Glycation of Lysozyme by Glycolaldehyde Provides ... - ACS Publications

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Glycation of lysozyme by glycolaldehyde provides new mechanistic insights in diabetes-related protein aggregation Laura Mariño, Carlos Andrés Maya-Aguirre, Kris Pauwels, Bartolomé Vilanova, Joaquin Ortega-Castro, Juan Frau, Josefa Donoso, and Miquel Adrover ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01103 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 5, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

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

ACS Chemical Biology

Glycation of lysozyme by glycolaldehyde provides new mechanistic insights in diabetes-related protein aggregation Laura Mariño1,2, Carlos Andrés Maya-Aguirre2, Kris Pauwels3,4, Bartolomé Vilanova1,2, Joaquin Ortega-Castro1,2, Juan Frau1,2, Josefa Donoso1,2 and Miquel Adrover1,2*

1University

Institute of Health Sciences (UNICS-IdisPa). Ctra. Valldemossa 79, E07010, Palma de Mallorca, Spain.

2Departament

de Química, Universitat de les Illes Balears, Ctra. Valldemossa km 7.5, E-07122, Palma de Mallorca, Spain.

3Structural

Biology Brussels, Vrije Universiteit Brussels, Pleinlaan 2, 1050 Brussels, Belgium.

4VIB

Structural Biology Research Centre, Vlaams Instituut voor Biotechnologie, Pleinlaan 2, 1050 Brussels, Belgium.

*Correspondence to: Miquel Adrover, University of Balearic Islands, Phone: +34 971 173491; Fax +34 971 173426; e-mail: [email protected]

1

ACS Paragon Plus Environment


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

Page 2 of 32

ABSTRACT Glycation occurs in vivo as a result of the non-enzymatic reaction of carbohydrates (and/or their autoxidation products) with proteins, DNA or lipids. Protein glycation causes loss-of-function and consequently, the development of diabetic-related diseases. Glycation also boosts protein aggregation, which can be directly related with the higher prevalence of aggregating diseases in diabetic people. However, the molecular mechanism connecting glycation with aggregation still remains unclear. Previously we described mechanistically how glycation of hen egg-white lysozyme (HEWL) with ribose induced its aggregation. Here we address the question whether the ribose-induced aggregation is a general process or it depends on the chemical nature of the glycating agent. Glycation of HEWL with glycolaldehyde occurs through two different scenarios depending on the HEWL concentration regime (both within the µM range). At low HEWL concentration non-crosslinking fluorescent advanced glycation end-products (AGEs) are formed on Lys side chains, which do not change the protein structure but inhibit its enzymatic activity. These AGEs have a little impact on HEWL surface hydrophobicity and therefore, a negligible effect on its aggregation propensity. Upon increasing HEWL concentration, the glycation mechanism shift towards the formation of intermolecular crosslinks, which trigger a polymerization cascade involving the formation of insoluble spherical-like aggregates. These results notably differ with the aggregation-modulation mechanism of ribosylated HEWL directed by hydrophobic

interactions.

Additionally,

their

comparison

constitutes

the

first

experimental evidence showing that the mechanism underlying the aggregation of a glycated protein depends on the chemical nature of the glycating agent.

