Mechanistic Insights in Glycation-Induced Protein ... - ACS Publications

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Mechanistic Insights in Glycation-Induced Protein Aggregation Miquel Adrover,*,† Laura Mariño,† Pilar Sanchis,†,‡ Kris Pauwels,§,∥ Yvonne Kraan,⊥ Pierre Lebrun,§,∥ Bartolomé Vilanova,† Francisco Muñoz,† Kerensa Broersen,⊥ and Josefa Donoso† †

Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Departament de Química, Universitat de les Illes Balears, Carretera de Valldemossa km 7.5, E-07122 Palma de Mallorca, Spain ‡ Research Unit of Son Llatzer Hospital, Carretera de Manacor km 4, E-07198 Palma de Mallorca, Spain § Structural Biology Brussels, Vrije Universiteit Brussels, Pleinlaan 2, 1050 Brussels, Belgium ∥ VIB Department of Structural Biology, Vlaams Instituut voor Biotechnology, Pleinlaan 2, 1050 Brussels, Belgium ⊥ Faculty of Science and Technology, Nanobiophysics, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Drienerlolaan 2, 7500AE Enschede, The Netherlands S Supporting Information *

ABSTRACT: Protein glycation causes loss-of-function through a process that has been associated with several diabetic-related diseases. Additionally, glycation has been hypothesized as a promoter of protein aggregation, which could explain the observed link between hyperglycaemia and the development of several aggregating diseases. Despite its relevance in a range of diseases, the mechanism through which glycation induces aggregation remains unknown. Here we describe the molecular basis of how glycation is linked to aggregation by applying a variety of complementary techniques to study the nonenzymatic glycation of hen lysozyme with ribose (ribosylation) as the reducing carbohydrate. Ribosylation involves a chemical multistep conversion that induces chemical modifications on lysine side chains without altering the protein structure, but changing the protein charge and enlarging its hydrophobic surface. These features trigger lysozyme native-like aggregation by forming small oligomers that evolve into bigger insoluble particles. Moreover, lysozyme incubated with ribose reduces the viability of SH-SY5Y neuroblastoma cells. Our new insights contribute toward a better understanding of the link between glycation and aggregation.

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transthyretin,7 albumin,10 β2-microglobulin,11 or glucose oxidase.12 The increased aggregation tendency was associated with the chaotropic effect of PG, but this suggestion was only supported by low-resolution structural techniques. For instance, changes in intrinsic protein fluorescence upon glycation seemed to support structural rearrangements.10,13,14 On the other hand, circular dichroism (CD) spectroscopy revealed subtle spectral changes concomitant with glycation, which in some cases were attributed to secondary structure alterations,13,15−17 while in others they were not linked to such changes.10,18 So far, the glycation effect on the protein fold has not been studied at the residue level yet. Glycation often results in a heterogeneous population of final products, which hampers detailed structural studies. For this reason, X-ray crystallography has never been used to characterize glycated proteins, while NMR spectroscopy has only been marginally used to show that glycation mediated by glucose distorts the α-helicity

INTRODUCTION

Protein glycation (PG) involves the chemical reaction of reducing sugars with primary amino groups, in contrast to glycosylation, which comprises an enzyme-catalyzed and sitespecific addition of carbohydrate moieties. Spontaneous glycation encompasses the reversible formation of a Schiff base that converts into an Amadori product, which can then further rearrange to give advanced glycation end products (AGEs)1 (Figure S1, Supporting Information). Accumulation of AGEs has been suggested as one of the main responsible factors of diabetes-associated complications, such as retinopathy,2 nephropathy,3 or atherosclerosis.4 In addition, AGEs were also recently linked to neurodegenerative diseases.5,6 The molecular mechanism through which PG triggers these pathologies is poorly understood. Glycation can modify the chaperon activity of some proteins7 and cause a loss-of-function in proteins with diverse biological roles (e.g., enzymes,8 and scaffolding proteins9) through chemical alterations, but also as a result of an increased aggregation tendency, as was already observed in proteins such as © XXXX American Chemical Society

Received: July 23, 2014

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dx.doi.org/10.1021/bm501077j | Biomacromolecules XXXX, XXX, XXX−XXX

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Fluorescent AGEs Formation. Time-dependent fluorescent AGEs formation was monitored on a Cary Eclipse fluorescence spectrophotometer equipped with a Peltier temperature controlled cell holder. Centrifuged aliquots of the HEWL/ribose mixture at different incubation times were diluted to a final HEWL concentration of 10 μM. The excitation wavelength was 320 nm, and the emission was recorded from 360 to 540 nm at 25 °C. HEWL and ribose solutions were used as control. Reaction mixtures containing Ac-Lys (1.2 mM) or Ac-Arg (2.2 mM) and ribose (0.2M) were also analyzed. The assays were done in triplicate. Intrinsic Protein Fluorescence. Centrifuged aliquots of the HEWL/ribose mixture and of the control HEWL solution were collected at different incubation times and diluted to 10 μM protein concentration. The fluorescence emission spectra were recorded between 300 and 600 nm (λexc 280 nm) at 25 °C. Aliquots of the reaction mixture collected at 0 h and 6 days of incubation were also titrated with acrylamide (from 0 mM to 82 mM). The assays were performed in triplicate. To analyze the quenching effect of ribosylated AGEs on intrinsic protein and Trp fluorescence, a phosphate buffered solution containing Ac-Lys (5 mM) and ribose (0.2M) was incubated at 37 °C for 7 days and used to titrate solutions containing either native HEWL (6 μM) or Boc-Trp (36 μM). Samples were titrated from 0 mM to 180 μM AGEs concentration assuming that all Ac-Lys was ribosylated. This assay was repeated using AGE-1 or Nmethylglycine (25 mM) stock solutions as titrants. Isolation and Characterization of the Ribose-Derived AGEs Formed on Ac-Lys. A reaction mixture containing Ac-Lys (5 mM) and ribose (0.2M) was incubated at 37 °C during 7 days. The solution was analyzed on a Shimadzu-LC 10AT chromatograph equipped with a Shimadzu SPD-10AV UV/vis detector (the range 200−600 nm was monitored) and a Supelco Ascentis RP-Amide (25 × 0.46 cm, 5 μm) column. The compounds were separated by using AcN/water-5 mM potassium phosphate (pH 7.4) in a gradient of 0−38% AcN for 40 min (flow rate 1 mL/min). Fractions corresponding to the eluted peaks at tR ∼ 25 and ∼28 min were freeze-dried and redissolved in 0.5 mL of phosphate buffer (0.2M) pD 7.4. Samples were then characterized by NMR and matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) spectroscopy. 1D-1H, 1D-13C, 1H,1H-COSY, 1H,1HTOCSY, 1H,13C-HSQC, 1H,13C-HMBC, and 1H,1H-ROESY experiments were carried out on a Bruker Avance III operating at 600 MHz and equipped with a 5-m 13C,15N,1H triple resonance cryoprobe. Experimental data corresponding to the compound eluted at tR ∼ 25 min. UV/vis: λmax 303 nm; fluorescence: λexc 342 nm/λem 420 nm. Experimental data corresponding to the compound eluted at tR ∼ 28 min. 1H NMR (600 MHz, D2O): δ 9.74 (s, H−C(15)), 7.31 (d, H− C(9), 2JH9−H10 = 2.27 Hz), 6.47 (d, H−C(10)), 5.29 (t, H−C(12), 3 JH12−H13 = 6.00 Hz), 4.41 (m, H2−C(8)), 4.19 (q, H−C(3), 4 JH3−H5a,H5b = 4.47 Hz), 3.87 (d, H−C(13)), 2.09 (s, H−C(1)), 1.86 (m, H−C(7)), 1.87/1.72 (m, H−C(5)), 1.38 (m, H−C(6)); 13C NMR (600 MHz, D2O): δ 179.5 C(15), 178.2 C(4), 172.64 C(2), 138.0 C(11), 132.3 C(9), 125.9 C(14), 107.6 C(10), 66.7 C(12), 65.4 C(13), 54.5 C(3), 48.2 C(8), 30.5 C(5), 29.4 C(7), 21.7 C(6), 21.3 C(1); MS (m/z): [M + Na]+ calcd. for C15H22N2O6, 349.15; found, 349.13 (100%); UV/vis: λmax 297 nm; fluorescence: λexc 342 nm/λem 420 nm. The compound was identified as (2S)-2-acetamido-6-(3-(1,2dihydroxyethyl)-2-formyl-1H-pyrrol-1-yl)hexanoic acid (AGE-1). Enzymatic Digestions. Dialyzed aliquots (10 μL) of the HEWL/ ribose mixture were combined with 15 μL of NH4HCO3 buffer (50 mM) at pH 9.2 and 1.5 μL of an aqueous DTT solution (100 mM). The samples were incubated at 95 °C for 5 min and cooled to room temperature. Next, 3 μL of iodoacetamide solution (18 mg/mL) and 10 μL of a trypsin solution (20 μg/mL) were added. Mixtures were incubated overnight at 37 °C to finally add 0.5 μL of TFA. Dialyzed aliquots (5 μL) were also combined with 3 μL of endoproteinase GluC (3 μg). The mixtures were incubated at 25 °C for 15 h. Two microliters of a 100 mM DTT solution was added prior to mass analysis. MALDI-TOF Mass Spectrometry. Digested sample (1.5 μL) was combined with 1.5 μL of matrix solution (10 μg of α-cyano-4hydroxycinnamic acid in a solution 50% acetonitrile and 2.5% TFA). A

