldehyde for 15 min at room temperature, followed by washing with PBS three times and blocking with 5 % BSA for 15 min at room temperature. The cells were then incubated with antiRAGE antibody (1 μg/ml) at 4 °C overnight, followed by incubation with donkey anti-rabbit IgG H&L Dylight 594 for 2 h at room temperature and washing in PBS three times. FITC-conjugated RP-1 was then added and observed after for 2 h at room temperature.
The 96-well plates coated with RAGE were incubated overnight at 4 °C and washed six times with TBST. The microwells were blocked using BSA. Phage clones were added to the wells and incubated for 2 h at room temperature, followed by incubation with HRP-conjugated anti-M13 monoclonal antibody (1:5000) for 1 h. Then, the plates were washed, substrate (50 μl/well of 3,3′,5,5′-tetramethyl-benzidine [TMB]) was added, and the plates were kept at room temperature in the dark for 20 min. The reaction was terminated by adding 50 μl/well of H2SO4. The absorbance was measured at 370 nm. Peptide synthesis Candidate peptides were synthesized by China Peptides (Shanghai, China) using standard solid-phase Fmoc chemistry. The method of peptide RP-1 labeling with FITC was referred the instructions of FITC Labeling Kit (#53027, Pierce, Rockford, USA). Briefly, 40 μl of the borate buffer (0.67 M, pH 8.5) and 0.5 ml of 2 mg/ml peptide in PBS and FITC reagent were mixed thoroughly. Then the mixture was incubated in the darkness for 2 h at room temperature. The labeling solution was applied onto Sephadex-G25 column and the labeled protein (yellowish color) was pooled and stored at −80 °C. The synthesized peptides were identified by reverse-phase HPLC and mass spectrometry analysis. Competitive inhibition assay The competitive inhibition ELISA method was used to evaluate the competitive binding inhibition between the positive phage clone and its encoded peptide. Briefly, 96-well plates were coated with sRAGE and incubated overnight at 4 °C and washed six times with TBST. The microwells were blocked with BSA and then incubated with 100 μl of synthetic peptide encoded by the positive clone at various concentrations (0, 100, 200, 300, 400, 500, and 600 μM) in a 96-well plate. Then, 1×1010 pfu homologous positive phage clones were added and incubated for 2 h at room temperature, followed by incubation with HRP-conjugated anti-M13 monoclonal antibody (1:5000) for 1 h. The plates were then washed, substrate (50 μl/well TMB) was added, and the plates were kept at room temperature in the dark for 20 min. The reaction was terminated by adding 50 μl/well of H2SO4. The absorbance
Binding specificity of the RP-1 peptide to RAGE
Cell culturing, treatment with Aβ1–42, and cell viability assay SH-SY5Y cells were maintained in DMEM medium, supplemented with 10 % of FBS, and incubated in a humidified atmosphere of 5 % CO2 at 37 °C for 24 h. The SH-SY5Y cells were randomly divided into six groups: (1) control cells, (2) control cells incubated for 24 h in DMEM without serum in the presence of Aβ1–42, (3) control cells pretreated with RP-1 (0.1 μM) and then incubated for 24 h in DMEM without serum in the presence of Aβ1–42, (4) control cells pretreated with RP-1 (1 μM) and then incubated for 24 h in DMEM without serum in the presence of Aβ1–42, (5) control cells pretreated with RP-1 (10 μM) and then incubated for 24 h in
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DMEM without serum in the presence of Aβ1–42, and (6) control cells pretreated with RP-1 (100 μM) and then incubated for 24 h in DMEM without serum in the presence of Aβ1– 42. An MTT assay was used to evaluate the viability of the SHSY5Y cells. Following treatment with RP-1 and Aβ1–42 as described above (2.9), 20 μl of MTT were added to each well and incubated for 4 h at 37 °C. The absorbance was measured at 490 nm. Measurements of intracellular ROS, lipid peroxidation, and MMP The SH-SY5Y cells were treated with RP-1 and Aβ1–42 as described above (2.9). The level of ROS in the SH-SY5Y cells was measured based on 2,7-dichlorofluorescein-diacetate (DCFH-DA) as described previously (Khan et al. 2012). After treatment, the cells were incubated with 10 μM of DCFH-DA at 37 °C for 30 min. Then cells were harvested, rinsed, and re-suspended in PBS. Fluorescence levels were quantified using a fluorescence microplate, with excitation at 488 nm and emission at 525 nm. Malonyldialdehyde (MDA), which is used as an indicator of lipid peroxidation, was measured with a lipid peroxidation MDA assay kit. Mitochondrial membrane potential kit (JC-1) was used to analyze the mitochondrial membrane potential (MMP) of the cells. The SH-SY5Y cells were then stained with JC-1 and incubated at 37 °C for 20 min. After rinsing, the cells were resuspended in 0.5 of DMEM and analyzed with FACScan. Assay of superoxide dismutase (SOD), GPx, caspase3, and caspase9 activity The SH-SY5Y cells were treated with RP-1 and Aβ1–42 as described above. The activity of SOD, GPx, caspase3, and caspase9 was measured using commercial kits following the manufacturer’s instructions.