2


Page 3 of 32

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


INTRODUCTION Non-enzymatic glycation involves the chemical reactions occurring between reducing sugars and the different nucleophilic groups present in proteins, lipids or DNA. When the targeted biomolecule is a protein, reducing sugars mostly react with the N-terminal amino group and with the Lys side chains to initially form a Schiff base. This imine further rearranges into an Amadori product that is considered the key factor that enables the final formation of the advanced glycation end products (AGEs).1,2 Although the formation of the Schiff base and the Amadori compound constitute the central pathways along the protein glycation (PG) mechanism, the whole process becomes much more complex as a result of collateral autoxidative reactions of reducing sugars, Schiff bases and the Amadori compounds. These reactions yield highly reactive carbonyl species (RCS) and free radicals3,4 that can further react with other amino acid side chains (mainly Arg and Cys) contributing to the AGEs formation.1,2 Among the different RCS formed during PG, the concentration of methylglyoxal (MG), glyoxal (GA), 3-deoxyglucosone (3-DG) or glycolaldehyde (GLA) increases remarkably in the plasma of hyperglycaemic people. Hence, the concentration of MG in plasma of healthy individuals is of ~0.7µM, while in diabetic patients it rises up to ~2.2µM. A similarly trend was observed for GA and 3-DG, whose concentration was found to be ~1.1µM and ~0.16µM in the controls, while under hyperglycaemia they increased up to ~1.4µM and ~0.5µM, respectively.5 The GLA concentration in plasma of healthy or diabetic people has not been reported yet. Nonetheless, its physiological levels are estimated to range from 0.1 to 1mM.6 In any case, the plasma levels of these RCS are much lower than those of reducing sugars (e.g. the blood glucose in diabetic people is >7mM).7 However, they have been shown to induce PG at rates up to 20000-fold faster than glucose.8 Thus these species are important mediators of PG contributing to AGEs formation and therefore, enhancing the protein damage started by reducing sugars. Whereas the role of MG, GA and 3-DG in PG has been widely studied9-11 that of GLA has been analysed much less. GLA is metabolically formed in neutrophils from L-serine by the myeloperoxidase/H2O2/chlorine system.12 It is also endogenously formed from glucose3 and glucosamine autoxidation through the Namiki pathway.13 Thereafter the fate of GLA in the body is unknown, although it is assumed that GLA may influence the tumor growth via the intracellular formation of free radicals14 produced during the GLA enolization to GA.3,14 GLA also holds a notorious nucleophilicity that turns out into a high ability to induce PG15,16 and consequently, to deplete the protein function and increase the oxidative damage.17 For instance, the modification of extracellular matrix proteins by GLA results in difficulties in the interaction cell/matrix, promoting diabetic nephropathy.18 The formation of GLA-derived AGEs on bone matrix depletes its ability to induce bone formation.19 Albumin glycated with GLA (Alb-GLA) reduces its capacity to bind drugs,20 while that

3



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

Page 4 of 32

occurring on LDL stimulates the formation of lipid-laden cells on the artery wall.21 In addition, it has also been shown that the presence of GLA-modified proteins directly influences several metabolic pathways. Hence, Alb-GLA induces pro-apoptotic effects on pancreatic islet cells22 and causes increased leukocyte adherence, inducing blood-retinal barrier dysfunction.23 Additionally, it has been observed that Alb-GLA decreases leptin expression, which is directly linked to the reduction of the insulin sensitivity.24 Glycation of Lys by GLA starts with the formation of a Schiff base, whereas that occurring on Cys initially produces a thiohemiacetal.25 These intermediates rapidly evolve to the formation of different AGEs whose chemical nature is influenced by the protein environment. For instance, GLA is able to form CML16 but at a less extend than others RCS such as GA.26 GLA induces the formation of a protein bound GLA-pyr•+ free radical cation, which was detected on histone H1 but not on lysozyme or Alb-GLA.27 Moreover, several 3hydroxypyridinium derivatives have been identified in vivo as a result of GLA glycation, such as OP-Lys28 or GLA-pyridine,29 whereas GLA also takes an active role during the formation of vesperlysine A on lens crystallins.30 On the other hand, the thiohemiacetal formed on Cys evolves to the final formation of CMC1 (Figure 1). Although it has been reported the chemical reaction between GLA and Arg,26 AGEs arising from this process have not been identified yet. Regardless of the source of AGEs, their accumulation has been described as one of the main responsible factors of diabetes-associated complications, such as retinopathy, nephropathy or atherosclerosis.31 In addition, AGEs have been linked to neurodegenerative diseases,32,33 although the molecular mechanism through which PG triggers these pathologies is poorly understood. However, it is assumed that glycation increases the protein aggregation tendency, as it has been already proven for transthyretin,34 albumin,35 β2-microglobulin,36 glucose oxidase37 or lysozyme,38 among others. The enhanced aggregation tendency was directly linked to the chaotropic effect of PG, but this

has

been supported only marginally by low-resolution techniques

such as

fluorescence35,39,40 or circular dichroism.39,41 Consequently, the aggregation-modulation mechanism of glycation remains unclear. Different publications suggest that glycation stimulates the amyloid cross-β structure formation42,43 while others demonstrate that glycation does not change the secondary structure but induces the formation of molten globular amyloid-like deposits.35,40,44 More recent data point out that aggregation linked to glycation could occur under the protein native state.45,46 This hypothesis was experimentally proven in our recent work, were we combined different biophysical techniques and cell viability studies to analyze in detail the glycation of hen egg-white lysozyme (HEWL) with ribose. We observed that the formation of different AGEs on the HEWL surface did not alter the protein structure, but changed its surface hydrophobicity profile. This was the key factor that triggered its native-