of short model peptides.19 However, high-resolution structural studies on full-length proteins are required to describe the effects of glycation on the protein fold in detail. Apart from lacking high-resolution structural information, the aggregation−modulation mechanism of glycation also remains obscure. Some publications suggest that glycation induces amyloid cross-β structure formation,20,21 while others claim that glycation does not change the secondary structure but alters the tertiary structure forming globular amyloid-like deposits.10,14 Here we have studied the glycation of hen egg white lysozyme (HEWL) with ribose (ribosylation) as a model system to accurately describe the interplay between glycation, aggregation, and its effect on cell viability. HEWL is a globular protein with antibacterial activity22 that has already been used as a model to study PG since it possesses six Lys as potential glycation sites.8,14,17 Wild-type HEWL has a low aggregation tendency under physiological-like conditions, although it can form amyloid fibrils at low pH and elevated temperature.23 Therefore, HEWL is ideal to study if glycation per se induces aggregation. On the other hand, D-ribose has been recently found to have an abnormally high concentration in the urine of type II diabetics.24 In addition, its high reactivity structurally mimicking glucose has promoted its widespread use as a glycating reagent, especially to generate glycated aggregates.10,11,25−27 By using various complementary techniques, we show that HEWL ribosylation implies the formation of different AGEs on the protein surface, without altering the structure but changing its hydrophobic surface. These two aspects are responsible for native-like aggregation, demonstrating that misfolding is not a necessity to induce the formation of glycated aggregates.

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MATERIALS AND METHODS

Materials. All the reagents were purchased from Sigma-Aldrich or Acros Organics. Buffers reagents were ACS grade, except those used for cell viability studies, which were molecular biology grade. All solutions were prepared by using milli-Q water. Preparation of Ribosylated HEWL. Solutions of monomeric HEWL (0.4 mM) and ribose (0.4M) were freshly prepared in 0.2 M phosphate buffer at pH 7.4 and filtered with a 0.22 μm membrane. These solutions were combined in 1:1 ratio to initiate the ribosylation reaction. HEWL (0.2 mM) and ribose (0.2M) solutions were used as controls. Reaction mixture and control samples were incubated at 37 °C. Aliquots at different incubation times were collected for analysis. If required, aliquots (0.5 mL) were dialyzed against 5 mM phosphate buffer (pH 7.4) for 12 h at 4 °C to remove unreacted ribose prior to analysis and/or centrifuged. Arginine Modification. Free arginine levels were monitored as published previously.28 Aliquots (100 μL) of the HEWL/ribose mixture were mixed with 100 μL of a 2% sodium hypobromite solution (0.68 mL of bromine in 100 mL of 5% NaOH), 2 mL of thymol solution (0.02% in NaOH 1N), and 2.8 mL of milli-Q water. Mixtures were kept at room temperature for 1 h before determining their absorbance at 440 nm. A HEWL buffered solution was used as control. The amount of arginine in each aliquot was deduced from a calibration curve. All measurements were done in triplicate. Determination of Dicarbonyl Compounds. Aliquots (100 μL) of the HEWL/ribose mixture were mixed with 900 μL of phosphate buffer and 100 μL of 0.1% 2,3-diaminonaphthalene solution in methanol. The solution was then incubated overnight at 4 °C. Afterward, it was extracted by 4 mL of ethyl acetate, and the organic fraction was dried under vacuum. The dried extract was reconstituted with 200 μL of methanol and analyzed by fluorescence spectroscopy (λexc 271 nm; λem 503 nm).29 A 0.2 M ribose solution was used as control. Concentration of dicarbonyl compounds was calculated using a glyoxal calibration curve. All measurements were done in triplicate. B


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0.5 μL aliquot of this mixture was spotted onto a steel target plate (MTP 384), air-dried, and subjected to mass determination. Mass spectra were analyzed on a Bruker Autoflex III MALDI-TOF spectrometer equipped with a 200-Hz smart-beam pulsed N2 laser (λ 337 nm). The IS1 and IS2 voltages were 19 kV and 16.65 kV respectively, and the lens voltage was 8.2 kV. Measurements were performed using a positive reflector mode with matrix suppression below 400 Da. External calibration was performed using a standard peptide mixture. Mass spectra of digested samples were matched against Swiss-Prot databases using the Mascot search engine (Matrix Sciences). The assay was performed in duplicate. Dialyzed aliquots (5 μL) of nondigested and centrifuged HEWL/ribose mixture were combined with 5 μL of water and 10 μL of matrix solution. A 0.5 μL aliquot of this mixture was spotted onto the steel target plate, air-dried, and analyzed by MALDI-TOF using the same conditions. The spectra were calibrated externally using a protein calibration standard from Bruker. The experiments were performed in duplicate. Circular dichroism. CD spectra were recorded with a J-715 spectropolarimeter (JASCO). Far-UV CD spectra corresponding to the centrifuged HEWL/ribose mixture at different incubation times were recorded between 260 and 190 nm in a quartz cell of 0.02 cm path length. The scan speed was 50 nm/min with a response time of 2 s and a step resolution of 0.2 nm, while eight scans were accumulated. HEWL buffered solution was used as control. Thermal denaturation curves were recorded by monitoring the change in CD intensity at 220 nm while increasing the temperature within the range of 37−90 °C at a heating rate of 2 °C/min. Data were baseline corrected. Fourier Transform Infrared (FT-IR) Spectroscopy. Attenuated total reflection infrared spectra of dialyzed and centrifuged aliquots of the HEWL/ribose mixture were measured with 5 μL of each aliquot on a diamond attenuated total reflectance (ATR) plate (50 × 20 × 2 mm), used as internal reflection element, with an aperture angle of 45°. Phosphate buffer (5 mM) at pH 7.4 was used for background correction. Solid state FT-IR spectra were collected in KBr pellets (1 mg sample/100 mg KBr) for native HEWL, for HEWL amyloid fibrils, and for the insoluble ribosylated HEWL aggregates. Amyloid fibrils were generated by incubating a 5 mM HEWL solution at pH 2 during 7 d at 65 °C to be then dried under vacuum to obtain a pellet. Ribosylated HEWL aggregates were generated by incubating a buffered solution containing HEWL (5 mM) in the presence of ribose (0.2M) for 7 days at 37 °C. The solution was then centrifuged, and the pellet was washed 5 times with water before drying it under vacuum. All data were obtained using a Bruker Vertex 80v FT-IR spectrophotometer, equipped with a MCT photovoltaic detector (broad band 10000−400 cm−1; liquid N2 cooled), and results were analyzed using OPUS software with a scan from 800 to 3000 cm−1 at 3 cm−1 resolution at 120 scans per spectrum. NMR Spectroscopy. For NMR analysis, the HEWL/ribose mixture was prepared identically as previously depicted, but using a higher HEWL concentration (5 mM). While incubating at 37 °C for 7 days, 0.5 mL aliquots were taken at regular intervals, centrifuged, and the supernatant was then buffered exchanged into 20 mM sodium citrate buffer (pH 3.5) by using a HiTrap desalting column coupled to an Ä KTA FPLC system. The elution peak was concentrated to a volume of 0.5 mL using a Vivaspin 5k concentrator. NMR spectra were acquired from 0.5 mL buffered exchanged aliquots (20 mM sodium citrate at pH 3.5 with a protein concentration ∼5 mM) containing 10% (v/v) D2O. All the spectra were acquired at 37 °C on a Bruker Avance III operating at 600 MHz and equipped with a 5-m triple resonance cryoprobe. For each aliquot, 1H NMR, 1H,15N-HSQC and 1H,1H-NOESY (100 ms) spectra were recorded at natural abundance. Amide chemical shift perturbation was quantified as the average chemical shift change of the amide protons and nitrogens: Δδave=[1/2]·[((ΔδHN)2+(ΔδN)2)/25]1/2. Longitudinal (T1) and transverse (T2) relaxation measurements were carried out using a series of 9 experiments with relaxation delays ranging from 10 to 1800 ms and from 16 to 350 ms, respectively. T1 and T2 values for each residue were used to determine R1 and R2, which in turn were fed to the r2r1_tm program (Palmer’s group: http://www.hhmi.umbc.edu/toolkit/ analysis/palmer/r2r1_tm.html) to determine the local correlation