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Results Identification of phage clones that selectively bind to sRAGE In this study, phages that specifically bind to sRAGE were identified through four rounds of selection and one round of reverse biopanning. The output/input ratio of phages after each round of biopanning was used to determine the enrichment efficiency, which increased from 4.5×10–5 to 8.52×10–3 as shown in Table 1. This result indicates that phages capable of specifically binding to RAGE were significantly enriched. Reverse biopanning generated a high-affinity RAGE blocker that selectively competed with Aβ and combined with RAGE. DNA sequencing of selected phage clones After the fourth round of biopanning and one reverse biopanning, 40 phage clones were randomly selected. The DNA sequence and the deduced peptide sequence were analyzed and classified. Eight different phage clones and sequences were obtained, named P-1 to P-8 and RP-1 to RP-8, respectively (Table 2). Identification of the binding selectivity of the eight clones by ELISA The major fragment of the Aβ1–42 binding of V-RAGE was Aβ16–23 fragment (Gospodarska et al. 2011). Therefore, the amino acid sequences of the selected peptides were compared with Aβ16–23. As shown in Fig. 1 and Table 2, the eight clones detected by ELISA showed a strong binding ability to sRAGE compared to the control blocking buffer, and P-1 (the peptide was named RP-1) clone was the highest enriched and highest sequence similarity to Aβ16–23 (0.1538426, PAM250 Matrix). RP-1 (APDTKTQ) contains three amino acids that are identical to those of Aβ16–23 (KLVFFAED) peptides. Structurally, the theoretical of RP-1 is the nearest to that of Aβ16–23 peptides. These data suggested that RP-1 may be a candidate peptide. Therefore, RP-1 was chosen for further study.
Western blot analysis
Identification of synthesized RP-1 peptide
The SH-SY5Y cells were treated with RP-1 and Aβ1–42 as described above. Following collection of the SH-SY5Y cells, the cell homogenates were separated on 10 % SDS-PAGE gels and transferred to a PVDF membrane. The membrane was incubated with primary antibodies (P-PI3K, PI3K, P-AKT, AKT, Bax, Bcl-2, NF-κB, BACE1, RAGE, and β-actin) at 4 °C, followed by the addition of goat anti-rabbit IgG and HRP-linked antibody (1:1000) and incubation for 2 h at room temperature. Protein expression enhanced chemiluminescence was used for visualization.
The result of reverse-phase HPLC analysis showed that the purity of RP-1 peptide was 98 % (Fig. 2a). The result of mass spectrometry analysis showed that the molecular weight of synthesized RP-1 peptide was consistent with the theoretical value (Fig. 2b). Competitive inhibition assay To determine whether the synthetic peptide RP-1 and the corresponding phage clone P-1 competed for the same binding
Appl Microbiol Biotechnol (2016) 100:825–835 Table 1
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Enrichment of sRAGE binding phages for each panning round
Round
sRAGE (μg)
Aβ (μg)
Concentration of Tween 20 (v/v)
Input phage (pfu)
Output phage (pfu)
Recover (output/input)
1 2 3 4 Reverse biopanning
15 10 5 2.5 2.5
0 0 0 0 10
0.05 0.1 0.3 0.5 0.5
2.0×1011 3.4×1011 4.5×1011 4.0×1011 2.4×1011
9×106 3.1×107 1.3×108 3.4×109 1.4×109
4.5×10−5 9.2×10−5 3.0×10−4 8.52×10−3
site, a competitive inhibition assay was performed, using AP6 (QWTOSHP) as a control. When the synthetic RP-1 peptide was pre-incubated with sRAGE, the binding of the corresponding phage clone P-1 was inhibited in a dose-dependent manner, demonstrating that the positive phage clone bound to sRAGE by displacing the RP-1 peptide. At an RP-1 peptide concentration greater than 600 μM, the inhibition ratio was about 77 % (Fig. 3a), and the control peptide AP6 had no effect on the binding of the phage P-1, even at 600 μM. In addition, no significant competitive inhibition was observed for P-1 to phage clones P-22 and P-31 (Fig. 3b). These results indicated that the synthetic peptide RP-1 and the corresponding phage clone P-1 were competing for the same binding site, and the binding clone P-1 to sRAGE was mediated by the peptide RP-1.