4


Page 5 of 32

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


like aggregation, initially forming small soluble oligomers capable to reduce the viability of SH-SY5Y cells, which further assembled to bigger insoluble particles.38 Our previous study raised the fundamental question whether our observations could be extrapolated to other proteins and other glycating agents. To understand whether the glycation-induced aggregation of HEWL is a general process, or if it depends particularly on the glycating agent, we have dissected the effects of GLA-induced glycation on the behaviour of HEWL. The results evidence different aspects along the glycation/aggregation process that are really independent of the glycating reagent (i.e. the fact that monomeric HEWL retains its native structure upon glycation; the inhibitory effect of glycation on the enzymatic activity of HEWL; or the size and shape of the aggregates arising from glycated HEWL), but at the same time report features strongly dependent of the nature of the glycating compound (i.e. the chemical nature of the AGEs; the effect of these AGEs on the surface hydrophobicity profile of HEWL; the aggregation mechanism of glycated HEWL; the structural features of HEWL when forming glycated aggregates; or the dependence of the glycation/aggregation mechanism with the HEWL concentration).

5



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

Page 6 of 32

RESULTS Incubation of HEWL with GLA induces the formation of insoluble aggregates Initially, the glycation of HEWL with GLA was studied using a reaction mixture containing 20µM of HEWL and 50mM of GLA. Its incubation at 37ºC led to the formation of insoluble brownish aggregates (HEWL-GLA aggregates), which were visually observed already after 2h of incubation. Hence, we resorted to UV-Vis spectroscopy to study the kinetics of this aggregation process. The observed turbidity in function of time displayed the typical profile of a nucleation-dependent pathway with a lag-phase of ~1.5h, which enlarged up to ~2.5h and ~4h when the HEWL concentration was reduced to 10µM and 5µM, respectively (Figure 2a). The turbidity followed a HEWL-concentration-dependent exponential growth, to level off and finally decrease as a result of the aggregate sedimentation. Therefore, the glycation of HEWL with GLA induces the rapid formation of insoluble aggregates following a nucleation-like mechanism. Aggregates are formed through a HEWL-concentration dependent process The formation of HEWL-GLA aggregates could be detected turbidimetrically only at [HEWL]>2µM (Figure 2a), which suggested that their formation could fit to a concentration-dependent mechanism. To prove this hypothesis we collected aliquots of different HEWL/GLA mixtures at different incubation times. Samples were centrifuged to remove the HEWL-GLA aggregates, and the UV-Vis spectra of the supernatants were acquired. At 2µM HEWL, the At/A0 ratio (281nm) increased as a result of the formation of UV-active AGEs (Figure 2b). Moreover, its fluorescence spectra (λexc 325nm) recorded after 20h of incubation, before and after centrifugation perfectly overlapped, which excludes any light scattering event that would be indicative of the presence of insoluble particles (Figure S1a). Despite the UV-active AGEs formation, the ∆(At/A0)/∆t decreased upon increasing the HEWL concentration from 2 to 10µM, becoming even negative at higher HEWL concentrations (Figure 2b). This is due to the removal of insoluble protein aggregates during the centrifugation step. These results show that the formation of HEWL-GLA aggregates at [HEWL]≤2µM is undetectable. Yet, the equilibrium between soluble glycated HEWL and insoluble HEWL-GLA

aggregates

shift

towards

the

latter

upon

increasing

the

HEWL

concentration. The formation of spherical-like HEWL-GLA aggregates is covalently driven and linked to structural rearrangements AFM was used to visualize the morphology of the HEWL-GLA aggregates. The micrographs of a HEWL/GLA reaction mixture, previously incubated for 6h at 37ºC,