time for each residue. Upon averaging, the overall τc values could be determined. Water suppression was achieved by the watergate pulse sequence. Proton chemical shifts were referenced to the water signal at 37 °C. 15N chemical shifts were referenced indirectly using the 1H,X frequency ratios of the zero-point. The chemical shift values for HEWL resonances at pH 3.5 and 37 °C were taken from previously published work.30 All the spectra were processed using NMRPipe/ NMRDraw, analyzed, and plotted by Xeasy/Cara and Sparky software. Isoelectric Focusing. The isoelectric points of HEWL at different ribosylation times were determined using a Phast System (Pharmacia). Aliquots of the HEWL/ribose mixture were taken at different incubation times and diluted up to 0.1 mM protein concentration. Volumes of 4 μL were loaded onto PhastGel IEF 3−9 gel (PhastGel), which was fixed in 20% trichloric acid, washed, and stained with PhastGel Blue R. The isoelectric points were referenced to a calibration sample (Cat. No. 17047101, Pharmacia). The experiment was performed in duplicate. Native-Polyacrylamide (PAGE) Gel Electrophoresis. Protein samples were analyzed in native-PAGE with 4−20% Mini-Protean TGX gels (Bio-Rad). For acid native gel the electrolyte for electrode reservoirs was acetic acid/β-alanine at pH 4.3, whereas the sample buffer (×2) contained 0.01% methylene green in 20% (w/v) of glycerol and potassium acetate at 25 mM (pH 6.8). Electrophoresis was run from anode to cathode for 4 h at 80 V. The basic native gel was run by using an electrophoresis buffer that simultaneously was 192 mM in glycine and 25 mM in Tris (pH 8.3), while the sample buffer (×2) contained 0.02% bromophenol blue in 20% (w/v) of glycerol and 625 mM Tris (pH 6.8). Electrophoresis was run from cathode to anode for 2.5 h at 100 V. Proteins were stained with Coomassie blue R-250 (Bio-Rad). Sodium Dodecyl Sulfate (SDS)-PAGE Electrophoresis. Protein samples were subjected to SDS-PAGE analysis by using 4−20% MiniProtean TGX precast gels (Bio-Rad). Ten microliters were mixed with 5 μL of Laemmli sample buffer (Bio-Rad) and 5 μL of an aqueous DTT solution (1M). Afterward, the mixture was heated at 100 °C for 10 min before loading it onto the gel. Proteins were visualized with Coomassie blue R-250 (Bio-Rad). The experiment was performed in duplicate. Determination of Surface Hydrophobicity (H0). Aliquots of the reaction mixtures containing HEWL (0.2 mM) or Ac-Lys (1.2 mM) and ribose (0.2M) were collected at different incubation times, centrifuged, and diluted in phosphate buffer to obtain a final Lys concentration of 60 μM. The fluorescence spectra of the resulting solutions were recorded between 400 and 600 nm (λexc 385 nm) before and after the addition of 10 μL of a 7.5 mM solution of 1anilinonaphthalene-8-sulfonic acid (ANS). The surface hydrophobicity (H0) values of HEWL at different ribosylation times were determined upon ANS addition according to Haskard and Li-Chan.31 Different volumes (5, 10, 15, 20, 25, and 30 μL) of the HEWL/ribose mixture taken at different incubation times were diluted in 3 mL of 5 mM phosphate buffer (pH 7.0) and mixed with 20 μL of an ANS solution (8 mM) prepared in the same buffer. The fluorescence intensity of the mixtures was measured at 470 nm (λexc 390 nm). The fluorescence intensity of each sample without ANS was also measured and then subtracted to the fluorescent intensity obtained in the presence of ANS. The initial slope of the fluorescent intensity versus protein concentration plot was used to obtain the H0 value. These measurements were repeated using a solution containing HEWL (0.2 mM) as control. Each experiment was performed in duplicate. Size-Exclusion Chromatography. Aliquots (100 μL) of the HEWL/ribose mixture at different incubation times were analyzed by size-exclusion chromatography at room temperature using a Superdex75 HR 10/300 column (GE Healthcare) equilibrated with 50 mM sodium phosphate and 750 mM NaCl at pH 6.5. Aliquots were injected using an Ä kta basic FPLC system at a flow rate of 0.5 mL/min coupled to a UV/vis detector (280 nm). Small Angle X-ray Scattering (SAXS). SAXS experiments were performed on the SWING beamline at the SOLEIL synchrotron (λ 1.03 Å). The Aviex charge-coupled device detector was positioned at 1507.5 mm. 60 μL of a HEWL/ribose mixture that was previously C