do not bound to 3T3 cells (Fig. 4b). Furthermore, RP-1 and RAGE were co-localized on the surface of the SH-SY5Y cells (Fig. 4c). RP-1 increased cell viability against Aβ1–42-induced toxicity in SH-SY5Y cells The viability of the SH-SY5Y cells significantly decreased in the presence 4 μM Aβ1–42 in DMEM. RP-1 pretreatment for 2 h enhanced the cell viability of SH-SY5Y cells exposed to Aβ1–42 (Fig. 5a). The MMP was also detected in the RP-1 treatment groups, and the MMP was reduced (Fig. 5b) in the RP-1 treatment group.
Binding specificity of the RP-1 peptide to RAGE
Effects of RP-1 on Aβ1–42-induced caspase3 and caspase9 activation in SH-SY5Y cells
We examined the expression of RAGE in SH-SY5Y cells using Western blot analysis. As shown in Fig. 4a, the SHSY5Y cells express high level of RAGE, while the control 3T3 cells do not express RAGE. Confocal microscopy performed to observe the binding of RP-1 in the SH-SY5Y cells indicated that RP-1 specifically bound to SH-SY5Y cells and
Mitochondrial dysfunction, caspase-dependent toxicity, and dysfunction of downstream signaling pathways documented critical apoptotic events during AD processes (Ghavami et al. 2014). The cytosolic activity of caspase3 and caspase9 was detected as mitochondrial-related apoptotic molecular. The activities of both caspase3 and caspase9 increased in the
Table 2
Amino acid sequences of selected phage clones from phage library
Clone
Peptide
Sequence (N–C)
Similarities to Aβ16–23
Theoretical PI
GRAVY
Frequency
P-1/4/9/11/18/23/24/28/30/32/33/34/37/40 P-8/10/35/39 P-22/38 P-12/17/21/36 P-31 P-7/13/14/15/20
RP-1 RP-2 RP-3 RP-4 RP-5 RP-6
APDTKTQ SPRVGAT RHFHFPA HLHAHKL HPMSAPR HASKQLL
0.1538426 0.1538426 0.250000 0.1538426 0.0714286 0.0476190
5.88 9.47 9.76 8.77 9.76 8.76
–1.729 –0.286 –0.729 –0.586 –1.147 –0.286
14 4 2 4 1 5
P-2/3/5/6/25/26 P-16/19/27/29
RP-7 RP-8 Aβ16–23 Aβ1–42
ATRQPNH LSLSTTS KLVFFAED DAEFRHD SGYEVHH QKLVFFAE DVGSNKG AIIGLMVG GVVIA
0.0952381 0.0714286
9.80 5.52 4.37 5.31
–2.171 0.543 0.682 0.205
6 4
830
Appl Microbiol Biotechnol (2016) 100:825–835
Fig. 1 ELISA of binding ability of phage clone to sRAGE. *P
APPLIED MICROBIAL AND CELL PHYSIOLOGY
A specific RAGE-binding peptide biopanning from phage display random peptide library that ameliorates symptoms in amyloid β peptide-mediated neuronal disorder Cuizan Cai 1 & Xiaoyong Dai 2 & Yujie Zhu 1 & Mengyang Lian 1 & Fei Xiao 3 & Fangyuan Dong 1 & Qihao Zhang 2 & Yadong Huang 2 & Qing Zheng 1
Received: 9 June 2015 / Revised: 1 September 2015 / Accepted: 10 September 2015 / Published online: 24 October 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Alzheimer’s disease (AD) is an age-related neurodegenerative disorder in which amyloid β (Aβ) peptide accumulates in the brain. The receptor for advanced glycation end product (RAGE) is a cellular binding site for Aβ peptide and mediates amyloid β-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a specific high-affinity RAGE inhibitor (APDTKTQ named RP-1) from a phage display library. RP-1 bound to RAGE and inhibited Aβ peptide-induced cellular stress in human neuroblastoma SH-SYSY cells in vitro. Three amino acids in RP-1 are identical to those in the Aβ peptide. RP-1 shows high homology to the 16–23 (KLVFFAED) regions in Aβ peptide and highaffinity RAGE. Functional analyses indicated that RP-1 significantly reduced the level of reactive oxygen species (ROS) and ROS products and that it enhanced catalase and glutathione peroxidase (GPx) activity. Furthermore, it inactivated caspase3 and caspase9 and inhibited the upregulation of RAGE, nuclear factor-κB (NF-κB), and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) protein expression. In addition, RP-1 activated the PI3K/AKT signaling pathway, inhibiting the interaction between Bax and Bcl-2. Our data suggest that RP-1 is a potent RAGE blocker that effectively controls the progression of Aβ peptide-mediated brain Cuizan Cai and Xiaoyong Dai contributed equally to this work. * Qing Zheng [email protected] 1
College of Pharmacy, Jinan University, 510632 Guangzhou, Guangdong, People’s Republic of China