6


Page 7 of 32

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


revealed the formation of a heterogeneous population of spherical-like particles (Figure 2c) with an average diameter of 12.2±4.7nm. Their amount exponentially increased with the incubation time, whereas their averaged size was mostly maintained (i.e. 11.7±6.7nm and 8.1±4.1nm after 10h and 12h of incubation, respectively). The formation of HEWL-GLA aggregates, as well as their population increase with the incubation time, was additionally monitored at “real-time” through AFM imaging. The use of AFM in liquid media also proved that spherical aggregates tend to cluster one to the others in aqueous media (Figure S1b-g), which suggests that some kind of event (e.g. hydrophobic interaction and/or covalent crosslinks) must be taking place between them. Although HEWL-GLA aggregates share a similar morphology, it is not clear whether they share a common molecular oriented pattern, as it has already been described for other polypeptide assemblies such as cross-β oligomers47 or amyloid fibrils.48 The Xray powder diffractogram of HEWL-GLA aggregates displayed a broad band typical of an amorphous state, similar to that observed for native HEWL. However, the profile of HEWL-GLA aggregates also evidenced the presence of weak (but nonetheless detectable) scattered peaks. This indicates that aggregation of glycated HEWL could occur through two different mechanisms: a) a predominant amorphous aggregation; and b) a minor molecular-oriented association. Anyway, any of the scattered peaks overlapped with the prominent scattered peaks arising from HEWL amyloid fibrils, thus indicating that a repeated cross-β structure is irrelevant in the architecture of HEWL-GLA aggregates (Figure 3a). The lack of a predominant ordered pattern does not rule out that glycated HEWL could undergo conformational changes before or during its aggregation. In fact, the amide I band in the FT-IR spectrum of HEWL-GLA aggregates did not overlap with that of native HEWL, thus proving that aggregates do not completely retain the HEWL native structure. This structural rearrangement must be different of a cross-β one, as proven by the absence of the shoulder at ~1625cm-1 typical of HEWL amyloid fibrils49 (Figure S2a). The application of second derivative FT-IR spectroscopy on the amide I bands revealed that HEWL-GLA aggregates display enriched α-helical and disordered conformations at expenses of their β-sheet content (Figures 3b, S2b and Table S1). We then tried to complement the structural characterization of HEWL-GLA aggregates through the determination of their polymeric state. Therefore, insoluble HEWL-GLA aggregates were mixed with different structure-breaking organic solvents (i.e. DMSO, TFA, HFIP and a TFA:HFIP (1:1) mixture), which are known to dissociate aggregates by weakening either hydrophobic interactions or hydrogen bond networks.50 However, any of the used solvent was able to solubilize the HEWL-GLA aggregates (Figures 3c and S2c,d). The fact that a TFA:HFIP (1:1) solution can solubilize native HEWL 7



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

Page 8 of 32

amyloid fibrils but not glycated HEWL amyloid fibrils (generated incubating native HEWL amyloid fibrils with GLA) (Figure 3d) indicates that these latter are further stabilized by intermolecular crosslinking AGEs, as it was suggested before.51 Therefore, we argue that HEWL-GLA insoluble aggregates must be also stabilized by intermolecular crosslinks that could be formed before, during or after aggregation. Our data proves that HEWL glycation mediated by GLA stimulates the formation of insoluble spherical-like particles, which lack a common molecular oriented pattern. This occurs through a mechanism involving a secondary structural rearrangement of HEWL and the plausible formation of intermolecular crosslinks that strengthen the stability of the HEWL-GLA aggregates. HEWL-GLA aggregates do not arise from the assembly of pre-existing soluble polymeric intermediates So far, our results do not answer any question concerning the molecular mechanism that yields insoluble HEWL-GLA aggregates. Therefore, we hypothesize that they could arise either from a monomeric crosslinking cascade triggered by GLA, or as a result of the association of soluble polymeric intermediates, a process commonly described in most of the aggregation mechanisms.52 To shield light on this aspect we initially quest for soluble HEWL-GLA polymeric intermediates. SEC peaks of monomeric HEWL broadened upon incubation, potentially as a result of the heterogeneity, and shifted toward lower elution volumes suggesting either an increase in the hydrodynamic radius or a change in the column affinity linked to glycation (Figure 4a). Nonetheless, SEC chromatograms did not evidence any additional peak at lower elution volume, which would be distinctive of soluble assemblies. The absence of high molecular intermediates was additionally confirmed by MALDI-TOF MS analysis since the obtained mass spectra lacked of peaks attributable to soluble polymeric species (Figure S3). These data suggest that HEWL-GLA insoluble aggregates must be formed through a mechanism directed by a rapid crosslinking process of HEWL monomers that occurs throughout its glycation, and not as a result of the association of soluble polymeric intermediates. Glycation of monomeric HEWL with GLA does not alter its solution structure The changes in HEWL native structure when forming the HEWL-GLA aggregates