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Figure 1. HEWL lysine side chains are the main ribosylation targets. (a) Relative peptide fragment intensity characteristic of K1 (V2−K13; black), K13 (R14-R21; red) and K116 (G117-R125; green) in HEWL after trypsin digestion and MALDI/TOF analysis at different ribosylation times. The peptide intensity values were internally referenced to the intensity of the N46-R61 fragment (m/z 1753.8). The insert contains the half-life times of different peptide fragments typical of each Lys. (b) Temporal variation of free guanidinium content of a solution containing HEWL incubated alone (○) or in the presence of ribose (●) at 37 °C. (c) Dicarbonyl content determined at different incubation times for a 0.2 M ribose solution incubated at pH 7.4 and 37 °C alone (○) or in the presence of 0.2 mM HEWL (●). (d) Intermolecular interactions of K1 (left) and K13 (right) ammonium group with nearby carboxylate groups in HEWL crystal structure (PDB 1LSE). incubated with or without ribose for 7 days was injected onto a sizeexclusion column (SEC-3, 300 Å; Agilent), using an Agilent HPLC system, and eluted directly into the SAXS flow-through capillary cell at a flow rate of 0.2 mL/min and at 15 °C. The elution buffer contained 20 mM Tris-HCl (pH 7.4), 150 mM NaCl and 3% glycerol. SAXS data were collected continuously, with a frame duration of 1.0 s and a dead time between frames of 0.5 s. Selected frames corresponding to the main protein elution peak were averaged using FOXTROT.32 A large number of frames were collected during the void volume of the elution and averaged to account for buffer scattering, which was subsequently subtracted. Data reduction to absolute units, frame averaging and subtraction were done using FOXTROT.32 All data processing, analysis and modeling steps were carried out with the ATSAS suite.33 The radius of gyration RG was derived by the Guinier approximation I(q) = I(0) exp(−q2RG2/3) for qRG < 1.3 using PRIMUS QT.34 GNOM was used to compute the pair-distance distribution functions, P(r), and to obtain the maximum dimension of the macromolecule (Dmax). Normalized Kratky plots (i.e., (qRG)2I(q)/I(0) versus qRG) were used to assess the conformation of the protein. Finally, 10 ab initio models were generated with gasbori.35 The best model was selected based on χ2 values and Normalized Spatial Discrepancy (NSD) value compared to the average by DAMAVER.36 Atomic Force Microscopy (AFM). Centrifuged aliquots from the HEWL/ribose mixture were collected after 0 h and 10 days of incubation at 37 °C and diluted 40-fold with water. A volume of 50 μL of the resulting dilutions were placed onto the mica surface and incubated for 5 min at room temperature before drying with N2 gas. The mica was rinsed 10 times with 20 mL milli-Q water and dried with N2 gas before observation under a Veeco Multimode atomic force microscope equipped with a NanoScope IV controller. The particle sizes were measured using the NanoScope SPM v5 software. Transmission Electron Microscopy (TEM). An aliquot (100 μL) of a reaction mixture containing HEWL (5 mM) and ribose (0.2M) in 0.2 M phosphate buffer at pH 7.4, which was previously incubated at 37 °C for 7 days, was taken and centrifuged. The insoluble fraction was

rinsed with 5 mL mili-Q water and then resuspended in 0.5 mL of mili-Q water. A droplet of 5 μL of the resulting sample was placed on freshly prepared carbon-coated copper grids and left to adsorb for 60 s. After adsorption to the grid surface, the sample was washed briefly in milli-Q and stained with 1% (w/v) uranylacetate for 30 s. Micrographs of negatively stained areas were taken with a Hitachi transmission electron microscope (H-600) operating at 75 kV. X-ray Powder Diffraction. X-ray powder diffraction measurements were carried out on HEWL amyloid fibrils, on ribosylated HEWL aggregates, and on native HEWL (see the FT-IR section for the procedure followed to generate these species) by using a Siemens D5000 theta/theta diffractometer and CuKα radiation. Powder diffraction data were collected by step scanning from 2 to 60° (2θ) with a step size of 0.05° and a count time of 3s/step. Cell Viability. Aliquots from the HEWL/ribose mixture were taken after 0 and 14 days of incubation and buffer exchanged against serumdeprived Dulbecco’s modified Eagle’s medium (DMEM/F-12) supplemented with 100 IU/ml penicillin and 100 μg/mL streptomycin using Zeba spin columns. The resulting samples were diluted to different HEWL concentration prior to cell treatment. The human neuroblastoma cell line SH-SY5Y (ATCC number CRL-226) was used for cell viability assays. Cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum (HyClone, ThermoScientific), 100 IU/ml penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37 °C in a humidified 5% CO2 atm. Cell viability assays were performed in 96-well plate after plating 25 000 cells per well in the culture media. Medium was removed from the sample and replaced with HEWL/ribose aliquots at various HEWL concentrations (0−30 μM). After 24 h treatment, cell viability was analyzed using the Cell Titer Blue Cell Viability assay (Promega). After 4 h incubation with Cell Titer dye, color conversion was analyzed by measuring the fluorescence intensity of the samples (λexc 544 nm; λem 590 nm). Enzymatic Activity. Aliquots from solutions containing HEWL incubated alone or in the presence of ribose were taken at different incubation times, and the rate of lysis of Micrococcus lysodeikticus cells D


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Figure 2. Formation of advanced ribosylation end products on lysine side chains. (a) Temporal changes in maximal fluorescent intensity (λexc 320 nm) for solutions containing HEWL/ribose (●), HEWL alone (○) and ribose alone (▼). (b) HPLC chromatogram of the Ac-Lys/ribose reaction mixture after 7 days of incubation in phosphate buffer at pH 7.4 and 37 °C. The insert contains the chemical structure of the characterized AGE-1. Each atom was arbitrarily numbered. (c) Overlapping of the fluorescence emission spectrum of AGE-1 (red) with the fluorescent spectrum of the HEWL/ribose reaction mixture after 7 days of incubation at 37 °C (black). The spectra were obtained at λexc 320 nm and normalized for comparison purposes. (d) MALDI-TOF spectra of the 10−20 kD region of a reaction mixture containing HEWL and ribose at different incubation times at 37 °C and at pH 7.4. were measured. Samples containing HEWL were diluted to 20 μg/mL in water, and 100 μL of this sample was then mixed with 900 μL of a fresh suspension of 0.3 mg/mL cells prepared in 0.1 M phosphate buffer at pH 7.0. The lysis was determined by measuring the change in absorbance at 450 nm during 5 min.

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show that each Lys has an intrinsic reactivity that is under the influence of its protein context. As glycation can give rise to a heterogeneous range of reaction products, we also characterized the ribosylation products formed on Lys side chains. Progressed stages of the HEWL ribosylation involved the formation of fluorescent products (Figure 2a and Figure S2), which were exclusively formed on the ε-amino groups as proved by the control experiments performed with Ac-Lys/ribose and Ac-Arg/ribose mixtures (Figure S3). Fluorescent AGEs were chemically characterized using an Ac-Lys/ribose reaction model (previously incubated at 37 °C for 7 days) and HPLC-UV analysis. The peak appearing at tR ∼ 28 min contained a single compound characterized as AGE-1 (Figure 2b) with an emission spectrum similar to that observed for the AGEs formed on ribosylated HEWL (Figure 2c). This observation strongly suggests that AGE-1 could be an end-product formed on HEWL. The peak eluting at tR ∼ 25 min was comprised of a mixture of compounds that could not be separated, and the low concentration precluded their characterization (Figure S4). However, at least one of the compounds was fluorescent (Figure S5). To determine whether AGE-1 was also formed on HEWL, we used MALDI-TOF MS analysis. Signals of the full-length monomeric HEWL became broader and shifted on average with a Δm/z of 837 Da at prolonged ribosylation times as a result of the formation of a heterogeneous mixture (Figure 2d). This molecular weight increment agrees with the assumption

RESULTS

Ribosylation Induces AGE-1 and Nε-(carboxymethyl)lysine (CML) Formation on HEWL Lysine Side Chains. Ribose was able to chemically modify the Lys side chains of HEWL (Figure 1a), although it also modified ∼13% of the Arg side chains (after ∼7 days of reaction) (Figure 1b). The latter process likely occurred through the reaction of Arg with dicarbonyl compounds,8 which were produced in substantial amounts under the used experimental conditions (Figure 1c). To assess the ribosylation sensitivity of every individual Lys, we used trypsin digestion and MALDI-TOF MS analysis, which allowed us to analyze the temporal intensity variation of different fragments typical of each Lys (Figure 1a and Table S1). The results show that the ribosylation sensitivity of each hot-spot is affected by its protein surroundings but it does not correlate with the solvent accessible surface area (SASA) or with the pKa values (Table S1). Inspection of the HEWL crystal structure (PDB 1LSE) revealed that those residues with shorter half-lifetime, K1 and K13, are located in close proximity of a carboxylate group. Therefore, this group might catalytically enhance the Lys ribosylation sensitivity (Figure 1d). Our results E