2
Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, 510632 Guangzhou, People’s Republic of China
3
Department of Pharmacology, School of Medicine, Jinan University, 510632 Guangzhou, Guangdong, People’s Republic of China
disorders and that it may have potential as a diseasemodifying agent for AD. Keywords Alzheimer’s disease . RAGE . Phage display . Aβ
Introduction Alzheimer’s disease (AD) is the result of neuronal degeneration associated with senile plaques in the brain (Vickers et al. 2000). Amyloid β (Aβ) is found in extracellular senile plaque cores and is recognized as one of the vital neuropathological characteristic of AD (Rohn et al. 2001). Aβ peptides are the result of proteolysis of the amyloid precursor protein by sequential enzymatic actions of the beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and γ-secretase (Haass and Selkoe 2007). There are many mechanisms involved in Aβ-mediated neurotoxicity, but there is evidence showing that oxidative stress plays a key role in Aβ-mediated neurotoxicity (Miranda et al. 2000). Studies have shown that Aβ increases intracellular levels of reactive oxygen species and induces oxidative damage in cellular macromolecules, such as DNA proteins and lipids (Chonpathompikunlert et al. 2011; Pratico 2008; Behl and Moosmann 2002). Previous studies also demonstrated that overexpression of antioxidant enzymes and pretreatment with an antioxidant compound attenuated Aβinduced apoptotic cell death (Aruoma et al. 2003; Anderton 1994). The PI3K signaling pathway plays a central role in neuronal survival (Koh et al. 2003; Franke et al. 2003), with studies showing that activation of PI3K prevents Aβ-induced neuronal death (Liu et al. 2010; Zhao et al. 2011; Zeng et al. 2011; Lou et al. 2011). Previous data also demonstrated that luteolin blocks the PI3K-AKT-NF-κB-ERK1/2 pathway and attenuates the increase in ROS and elevates the Bcl-2/Bax
826
ratio, leading to decreased apoptotic cell death (Zhou et al. 2011). As reported in the literature, the receptor for advanced glycation end products (RAGE) plays an important role as a cell-surface receptor for Aβ at the blood-brain barrier and in neurons and microglia (Yan et al. 1996, 2010; Zlokovic 2011; Origlia et al. 2010). The extracellular V domain of RAGE is the key domain in Aβ binding to RAGE (Sturchler et al. 2008). In the Aβ peptide, the major binding site is localized to an eight amino acid stretch of residues in positions 16–23 of the KLVFFAED sequence, which consists of a series of hydrophobic residues flanked by two negatively charged residues at the peptide c-terminus (Gospodarska et al. 2011; Jargilo et al. 2013). Research had shown that RAGE mediates Aβ-induced oxidant stress in neurons, causing mitochondrial dysfunction and Aβ intraneuronal transport (Deane et al. 2012; Liu et al. 2012). In microglia, it amplified the Aβmediated inflammatory response and activated the nuclear factor-κB (NF-κB) pathway. In APP transgenic mice, targeted expression of RAGE in neurons accelerated cognitive decline and Aβ-induced neuronal perturbation (Zlokovic 2005). In an Aβ-rich environment, RAGE expression increased (Rong et al. 2005), and RAGE regulated the generation of the betasite amyloid precursor protein-cleaving enzyme 1 (BACE1) protein and Aβ (Cho et al. 2009). It was also shown to contribute to stroke pathology through dependent upregulation of inflammatory cytokines and NF-κB (Rong et al. 2005). Therefore, there is a need to develop new efficacious highaffinity Aβ/RAGE blockers that are safe and nontoxic. To develop such an agent, we first used a phage display library to obtain a novel peptide that bound to RAGE with high affinity. RAGE is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules, which include advanced glycation end product proteins S100/calgranulins, Aβ, and amphotericin (Koch et al. 2010). Second, we used reverse screening to obtain a high-affinity peptide, which specifically blocked the Aβ/RAGE interaction, inhibited Aβinduced cellular stress in SH-SY5Y cells, and was not toxic to cells.