raises

the

intriguing

question

of

whether

this

conformational

rearrangement occurs already at monomeric level concomitant with glycation or on the contrary, it is a side event that ensues during the crosslinking process.

8


Page 9 of 32

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


To shield light on these aspects we initially studied the glycation effect on the structure of monomeric soluble HEWL. The incubation of a HEWL/GLA reaction mixture scarcely altered its far UV-CD spectrum profile -which always exhibited the two minima characteristic of an α-helical conformation (Figure 4b and Table S2)-, thus proving that GLA-mediated glycation barely affects the secondary structure of monomeric soluble HEWL. The slight decrease in the spectrum intensity, which also occurred when HEWL was incubated alone (Figure S4a), can be attributed to a minor level of aggregation as suggest the modest increase in the voltage, typically of light scattering events (Figure S4b,c). On the other hand, intrinsic Trp fluorescence has been commonly used to report on protein tertiary structure changes linked to glycation.35,37,40 In fact, GLA caused a notorious temporal decrease in the fluorescence spectra of HEWL at a pseudo-first order rate of 0.37±0.02h-1 (Figures 4c and S5). However, this reduction in the quantum yield was not concomitant with the red-shift typical of an unfolding event. Accordingly, the Stern–Volmer plots collected upon titration of native and GLAmodified HEWL with acrylamide were roughly similar (Ksv ∼11M–1) (Figure 4d). These results discard a significant change in the solvent accessible surface area of Trp concomitant with glycation. Additionally, the 1H-NMR spectra of a reaction mixture containing HEWL (2µM) and GLA (50mM) were collected at different incubation times. We observed that ring shifted resonances, arising from aliphatic groups close to aromatic rings, which are highly sensitive to changes in tertiary structure, do not change their chemical shifts upon glycation (Figure 4e). These peaks scarcely changed their intensity, which was not the case when using a 80µM HEWL solution, as a result of the simultaneous aggregation (Figure S6). Consequently, glycation does not alter the aliphatic-aromatic structural contacts defining the structure of HEWL. As a result, the shifting of the SEC monomeric peaks upon glycation (Figure 4a) must be due to a change in the affinity of HEWL for the chromatographic resin rather than a change in the protein hydrodynamic size. Far UV-CD, NMR and intrinsic protein fluorescence spectroscopies have demonstrated that the glycation of HEWL with GLA does not change the secondary or the tertiary structures as long as HEWL retains its monomeric soluble state. Consequently, the conformational rearrangement observed in the HEWL-GLA insoluble aggregates must occur during the AGE-related intermolecular crosslinking/aggregation processes. GLA induces the formation of fluorescent AGEs on HEWL lysine side chains Our results bring us to a close description of the mechanism through which GLA turns soluble monomeric HEWL into insoluble assemblies. However, they do not 9