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Table 1. Ribosylated Products Identified on K1 and K13 in HEWL after Digestion with Glu-C and MALDI-TOF Peptide Mapping incubation timee a

peptide fragment

m/z fragment

increase in the MW peptide fragment (Da)

type of modification

0h

9h

70 h

166 h

K1

NH2_KVFGRCE-L

K13

E-LAAAMKRHGLD-N

838.4 970.2 897.3 976.4 1028.2 1034.3 954.8 1182.6 1314.4 1241.2 1320.6

Δm = 0 Δm = 132 Δm = 59 Δm = 138 Δm = 190 Δm = 196 Δm = 116 Δm = 0 Δm = 132 Δm = 59 Δm = 138

native fragment AMDb CMLc AGE-1d AMDb+CMLc AGE-1d+CMLc CMLc+CMLc Native fragment AMDb CMLc AGE-1d

++++ − − − − − − ++++ − − −

+ +++ − − − − − − + − −

− + + + − − − − − + −

− − + ++ + + + − − ++ +++

residue

The ribosylation hot-spots are highlighted in bold. The hyphen indicates the Glu-C cleavage site. bAmadori compound. cNε-carboxymethyl-lysine (CML). dFor structural details, see Figure 2b. e“++++” indicates that the peptide represents 75−100% of the total peptide content, “+++” 50−75% of the total peptide content, while “++” indicates that the peptide represents 25−50% of the total peptide content and “+” represents 0−25%. “−” indicates that the peptide fragment was not observed. The relative quantification of the peak intensity was performed with the S36-D52 fragment (m/z 1842.7) as internal reference. a

Figure 3. Ribosylation effect on the size of HEWL. (a) Ratios between the intensity of the MALDI peaks corresponding to the monomeric and the dimeric HEWL at different ribosylation times. Error bars were obtained from five replicates. (b) Elution profiles from a Superdex-75 HR 10/300 column of the HEWL/ribose reaction mixture at different incubation times. (c) Kratky plot of the SAXS signals for the monomeric HEWL isolated from the SEC elution peak after 7 days of incubation at 37 °C and at pH 7.4 in the absence (red) and in the presence (blue) of ribose. (d) Ab initio models based on the experimental SAXS data of HEWL after 0 h (left) and 7 days (right) of incubation at 37 °C and at pH 7.4. The colored spheres represent the calculated models that are superposed with the cartoon representation of the HEWL model (PDB 1LSE).

that AGE-1 (Δm/z 138 Da) is the final product formed on the six Lys. To verify this hypothesis, we digested HEWL at different ribosylation times with endoproteinase Glu-C. We used the N-terminal K1-E7 (m/z 838.4) and the L8-D18 (m/z 1182.6) fragments for the identification and the relative quantification of the compounds formed on K1, on the Nterminus amino group and on K13. Ribosylation resulted in the appearance of new peaks that were absent in the spectrum of native HEWL. Those peaks were assigned to the Amadori adduct, CML, and the AGE-1, consistent with mass increases of

132, 59, and 138 Da, respectively. For the K1-E7 fragment, double ribosylated fragments were also identified, corresponding to the simultaneous formation of end-products on the Nterminus and on K1 side chain (Table 1). For K1 and K13, the native peptide fragment decreased with the formation of the Amadori compound, which degraded to mainly form AGE-1 and CML. These results show that AGE-1 and CML are the main ribosylation end-products formed on HEWL. Ribosylation Has Little Impact on HEWL Hydrodynamic Radius. MALDI-TOF MS analysis also suggested F


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Figure 4. Ribosylation effect on the secondary and tertiary structure of HEWL. (a) CD spectra of HEWL incubated in the absence and presence of ribose for 0 and 11 days at 37 °C and at pH 7.4. (b) 2D 1H,1H-NOESY spectra of the region containing the amide resonances (5−11 ppm) of HEWL after 0 h (red) and after 6 days (blue) of ribosylation. (c) 1H NMR spectra of HEWL at different ribosylation times at pH 3.5 and at 37 °C. Only the regions corresponding to the Trp-indol, the amide, and the highly shifted resonances are shown. (d) Overlapping of long-range 1H,1HNOEs typical of different regions in HEWL structure at 0 h (red) and 6 days (blue) of ribosylation.

the formation of low-n oligomers as evidenced by the decrease of the monomer/dimer and monomer/trimer ratios upon ribosylation (Figure 3a and Figure S6). To isolate and further study the monomeric ribosylated HEWL, size-exclusion chromatograms (SEC) were collected at different ribosylation times (Figure 3b). The peaks broadened upon ribosylation, potentially as a result of the heterogeneity, and shifted toward lower elution volumes, which suggested an increase in the hydrodynamic radius. The peaks also displayed a distinct shoulder appeared at the starting edge of the main elution peak that could correspond to the formation of low-n oligomers. To analyze the effect of ribosylation on the hydrodynamic behavior of monomeric HEWL, we isolated the main SEC elution peak after incubation for 7 days with or without ribose. SAXS scattering curves of both samples showed a nearly identical profile (Figure S7), and their linear Guinier plot of the low q region indicated the absence of aggregates (Figure S8). The hydrodynamic size of native HEWL compared well with that of the ribosylated HEWL, as evidenced by the normalized Kratky plot (Figure 3c), and the global shape of the ab initio models based on the SAXS data fit very well with the HEWL crystal structure (Figure 3d). Nevertheless, subtle differences could be detected in the measured size-related parameters (Table S2), likely due to a change in the hydration layer. Ribosylation Does Not Change HEWL Structure. Although the SAXS results indicated that monomeric ribosylated HEWL has native-like hydrodynamic behavior,

they do not provide information about the ribosylation effect on the protein structure. To determine whether AGE formation could modify the secondary structure of monomeric HEWL and its soluble low-n oligomers, FT-IR, far UV-CD, and NMR experiments were performed. CD spectra exhibited the two minima characteristic of an α-helical conformation (Figure 4a), while the amide I band in the FT-IR spectra was still centered at 1649 cm−1, further indicating the prevalence of an α-helical native fold (Figure S9). NMR spectroscopy showed that the strong native HNi−HNi+1 NOEs, characteristic of helical regions, were still present at prolonged ribosylation times (Figure 4b). Collectively, these results demonstrate that ribosylation does not change the secondary structure of HEWL. Ring shifted resonances, arising from aliphatic groups close to aromatic rings, are very sensitive to the changes in tertiary structure. The 1H NMR spectra recorded at different incubation times show that these resonances do not change their values upon ribosylation, as it also happens with the TrpHε and with the amide signals (Figure 4c). The only change arises from an increase in signal width as a result of an enlargement of the rotational correlation time, also indicating heterogeneity and/or oligomerization.37 Moreover, the intensity of long-range NOEs, typical of different HEWL regions defining its tertiary structure, is not affected by ribosylation (Figure 4d). In addition, Stern−Volmer plots collected upon titration of ribosylated and nonribosylated HEWL with acrylamide revealed a similar quenching constant (Ksv ∼ 1.6 G


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Figure 5. Fluorescence quenching effect of advanced ribosylation end products on HEWL intrinsic fluorescence. (a) Fluorescence spectra of HEWL at different ribosylation times (λexc 280 nm). (b) Changes in the intrinsic fluorescence spectra of native HEWL (λexc 280 nm) upon addition of different aliquots of an Ac-Lys/ribose reaction mixture, which was previously incubated at 37 °C during 7 days. (c) Changes in the intrinsic fluorescence spectra of native HEWL (λexc 280 nm) upon addition of different concentrations of purified AGE-1. (d) Changes in the intrinsic fluorescence spectra of native HEWL (λexc 280 nm) upon addition of different concentrations of N-methylglycine.