Materials and methods Materials A ph.D.-T™ Phage Display Peptide Library Kit and Escherichia coli ER2738 were purchased from New England Biolabs (Beverly, MA, USA). Human neuroblastoma SH-SY5Y cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA, CRL-2266). A caspase3 activity assay kit, caspase9 activity assay kit, reactive oxygen species (ROS) assay kit, cellular glutathione peroxidase (GPx) assay kit, lipid peroxidation MDA assay
Appl Microbiol Biotechnol (2016) 100:825–835
kit, and JC-1 mitochondrial membrane potential (MMP) assay kit were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Anti-RAGE antibody (Abcam, Cambridge, UK) PI3K antibody, P-PI3K antibody, AKT antibody, P-AKT antibody, NF-κB antibody, Bax antibody, β-actin antibody, and goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Bcl-2 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), sheep anti-rabbit IgG H&L Dylight 488 and Northernlights anti-rabbit IgG NL 557 were purchased from R&D (R&D, USA). Human soluble receptor for advanced glycation end products (sRAGE) were obtained from R&D (R&D, USA). Amyloid β 1-42 (Aβ1–42) were purchased from Sigma (Shanghai, China). Phage display biopanning procedures sRAGE-coated 96-well microtiterplates were incubated overnight at 4 °C and then blocked with bovine serum albumin (BSA) for 2 h at room temperature. The plate wells were then washed six times with 0.05 % Tween-20 in TBS (TBST). Then, 10 μl of original library (2×1011 plaque-forming units, PFUS) were diluted in 100 μl of TBST and added to plate wells for 2 h at room temperature, with gentle agitation. Removing the unbound phages uses 0.05 % TBST and then bound phages with 0.1 M glycine-HCl (pH 2.2) were eluted and neutralized with 1 M Tris–HCl (pH 9.1). The bound phages were amplified by infecting them with liquid E. coli ER2738 culture. After being purified and concentrated, these amplified phages were used for additional rounds of biopanning. Reverse biopanning To obtain an efficacious high-affinity Aβ/RAGE blocker, we performed inversion screening. sRAGE-coated 96-well microtiterplates were incubated overnight at 4 °C and then blocked with BSA for 2 h at room temperature. The plate wells were then washed six times with 0.05 % TBST, followed by blocking with 10 μg of Aβ1–42 for 2 h at room temperature and washing six times with TBST. The phages from the fourth round (phage display biopanning procedures) were added to microtiterplates and the unbound positive phages were collected. DNA sequencing of the selected phage clones After the inversion biopanning, 40 phage clones were randomly selected and the ssDNA was prepared as described by the standard protocol (NEB). Briefly, the phage clones were precipitated with polyethylene glycol (PEG)/NaCl for 10 min at room temperature. After centrifuged at 12,000 rpm for
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10 min, the pellet was resuspended in 100 μl iodide buffer, and DNA was precipitated with 250 μl ethanol for 10 min at room temperature. After washing with 70 % ethanol, the DNA was resuspended in 30 μl TE buffer 10 mM Tris–HCl (pH 8.0), 1 mM EDTA. DNA sequencing was performed with −96 gIII primer by Invitrogen Corporation (Shanghai, China).
was measured at 370 nm. An unrelated peptide was used as a negative control. The competitive inhibition of binding of other phage clones (clone P-22, P-31) to sRAGE by the RP-1 peptide was conducted using the same method.