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

provide any information on the chemistry underlying the glycation process, which seems to directly induce the aggregate formation. The comparison of the MALDI-TOF spectra of HEWL before and after incubation with GLA evidences a notorious increase in the molecular weight, ascribable to formation of covalently linked AGEs, and an increase in the peak width as a result of the heterogeneity typically occurring during in vitro glycation41 (Figure 5a). Albeit, to better understand how GLA can chemically modify HEWL, we initially used fluorescence spectroscopy to demonstrate the formation of GLA-derived fluorescent AGEs on HEWL (Figures 5b and S7). However, over the 17 glycation hot-spots that holds HEWL (6 Lys and 11 Arg) (Figure S8a), fluorescent AGEs were only formed on the ε-amino groups of HEWL, as proved control experiments carried out on Ac-Lys/GLA and Ac-Arg/GLA reaction mixtures (Figure S9). The formation of these fluorescent compounds, most likely occurring on the N-terminal Lys (K1, K13 and K33 according to the in silico prediction derived from the NetGlycate server53 [Figure S8b]), directly arise from the rearrangement of an initial Schiff base, given that the presence of NaBH3CN in the reaction media, a reagent that selectively reduces imino groups at neutral pH,54 completely inhibited the formation of UV-active AGEs (Figure S10). Moreover, we observed that the protein environment had a catalytic effect on fluorescent AGEs formation since their pseudo-first order rate constant of formation was higher (0.32±0.01h-1) than that for the AGEs formed on Ac-Lys (0.16±0.01h-1)(Figures S7b and S9d). AGEs formed on Ac-Lys displayed an overall fluorescence spectrum profile nearly identical to that obtained from those formed on HEWL (Figure S11). This suggests that the protein environment, although affecting the kinetics of AGEs formation, seems to have a little effect on their chemical nature. Consequently, we used AcLys/GLA reaction mixture as a model to chemically characterize the AGEs formed on HEWL. HPLC-UV analysis of an Ac-Lys/GLA reaction mixture obtained after 48h of incubation evidenced the appearance of a single peak (tR ~12min) (Figure S12a,b), which content was isolated and studied by NMR and MALDI-TOF (Figure S12c-e). It included a heterogeneous mixture of products that could not be separated -even using 0% AcN (Figure S12a)- neither identified. Because these experimental hindrances we afforded for the search of m/z peaks in the MALDI-TOF spectra of the Ac-Lys/GLA reaction mixture that would be indicative of the formation of previously characterized GLA-derived AGEs. After 24h of incubation we observed the appearance of many new peaks that were absent prior incubation, thus suggesting the formation of a heterogeneous mixture of AGEs (Figures 5c and S13). However, only the peaks with m/z 297.2, 319.1 and 335.1 could be assigned to the [M+H]+, [M+Na]+ and [M+K]+ signals of Ac-GLA-pyridine (Figures 1 and 5c). Any of the observed m/z signals was

10


Page 10 of 32

Page 11 of 32

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


indicative of the formation of Ac-Lys-CML ([M+H]+~247) or of dimeric crosslinked AcLys-AGEs (Figure S13). The formation of GLA-pyridine on the 6 Lys that holds HEWL, would imply an increase in the protein m/z ratio of ~642 (∆m/z~107 for each Lys). Therefore, the increase in the m/z ratio of ~638 in glycated HEWL (Figure 5a) suggests that GLA-pyridine is the main AGE formed on monomeric HEWL. Crosslinking AGEs (but not non-crosslinked AGEs) are the key factor for the formation of HEWL-GLA aggregates If the intermolecular crosslinking cascade would be the mechanism through which HEWL-GLA aggregates are formed, non-crosslinked GLA-pyridine should inhibits the assembly of the insoluble HEWL-GLA aggregates. To additionally prove this crosslinking mechanism, we produced soluble monomeric glycated HEWL incubating a 2µM HEWL solution with GLA. Afterwards, the non-reacted GLA was removed through a dialysis step, and the remaining protein was then concentrated up to a 20µM. While the incubation of a 20µM native HEWL solution with GLA involved the formation of insoluble HEWL-GLA aggregates (Figure 2), the incubation of the 20µM monomeric glycated HEWL, either in absence or in presence of GLA, did not yield the formation of the insoluble aggregates (Figure S14). These experimental results constitutes an additional prove that HEWL-GLA aggregates directly arise from the formation of intermolecular crosslinking AGEs, and demonstrates that non-crosslinked AGEs are capable to inhibit aggregation of HEWL glycated with GLA. GLA-derived fluorescent AGEs affect the HEWL intrinsic fluorescence Interestingly, we also observed that the pseudo-first order rate constants of formation of fluorescent AGEs on HEWL (0.32±0.01h-1; Figure S7b) was similar to that corresponding to the decrease of its intrinsic fluorescence (0.37±0.02h-1; Figure S5b), which suggest that both processes could be related. In fact, the emission fluorescence wavelength of HEWL ranges from 300 to 400nm (Figure 4c), while the maximal excitation wavelength of GLA-derived AGEs is ~325nm suggesting a possible inner filter effect between them. Titration of native HEWL and Boc-Trp solutions with an AcLys/GLA reaction mixture (previously incubated during 24h at 37ºC) induced a similar decrease in their fluorescence intensities (Figures 6a and S15a) and the simultaneous appearance of a band centred at ~460nm attributed to the added AGEs (Figure S15c,d), which also appeared in the intrinsic HEWL spectra collected at advanced glycation times (Figure 4c). Additionally, the corresponding Stern-Volmer plots exhibited a similar upward curvature profile (Figure 6b and S15b) typically indicating a combination of static and dynamic quenching processes.55 Thus, the overlap of the 11