Figure 6. Ribosylation effect on the dynamics and the thermal stability of HEWL. (a) 15N longitudinal (T1) and transversal (T2) relaxation times along the sequence after 0 h (black) and 72 h (red) of ribosylation. Regions with higher differences are highlighted with a square. The secondary structure is indicated on the plot. (b) CD intensity signal at 220 nm monitored in function of the temperature for the HEWL/ribose reaction mixture after 0 h (●) and 144 h (○) of incubation at 37 °C.

× 10−5 M−1), confirming that the SASA of Trp does not change upon ribosylation (Figure S10). AGE-1 and CML Quench HEWL Intrinsic Fluorescence. Our results provide evidence that ribosylation does not alter the tertiary structure of HEWL. Despite that, all these data seem to

be inconsistent with the decreasing fluorescence of HEWL upon ribosylation (Figure 5a). Intrinsic tryptophan fluorescence is commonly used to report on tertiary structure changes upon glycation and to suggest molten globule formation in glycated proteins.10,12,14,38 However, we observe that the H


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Figure 7. Residual mapping of the ribosylation effect on the structure of HEWL. (a) Overlap of the regions between 6.5 and 8.3 ppm (1H dimension) and between 113 and 123 ppm (15N dimension) of the 15N-HSQC spectra of HEWL after 0 h (red) and 6 days (blue) of ribosylation. Three different behaviors were exposed: cross-peaks that hardly modify their chemical shifts upon ribosylation are labeled in orange; cross-peaks that exhibit a slow exchange regime within the NMR time scale between the native and ribosylated conformations are labeled in green; cross-peaks that change their chemical shifts upon ribosylation are labeled in black. (b) Chemical shift histogram of the weighted amide chemical shift perturbation with ribosylation. Chemical shift variation was subtracted from the 15N-HSQC spectrum of HEWL after 0 h and 6 days of ribosylation. Residues that changed their chemical shifts upon ribosylation were colored in orange, except Lys, which was colored in blue. Residues exhibiting a slow exchange regime were colored in purple. The secondary structure is indicated on the plot as reference. (c) HEWL structure (PDB 1LSE) showing the side chains of residues with Δδave > 0.02 ppm. Color code is the same than that depicted in panel b. Pymol was used to build the figure.

that AGE-1 and CML are able to quench protein intrinsic fluorescence. Ribosylation Effect on the Dynamics and Thermal Stability of HEWL. Although the HEWL conformation is unaltered, the formation of AGE-1 and CML on Lys side chains affects protein dynamics and stability. The ribosylation effect on HEWL dynamics was assessed by NMR spectroscopy at early ribosylation times (72 h) to diminish the signal broadening effect on the relaxation measurements.40 The T1 values did not change upon ribosylation evidencing that AGEs do not influence the overall rotational tumbling. The T2 values for the ribosylated HEWL are fairly close to those determined for the native HEWL (Figure 6a). However, some low order regions exhibited lower motions upon ribosylation. These are the V2-G4 and G22-L25 stretches, which both are located in close proximity of a modified Lys, and the R45-G49 loop. Also, the A90-A95 α-helical region reduced its motion, likely as a consequence of K96 and K97 ribosylation. T1 and T2 values were used to calculate τc, being 5.6 ± 0.4 for the native and 6.2 ± 0.5 ns for the ribosylated states, which provides further support that ribosylation practically does not alter the dimensions of HEWL. On the other hand, CD spectroscopy was used to demonstrate that ribosylation diminished the protein thermal stability while decreasing the transition cooperativity (Figure 6b). The loss in cooperativity is likely to be associated with the increase in the sample heterogeneity. Collectively, these results show that the formation of AGE-1

reduction in the quantum yield is not concomitant with the redshift typical of an unfolding event, which agrees with our NMR results, and indicates that this decrease could possibly be attributed to alternative factors than structural changes. To understand what causes the observed reduction in fluorescence quantum yield, we titrated native HEWL and BocTrp with ribosylated Ac-Lys. In both assays, the fluorescence decreased in a nearly identical manner, and their Stern−Volmer plots exhibited an identical upward curvature profile (Figure 5b and Figure S11) typically indicating a combination of static and dynamic quenching processes.39 The overlap of the excitation wavelength of the AGEs and the emission wavelength of HEWL suggest that AGE-induced quenching is the predominant mechanism of the decreasing quantum yield upon glycation and that energy transfer is occurring.39 To assess the specific role of AGE-1 and CML in the quenching process, we used the isolated AGE-1 and Nmethylglycine (as a CML analogue) to titrate native HEWL and Boc-Trp solutions. The intrinsic HEWL and Boc-Trp fluorescence decreased upon AGE-1 addition through a process that likely involves energy transfer (Figure 5c and Figure S12). N-methylglycine also quenched HEWL intrinsic fluorescence, exhibiting a downward Stern−Volmer plot, characteristic of the coexistence of fluorophores with different solvent accessibility39 (Figure 5d and Figure S13). These results reinforce the lack of tertiary structure rearrangement upon ribosylation and prove I


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Figure 8. Changes in HEWL surface features concomitant with ribosylation. (a) Isoelectric focusing electrophoresis of HEWL at different ribosylation times. The number after the LR label indicates the ribosylation time in days. M = marker. (b) Acid native-PAGE gel of HEWL at different ribosylation times. Line 1 (0 h), Line 2 (7 h), Line 3 (26 h), Line 4 (48 h), Line 5 (72 h), Line 6 (102 h), Line 7 (118 h), Line 8 (150 h) and Line 9 (168 h). (c) Temporal changes in the surface hydrophobicity (H0) of HEWL when it was incubated alone (○) or with ribose (●). (d) Surface hydrophobicity of native (left) and ribosylated (right) HEWL (PDB 1LSE). The ribosylated structural model was built by attaching the characterized AGE-1 on each Lys side chain and on the N-terminal amino group. Protein surfaces are colored such that blue to white to red represents increase in hydrophobicity, regarding the octanol−water partition coefficient (log P) determined for the methyl ester amino acids and AGE-1 using the online server log P (http://www.molinspiration.com/services/logp.html). UCSF Chimera (1.5.1) software was used to build the figures.

AGEs modified the chemical environment of those residues near Lys, which resulted in a change in the overall physicochemical features of the protein surface. Ribosylation Alters the HEWL Surface Features. We then investigated how the chemical environment change of those regions nearby Lys affected the surface properties of HEWL. The formation of AGE-1 and/or CML theoretically converts the cationic Lys into a neutral or an anionic residue. Ribosylation of HEWL was indeed found to gradually decrease the pI from >9.3 up to values ∼4 (Figure 8a) and alter protein mobility in basic (Figure S15) and acidic (Figure 8b) nativePAGE gels concomitant with an enhancement of the smears on the gels, also suggestive of increased heterogeneity. Positive charges are crucial of HEWL enzymatic activity, but also the motions of the low ordered regions.41 Ribosylation modified these two aspects explaining the drop in HEWL activity (Figure S16). From the observed pI decrease we suspected that ribosylation modified the hydrophobicity of the HEWL surface. To analyze whether the protein surface hydrophobicity (H0) was altered upon ribosylation, we used the method of Haskard and Li-Chan that is based on the fluorescent probe ANS.31 Ribosylation increased the H0 by ∼64% during the first 70 h of incubation, but further incubation did not induce additional H0 change (Figure 8c). This observation was also supported by the comparison of the surface hydrophobic maps of native with ribosylated HEWL, which show the formation of extended