ELISA assay of high-affinity sRAGE-binding phages
Western blot analysis was used to analyze the expression of RAGE in SH-SY5Y cells. Briefly, the SH-SY5Y cells were maintained in DMEM medium, supplemented with 10 % of FBS, and incubated in a humidified atmosphere of 5 % CO2 at 37 °C for 24 h. SH-SY5Y cell homogenates were separated on 10 % SDS-PAGE gels, then transferred to a PVDF membrane, and incubated with the primary anti-RAGE antibody overnight at 4 °C. Goat anti-rabbit IgG and HRP-linked antibody (1:1000) were then added and incubated for 2 h at room temperature. Expression of RAGE in SH-SY5Y cells enhanced chemiluminescence was used for visualization, and confocal microscopy was used to directly observe the binding of the synthetic RP-1 peptide to the SH-SY5Y cells. Briefly, the SHSY5Y cells were washed three times with PBS and fixed with 5 % paraformaldehyde for 15 min at room temperature, followed by washing with PBS three times and blocking with 5 % BSA for 15 min at room temperature. Next, the cells were incubated with FITC-conjugated RP-1 for 2 h at room temperature. The colocalization of the positive peptide and RAGE on the surface of the SH-SY5Y cells were observed with confocal microscopy. Briefly, the SH-SY5Y cells were first washed three times with PBS and fixed with 5 % paraformaldehyde for 15 min at room temperature, followed by washing with PBS three times and blocking with 5 % BSA for 15 min at room temperature. The cells were then incubated with antiRAGE antibody (1 μg/ml) at 4 °C overnight, followed by incubation with donkey anti-rabbit IgG H&L Dylight 594 for 2 h at room temperature and washing in PBS three times. FITC-conjugated RP-1 was then added and observed after for 2 h at room temperature.
The 96-well plates coated with RAGE were incubated overnight at 4 °C and washed six times with TBST. The microwells were blocked using BSA. Phage clones were added to the wells and incubated for 2 h at room temperature, followed by incubation with HRP-conjugated anti-M13 monoclonal antibody (1:5000) for 1 h. Then, the plates were washed, substrate (50 μl/well of 3,3′,5,5′-tetramethyl-benzidine [TMB]) was added, and the plates were kept at room temperature in the dark for 20 min. The reaction was terminated by adding 50 μl/well of H2SO4. The absorbance was measured at 370 nm. Peptide synthesis Candidate peptides were synthesized by China Peptides (Shanghai, China) using standard solid-phase Fmoc chemistry. The method of peptide RP-1 labeling with FITC was referred the instructions of FITC Labeling Kit (#53027, Pierce, Rockford, USA). Briefly, 40 μl of the borate buffer (0.67 M, pH 8.5) and 0.5 ml of 2 mg/ml peptide in PBS and FITC reagent were mixed thoroughly. Then the mixture was incubated in the darkness for 2 h at room temperature. The labeling solution was applied onto Sephadex-G25 column and the labeled protein (yellowish color) was pooled and stored at −80 °C. The synthesized peptides were identified by reverse-phase HPLC and mass spectrometry analysis. Competitive inhibition assay The competitive inhibition ELISA method was used to evaluate the competitive binding inhibition between the positive phage clone and its encoded peptide. Briefly, 96-well plates were coated with sRAGE and incubated overnight at 4 °C and washed six times with TBST. The microwells were blocked with BSA and then incubated with 100 μl of synthetic peptide encoded by the positive clone at various concentrations (0, 100, 200, 300, 400, 500, and 600 μM) in a 96-well plate. Then, 1×1010 pfu homologous positive phage clones were added and incubated for 2 h at room temperature, followed by incubation with HRP-conjugated anti-M13 monoclonal antibody (1:5000) for 1 h. The plates were then washed, substrate (50 μl/well TMB) was added, and the plates were kept at room temperature in the dark for 20 min. The reaction was terminated by adding 50 μl/well of H2SO4. The absorbance
Binding specificity of the RP-1 peptide to RAGE
Cell culturing, treatment with Aβ1–42, and cell viability assay SH-SY5Y cells were maintained in DMEM medium, supplemented with 10 % of FBS, and incubated in a humidified atmosphere of 5 % CO2 at 37 °C for 24 h. The SH-SY5Y cells were randomly divided into six groups: (1) control cells, (2) control cells incubated for 24 h in DMEM without serum in the presence of Aβ1–42, (3) control cells pretreated with RP-1 (0.1 μM) and then incubated for 24 h in DMEM without serum in the presence of Aβ1–42, (4) control cells pretreated with RP-1 (1 μM) and then incubated for 24 h in DMEM without serum in the presence of Aβ1–42, (5) control cells pretreated with RP-1 (10 μM) and then incubated for 24 h in