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

excitation wavelength of the AGEs and the emission wavelength of HEWL indicates that AGE-induced quenching is the reason of the decrease in quantum yield of HEWL upon glycation with GLA and that energy transfer is occurring.55 These results conclusively prove that protein intrinsic fluorescence cannot be used to evaluate protein tertiary structural changes as a result of glycation. Glycation mediated by GLA depletes the enzymatic activity of HEWL Surface lysines are structurally far away from the HEWL enzymatic site, thus their glycation is not expected to influence the formation of the enzyme-substrate complex or to modify its hydrolytic activity (Figure S16). However surface positive charges and motions of the low ordered regions have been described as crucial for HEWL enzymatic activity.56 If that would be true, the glycation of Lys side chains and the subsequent formation of the zwitterionic GLA-pyridine should deplete its enzymatic activity despite retaining its native structure. Accordingly, we observed that incubation of monomeric HEWL with GLA completely abolished its enzymatic activity even after 2h of incubation (Figure 6c). This occurs without altering residues located at the catalytic pocket, but depleting the cationic charges at the protein surface, which must modify the stability of the enzyme-substrate complex.

12


Page 12 of 32

Page 13 of 32

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


DISCUSION Unraveling the molecular mechanism that increases the prevalence of neurodegenerative disorders -such as Alzheimer’s disease, Parkinson’s disease or frontotemporal dementia- in diabetic patients is becoming an urgent need. This is because diabetes is actually considered as one of the main threats to human health, which incidence is expected to rise beyond 592 million of people by 2035.57 PG has been described as a triggering factor of neurodegenerative disorders in diabetic people.32,33 However, the exact molecular mechanism through which this occurs is still unknown. Its understanding is of crucial interest since it would pave the way to develop efficient therapies. Several publications have suggested that AGEs enhance the protein fibrillogenic propensity,42,58 whereas others have hypothesized that PG induces the formation of molten globule structures35,40,44 thereby inhibiting the amyloid fibril formation.45,59 In contrast, Oliveira et al. reported evidences that aggregation linked to glycation occurs under the protein native state.45,46 To settle this controversy, we previously studied the molecular mechanism underlying the aggregation of HEWL glycated with ribose. Ribose-derived AGEs were formed on monomeric HEWL, but also induced the formation of covalently crosslinked dimers. Moreover, AGEs increased the HEWL surface hydrophobicity, which acted as the driving force that induced the coaggregation of glycated monomers and dimers into insoluble particles. In any case, soluble and insoluble aggregates retained the HEWL native structure, thus reinforcing the idea that glycated globular proteins might assemble into native-like aggregates.38 These results raised the crucial question whether glycated HEWL would behave in the same way when it is modified by a different agent. Hence, here we have studied the glycation-coupled aggregation of HEWL with GLA. Regardless the glycation potential of GLA in vitro15,16,27-29 and in vivo,17-19,30 its effect on protein aggregation has only scarcely been addressed by Xu et al.60 That study merely reported the phenomenological observation that GLA is able to induce aggregation on fibrinogen, yet insights on the molecular mechanism leading to the final aggregates and, on the structural implications of the process were lacking. In this study we have proved that Lys side chains of HEWL are the main targets for GLA modification. Their reaction starts with the formation of a Schiff base, which undergoes a protein-catalyzed rearrangement towards the formation of fluorescent AGEs. This occurs through a non-specific mechanism that yields a heterogeneous mixture of glycated forms, typically appearing when PG occurs in vitro,41 which hampered their detailed characterization. However, the analysis of an Ac-Lys/GLA reaction mixture, mimicking glycated HEWL, let us to suggest that GLA-pyridine29 is likely the main AGE formed on monomeric HEWL. MALDI-TOF/TOF data did not