and CML on HEWL alter the protein dynamics and its thermal stability, without changing the secondary and tertiary structural elements. Mapping the Ribosylation Effect at Residue Level. Trying to understand what causes these ribosylation-induced dynamical and stability changes, we used NMR to map the ribosylation effect at residue level. The 15N-HSQC spectrum of ribosylated HEWL shows that the cross-peaks overlay the spectrum of native HEWL (Figure S14). Nevertheless, line broadening is observed, leading to decrease of signal intensity and even disappearance of some cross-peaks (e.g., E7, V29 or A107). In-depth analysis of the chemical shift variation revealed different behaviors (Figure 7a). Some residues retained their native chemical shifts (Δδave ≤ 0.01 ppm; e.g. A11, I98, A122 or C127) while others, that mapped to regions near to Lys, modified their chemical shifts (Δδave > 0.01 ppm) showing that AGEs are the culprit of their environmental change (Figure 7b,c). Most of these residues exhibited a slow exchange regime within the NMR time scale, revealing both a native-like environment and a ribosylated one. This must be related with a reduction into the Lys side chain dynamics and the endproducts half-lifetime nearby to their neighboring residues. Lys cross-peaks showed a fast exchange regime consistent with what is expected from their modifications. A fast exchange regime was also observed for G4 and E7, likely due to the breakage of the K1-E7 salt-bridge, but also for M12, R14, and F34, all of them sequential neighbors of Lys. These results show that J


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Figure 9. Structural characterization of the HEWL ribosylated aggregates and their effect on cell viability. (a) Ab initio model based on the experimental SAXS data obtained from the species contained in the shoulder at the start of the main SEC elution peak acquired after 7 days of incubation of HEWL with ribose at 37 °C and at pH 7.4. The red sphere represents the calculated model that is superposed with the cartoon representation of two HEWL models (PDB 1LSE). (b) AFM micrograph of HEWL and ribose after 10 days of incubation at pH 7.4 and at 37 °C (monomeric + multimeric). The height scale bar is inset, while the length scale bar corresponds to 0.1 μm. (c) Electron micrographs of negatively stained protofibrillar aggregates from a reaction mixture containing HEWL (5 mM) and ribose (0.2M) in 0.2 M phosphate buffer at pH 7.4 that was previously incubated at 37 °C for 7 days (multimeric). The length scale bar corresponds to 0.35 μm. (d) FT-IR spectra of the amide I band of solid state HEWL fibrils, native HEWL and ribosylated HEWL oligomers (monomeric+multimeric). The maximum of the peaks were normalized to better compare the profiles. (e) Concentration-dependent analysis of cell survival upon addition of HEWL to SH-SY5Y neuroblastoma cells. HEWL was incubated in the presence and absence of ribose for 0 (monomeric) and 14d (monomeric+multimeric) at 37 °C and buffer exchanged against DMEM:F-12 medium prior to its addition.

hydrophobic regions, like the one ongoing from W63 to K13 (Figure 8d). ANS fluorescent spectra per se have been used in the past to detect solvent exposed hydrophobic regions and therefore, to describe the formation of partially folded conformations as a result of glycation.12−15,27 The intensity of the ANS fluorescent spectrum temporally increased and blue-shifted when HEWL was ribosylated, but this was also observed in an Ac-Lys/ribose reaction mixture (Figure S17). Therefore, these observed changes appeared to be the direct result of fluorescent AGEs formation instead of induced by conformational changes of the protein (Figures S17 and S18). Hence we concluded that ANS is not an appropriate reporter to investigate the solvent exposed hydrophobic regions associated with PG. Ribosylated HEWL Induces Native-Like Aggregate Formation. Although ribosylation does not induce fold changes of HEWL, the formation of newly surface exposed hydrophobic patches does raise the question whether ribosylation affects the self-assembly tendency of HEWL, as SEC results have suggested (Figure 3b). In agreement with the previously shown MALDI-TOF MS data, SDS-PAGE showed progressive formation of SDS-resistant dimers and trimers for HEWL incubated with ribose, while in its absence, only monomeric HEWL was detected (Figure S19). To gain structural insights on these soluble low-n oligomers, we used SAXS to analyze the content in the shoulder at the

start of the main SEC peak obtained after 7 days of ribosylation (Figure 3b). The normalized Kratky plot for the species in the shoulder exhibited a profile typical for multidomain or multimeric protein, while the global fold and flexibility seemed to be preserved (Figure S20). Moreover, the global shape of the ab initio models based on the SAXS data (Figure 9a), as well as the measured size-related parameters (Table S2), fitted very well with the definition of a native-like HEWL dimer. AFM and TEM were used to visualize the morphology of the soluble aggregates. After 10 days of incubation we detected sphericallike aggregates with an average diameter of 20 nm in the sample containing HEWL and ribose, which were absent in the controls (Figure 9b and Figure S21, Figure S24). In addition, visual inspection of the HEWL/ribose reaction mixture evidenced the appearance of insoluble brown-colored particles in a time- and concentration-dependent manner (Figure S22). However, we did not detect high-molecular aggregates in the SDS-PAGE analysis (Figure S19), which therefore must be non-SDS resistant assemblies. After 7 days of incubation, these insoluble aggregates were analyzed by TEM and AFM, which revealed the existence of assemblies that appeared heterogeneous in length, but with an apparent uniform diameter of ∼21 ± 3.0 nm (Figure 9c, Figure S25). The solid state FT-IR spectrum of the insoluble ribosylated aggregates showed an amide I band with a maximum at ∼1650 cm−1 typical of an α-helical conformation identical to that of K


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neuroblastoma cell viability, and we explored the factors that are responsible for ribose induced-aggregation including reactivity, structure, conformational flexibility, stability and hydrophobicity. HEWL ribosylation mainly occurred on the Lys side chains, whose sensitivity seemed to be enhanced by the close proximity of carboxylates, which can catalyze the Amadori rearrangement step.45 We identified AGE-1 formation, which has been characterized for the first time on a protein even though an analogue was previously detected on neopentylamine.46 CML, an AGE commonly found on human proteins,1 was also identified on ribosylated HEWL. The combined use of Glu-C digestion and MALDI-TOF MS analysis, together with fluorescent studies did not enable us to detect other previously characterized ribose-derived AGEs, such as α/β-NFC-147 or pentosidine (λem,max 385 nm).48 Until now it was assumed that AGEs induced a chaotropic effect on glycated proteins, which seemed plausible to explain from the changes in intrinsic protein fluorescence upon glycation.10,13,14 Nevertheless, our results based on a combination of complementary low- and high-resolution techniques prove that fluorescence changes are related to an AGEassociated energy transfer process rather than structural changes. We show that ribosylation does not induce structural alterations of HEWL, even though ribosylation changed the chemical nature of Lys, and the chemical environment of those residues located in close proximity to modified Lys. These changes, guided by the AGEs, necessarily affect the intramolecular interaction pattern,49 as evidenced by the reduction in the dynamics of low ordered regions nearby modified Lys, and the reduction in the HEWL Tm, as a possible result of the K1-E7 and K13−C-terminal carboxylate salt-bridges breakage. In agreement with our observations, molecular dynamics simulations were recently used to suggest that glycation reduces backbone flexibility,50 while disruption of salt bridges as a result of glycation was also reported for other proteins9,13,38 and directly linked with the reduction of the protein stability.51 We also report that ribosylation decreases the pI of HEWL, which has already been observed for other glycated proteins,52 while simultaneously increasing surface hydrophobicity, a finding that is consistent with earlier reports on the glycation of phaseolin.53 It is generally assumed that proteins have to undergo (partial) unfolding to allow the exposure of sufficient hydrophobic regions to induce aggregation. Although ribosylation does not affect HEWL fold, its aggregation is apparent from TEM, SEC, and AFM. Therefore, it appears that the increased surface hydrophobicity of HEWL through the covalent modification of the Lys residues most likely acts as the driving force that facilitates the assembly of HEWL into native-like aggregates, in decisive contrast to an interpretation based on protein unfolding. We report that ribosylated HEWL aggregates into native-like small spherical oligomers, which further evolve into insoluble native-like protofibrils. Oligomer formation was also observed for other ribosylated proteins including β2-microglobulin,11 albumin,10 tau protein,26 or α-synuclein.27 The formation of ribosylated aggregates raised concerns regarding their impact on cell viability, as it has been established that oligomers can induce toxicity in a wide range of neurodegenerative diseases. We found that the soluble fraction of HEWL incubated in the presence of ribose reduced the cell viability (LD50 ∼ 10 μM) of