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DMEM without serum in the presence of Aβ1–42, and (6) control cells pretreated with RP-1 (100 μM) and then incubated for 24 h in DMEM without serum in the presence of Aβ1– 42. An MTT assay was used to evaluate the viability of the SHSY5Y cells. Following treatment with RP-1 and Aβ1–42 as described above (2.9), 20 μl of MTT were added to each well and incubated for 4 h at 37 °C. The absorbance was measured at 490 nm. Measurements of intracellular ROS, lipid peroxidation, and MMP The SH-SY5Y cells were treated with RP-1 and Aβ1–42 as described above (2.9). The level of ROS in the SH-SY5Y cells was measured based on 2,7-dichlorofluorescein-diacetate (DCFH-DA) as described previously (Khan et al. 2012). After treatment, the cells were incubated with 10 μM of DCFH-DA at 37 °C for 30 min. Then cells were harvested, rinsed, and re-suspended in PBS. Fluorescence levels were quantified using a fluorescence microplate, with excitation at 488 nm and emission at 525 nm. Malonyldialdehyde (MDA), which is used as an indicator of lipid peroxidation, was measured with a lipid peroxidation MDA assay kit. Mitochondrial membrane potential kit (JC-1) was used to analyze the mitochondrial membrane potential (MMP) of the cells. The SH-SY5Y cells were then stained with JC-1 and incubated at 37 °C for 20 min. After rinsing, the cells were resuspended in 0.5 of DMEM and analyzed with FACScan. Assay of superoxide dismutase (SOD), GPx, caspase3, and caspase9 activity The SH-SY5Y cells were treated with RP-1 and Aβ1–42 as described above. The activity of SOD, GPx, caspase3, and caspase9 was measured using commercial kits following the manufacturer’s instructions.
Appl Microbiol Biotechnol (2016) 100:825–835
Results Identification of phage clones that selectively bind to sRAGE In this study, phages that specifically bind to sRAGE were identified through four rounds of selection and one round of reverse biopanning. The output/input ratio of phages after each round of biopanning was used to determine the enrichment efficiency, which increased from 4.5×10–5 to 8.52×10–3 as shown in Table 1. This result indicates that phages capable of specifically binding to RAGE were significantly enriched. Reverse biopanning generated a high-affinity RAGE blocker that selectively competed with Aβ and combined with RAGE. DNA sequencing of selected phage clones After the fourth round of biopanning and one reverse biopanning, 40 phage clones were randomly selected. The DNA sequence and the deduced peptide sequence were analyzed and classified. Eight different phage clones and sequences were obtained, named P-1 to P-8 and RP-1 to RP-8, respectively (Table 2). Identification of the binding selectivity of the eight clones by ELISA The major fragment of the Aβ1–42 binding of V-RAGE was Aβ16–23 fragment (Gospodarska et al. 2011). Therefore, the amino acid sequences of the selected peptides were compared with Aβ16–23. As shown in Fig. 1 and Table 2, the eight clones detected by ELISA showed a strong binding ability to sRAGE compared to the control blocking buffer, and P-1 (the peptide was named RP-1) clone was the highest enriched and highest sequence similarity to Aβ16–23 (0.1538426, PAM250 Matrix). RP-1 (APDTKTQ) contains three amino acids that are identical to those of Aβ16–23 (KLVFFAED) peptides. Structurally, the theoretical of RP-1 is the nearest to that of Aβ16–23 peptides. These data suggested that RP-1 may be a candidate peptide. Therefore, RP-1 was chosen for further study.
Western blot analysis
Identification of synthesized RP-1 peptide
The SH-SY5Y cells were treated with RP-1 and Aβ1–42 as described above. Following collection of the SH-SY5Y cells, the cell homogenates were separated on 10 % SDS-PAGE gels and transferred to a PVDF membrane. The membrane was incubated with primary antibodies (P-PI3K, PI3K, P-AKT, AKT, Bax, Bcl-2, NF-κB, BACE1, RAGE, and β-actin) at 4 °C, followed by the addition of goat anti-rabbit IgG and HRP-linked antibody (1:1000) and incubation for 2 h at room temperature. Protein expression enhanced chemiluminescence was used for visualization.
The result of reverse-phase HPLC analysis showed that the purity of RP-1 peptide was 98 % (Fig. 2a). The result of mass spectrometry analysis showed that the molecular weight of synthesized RP-1 peptide was consistent with the theoretical value (Fig. 2b). Competitive inhibition assay To determine whether the synthetic peptide RP-1 and the corresponding phage clone P-1 competed for the same binding
Appl Microbiol Biotechnol (2016) 100:825–835 Table 1
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Enrichment of sRAGE binding phages for each panning round
Round
sRAGE (μg)
Aβ (μg)
Concentration of Tween 20 (v/v)
Input phage (pfu)
Output phage (pfu)
Recover (output/input)
1 2 3 4 Reverse biopanning
15 10 5 2.5 2.5
0 0 0 0 10
0.05 0.1 0.3 0.5 0.5
2.0×1011 3.4×1011 4.5×1011 4.0×1011 2.4×1011
9×106 3.1×107 1.3×108 3.4×109 1.4×109
4.5×10−5 9.2×10−5 3.0×10−4 8.52×10−3
site, a competitive inhibition assay was performed, using AP6 (QWTOSHP) as a control. When the synthetic RP-1 peptide was pre-incubated with sRAGE, the binding of the corresponding phage clone P-1 was inhibited in a dose-dependent manner, demonstrating that the positive phage clone bound to sRAGE by displacing the RP-1 peptide. At an RP-1 peptide concentration greater than 600 μM, the inhibition ratio was about 77 % (Fig. 3a), and the control peptide AP6 had no effect on the binding of the phage P-1, even at 600 μM. In addition, no significant competitive inhibition was observed for P-1 to phage clones P-22 and P-31 (Fig. 3b). These results indicated that the synthetic peptide RP-1 and the corresponding phage clone P-1 were competing for the same binding site, and the binding clone P-1 to sRAGE was mediated by the peptide RP-1.
do not bound to 3T3 cells (Fig. 4b). Furthermore, RP-1 and RAGE were co-localized on the surface of the SH-SY5Y cells (Fig. 4c). RP-1 increased cell viability against Aβ1–42-induced toxicity in SH-SY5Y cells The viability of the SH-SY5Y cells significantly decreased in the presence 4 μM Aβ1–42 in DMEM. RP-1 pretreatment for 2 h enhanced the cell viability of SH-SY5Y cells exposed to Aβ1–42 (Fig. 5a). The MMP was also detected in the RP-1 treatment groups, and the MMP was reduced (Fig. 5b) in the RP-1 treatment group.
Binding specificity of the RP-1 peptide to RAGE
Effects of RP-1 on Aβ1–42-induced caspase3 and caspase9 activation in SH-SY5Y cells
We examined the expression of RAGE in SH-SY5Y cells using Western blot analysis. As shown in Fig. 4a, the SHSY5Y cells express high level of RAGE, while the control 3T3 cells do not express RAGE. Confocal microscopy performed to observe the binding of RP-1 in the SH-SY5Y cells indicated that RP-1 specifically bound to SH-SY5Y cells and
Mitochondrial dysfunction, caspase-dependent toxicity, and dysfunction of downstream signaling pathways documented critical apoptotic events during AD processes (Ghavami et al. 2014). The cytosolic activity of caspase3 and caspase9 was detected as mitochondrial-related apoptotic molecular. The activities of both caspase3 and caspase9 increased in the
Table 2
Amino acid sequences of selected phage clones from phage library
Clone
Peptide
Sequence (N–C)
Similarities to Aβ16–23
Theoretical PI
GRAVY
Frequency
P-1/4/9/11/18/23/24/28/30/32/33/34/37/40 P-8/10/35/39 P-22/38 P-12/17/21/36 P-31 P-7/13/14/15/20
RP-1 RP-2 RP-3 RP-4 RP-5 RP-6
APDTKTQ SPRVGAT RHFHFPA HLHAHKL HPMSAPR HASKQLL
0.1538426 0.1538426 0.250000 0.1538426 0.0714286 0.0476190
5.88 9.47 9.76 8.77 9.76 8.76
–1.729 –0.286 –0.729 –0.586 –1.147 –0.286
14 4 2 4 1 5
P-2/3/5/6/25/26 P-16/19/27/29
RP-7 RP-8 Aβ16–23 Aβ1–42
ATRQPNH LSLSTTS KLVFFAED DAEFRHD SGYEVHH QKLVFFAE DVGSNKG AIIGLMVG GVVIA
0.0952381 0.0714286
9.80 5.52 4.37 5.31
–2.171 0.543 0.682 0.205
6 4
830
Appl Microbiol Biotechnol (2016) 100:825–835
Fig. 1 ELISA of binding ability of phage clone to sRAGE. *P