native HEWL, but different from the one obtained for HEWL amyloid fibers, which exhibited a shoulder at ∼1625 cm−1 typical of cross-β aggregates42 (Figure 9d). Also, the X-ray powder diffraction profile of the insoluble ribosylated aggregates and native HEWL overlapped perfectly and showed a broad band typical of an amorphous state (Figure S23). This contrasts with the prominent scattered peaks observed in HEWL amyloid fibers43 typical of a highly ordered repeated pattern. These insoluble glycated aggregates were also produced from the incubation (during 10 days at 37 °C) of a solution containing monomeric glyated HEWL previously isolated by SEC after 6 days of incubation (Figure 3B). Hence, monomeric glycated HEWL can aggregate even in the absence of ribose. To characterize the polymeric state of these insoluble glycated aggregates we dissolved them (5 mg) in DMSO (150 μL). The mixture was sonicated for 10 min and the brownish soluble fraction was analyzed by SDS-PAGE (Figure S26A). The obtained results proved that insoluble glycated aggregates mainly arised from monomeric HEWL, but also dimers, trimers, and tetramers cross-linked in the absence of ribose can coaggregate. To better understand the role of covalent cross-linking in the whole glycation-linked aggregation process, we chemically cross-linked native HEWL with formaldehyde and glutaraldehyde. The resulting dialyzed samples (devoid of cross-linker) were then incubated in phosphate buffer at 37 °C for 7 days. SDS-PAGE analysis proved that both cross-linkers induced the formation of several multimeric species (Figure S26B). However, AFM data did not reveal the formation of insoluble aggregates, nor the soluble spherical-like aggregates as we observed for HEWL glycated with ribose (Figure S26C). Hence, these results prove that cross-linking alone is not sufficient to induce aggregation linked to glycation. Our results clearly show that change in the H0 of HEWL upon ribosylation is the key factor that triggers assembly into native-like aggregates through a nonamyloidogenic mechanism that implies the formation of low-n oligomers that further clumps into insoluble bigger particles. Incubation of HEWL in the Presence of Ribose Affects the Viability of SH-SY5Y Cells. Incubation of diverse proteins related to neurodegenerative disorders in the presence of glycating agents has been shown to induce gain-oftoxicity.27,44 Soluble fractions from HEWL incubated 14 days in the presence or absence of ribose were therefore tested for their impact on the viability of SH-SY5Y neuroblastoma cells. The number of viable cells decreased significantly upon incubation with ribosylated HEWL in a concentration-dependent manner with similar LD50 (∼10 μM) as, e.g., Aβ42. At the same time, HEWL incubated in the absence of ribose and HEWL combined with ribose prior to incubation showed no remarkable effect up to concentrations of 30 μM (Figure 9e). These results show that soluble HEWL incubated in the presence of ribose can profoundly influence neuroblastoma cell viability.

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DISCUSSION PG has been related to toxic aggregate formation and neurodegenerative disease.5,6,44 However, a comprehensive description of the molecular mechanism that correlates PG and aggregation is lacking. To settle this issue, we have studied the ribosylation-coupled aggregation of HEWL. We found that ribosylation induces HEWL aggregation into native-like aggregates and influences L


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UV/vis and fluorescence experiments, carried out electrophoretic experiments, and contributed to data analysis. L.M. carried out the characterization of the advanced ribosylation end-products, performed the MALDI-TOF experiments and contributed to its analysis, and performed the FT-IR studies. K.P. performed TEM, SEC, SAXS and CD data acquisition and interpretation, and notably contributed to writing the paper. Y.K. performed cell viability assays and isoelectric focusing electrophoresis. P.L. collected the SAXS data and performed its analysis and interpretation. B.V. performed AFM data acquisition and analysis. K.B. performed isoelectric focusing electrophoresis, analyzed the cell viability results, and contributed to writing the paper. F.M. and J.D. performed data analysis and contributed to writing the paper. All authors have given approval to the final version of the manuscript.

SH-SY5Y neuroblastoma cells, while HEWL incubated in the absence of ribose or nonincubated HEWL in the presence of ribose did not impact cell viability. These observations on HEWL provide for an interesting, yet unexplained comparison to the observation for ribosylated aggregates from amyloidogenic Parkinson’s disease-related α-synuclein27 and Alzheimer’s disease-related tau.26 Our results raise the fundamental question as how native-like glycated HEWL assemblies influence cell death. Simultaneously, we provide a detailed structural platform to further investigate the modus operandi of glycated proteins in influencing cell death.

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CONCLUSIONS Here we have combined interdisciplinary and complementary techniques to identify the formation of advanced ribosylation end products and assess their effect on the protein structure with HEWL as the polypeptide model. Glycation of HEWL by ribose induced an increase in surface hydrophobicity that suffices to trigger a native-like oligomerization mechanism. We have shown that even partial unfolding is not essential to initiate the aggregation process linked to glycation, while our data cannot rule out that glycation could induce misfolding in other proteins. By studying a glycated polypeptide at the residue level by NMR up to the morphology of its aggregates and their effect on cell viability, we could propose a new model that describes the molecular mechanism through which glycation induces protein aggregation. Our findings have fundamental implications for future therapeutic interventions in diabetes-related diseases: it is less important to focus on glycation-induced protein misfolding events, but it is essential to completely understand the glycation-induced loss of cell viability.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the excellent technical assistance from the Serveis Cientificotècnics at the UIB, especially to Dr. Gabriel Martorell for his generous assistance with NMR measurement, to Dr. Joan Cifre for his help with AFM and X-ray powder diffraction measurements and to Dr. Rosa Gomila for her aid with the MALDI-TOF set up and analysis. K.P. is supported by a FWO Pegasus long-term postdoctoral fellowship. We acknowledge access to the SWING beamline of the SOLEIL synchrotron facility (Paris, France).

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ASSOCIATED CONTENT

S Supporting Information *

Additional graphics: a general mechanism of protein glycation; temporal variation of fluorescence upon ribosylation; characteristic NMR and intrinsic fluorescence data of the isolated AGEs; temporal variation of MALDI-TOF spectra of HEWL upon ribosylation; SAXS curves and the corresponding Guinier plots of native monomeric and ribosylated monomeric HEWL; FTIR spectra of HEWL upon ribosylation; fluorescence quenching data analysis; 15N-HSQC of native and ribosylated HEWL; study of the ribosylation effect on the enzymatic activity of HEWL; basic native-PAGE and SDS-PAGE analysis of HEWL upon ribosylation; ANS fluorescence spectra analysis; SAXS data corresponding to the multimeric ribosylated HEWL; microcentrifuge tubes containing insoluble aggregates; AFM and TEM micrographs of HEWL at different ribosylation times; X-ray powder diffraction studies. Additional tables: trypsin digestion and MS analysis; SAXS derived size-related parameters. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +34-971-173491. Fax: +34- 971-173426. Author Contributions

M.A. designed and supervised the project, performed NMR, Xray diffraction, contributed to the data acquisition and interpretation, and wrote the paper. P.S. performed the DSC, M


Biomacromolecules

Article

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