13



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

Page 14 of 32

provide evidence for the formation of other GLA-derived AGEs, such as CML, GLApyr•+, OP-Lys or CMC (Figure 1). The formation of GLA-derived non-crosslinking AGEs did not change the secondary or the tertiary structures of soluble monomeric HEWL. We already described this lack of structural rearrangement when HEWL was glycated with ribose.38 However, a different reasoning was made by other authors who reported structural changes on HEWL glycated with glucose,40,61 fructose and even with ribose.61 Their assumption mainly arose from intrinsic fluorescent data, without taking into account the inner filter effects occurring between the emission wavelengths of HEWL and the excitation wavelengths of AGEs. Therefore, it becomes clear that glycation does not modify the three dimensional structure of soluble globular proteins (at least of HEWL) regardless of the chemical nature of the glycating agent. The replacement of cationic Lys, likely by zwitterionic GLA-pyridine abolished the HEWL enzymatic activity. This was also observed when HEWL was glycated with ribose,38 glucose40 or MG.62 Consequently, this process seems to be completely independent of the chemical nature of the resulting AGEs and constitutes an additional proof that Lys and/or their cationic charges, despite being far away of the active site, take an active role as fine-tuners of the stability of the HEWL-substrate complex. Structural, enzymatic and fluorescent studies on glycated HEWL could only be carried out when using a relatively low HEWL concentration (i.e. up to ~2µM). Upon increasing this concentration in the reaction mixture, we observed a gradual decrease in the soluble monomeric HEWL fraction concomitant with an increase in the amount of insoluble brownish aggregates. Their formation occurred through a concentrationdependent collagen,63 AFM

nucleation

mechanism,

already

described

during the

glycation of

but not observed during the formation of ribosylated HEWL aggregates.38

micrographs

revealed

that

HEWL-GLA

aggregates

were

built

from

a

heterogeneous population of spherical-like particles that had a similar morphology and size than those resulting from HEWL glycated with ribose,38,61 glucose or fructose.61 Their spherical-like morphology, as well as their averaged size (~12nm), were also similar to that observed from aggregates resulting from glycated albumin,35 α-synuclein64 or tau.65 Hence, we can argue that aggregates arising from glycated proteins share a similar spherical-like morphology and sizes, being therefore independent of the protein model and of the nature of the glycating agent. Insoluble aggregates formed from HEWL glycated with ribose comprise monomers and covalently linked dimers aggregating predominantly through a hydrophobic process driven by the replacement of cationic Lys (log P~-2.98) by AGE-1 (log P~0.11), among

14


Page 15 of 32

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


others.38 Therefore, we expected that this would also be the main mechanism triggering the formation of HEWL-GLA aggregates. However, we obtained experimental evidence

that

conclusively

reject

this

hypothesis:

i)

GLA-pyridine

has

low

hydrophobicity (log P~-5.51); ii) HEWL-GLA aggregates could not be resolubilized in any structure-breaking organic solvent (differently of ribosylated HEWL aggregates that were easily dissolved in DMSO38), as it also happened to glycated HEWL amyloid fibrils; iii) MALDI-TOF analysis and SEC data oppose the formation of soluble polymeric intermediates, which were observed during the glycation of HEWL with ribose;38 iv) monomeric glycated HEWL does not aggregate even in the presence of free GLA. All these findings demonstrate that a hydrophobic driven aggregation is lacking during the formation of HEWL-GLA aggregates, and strongly support that their formation occurs unambiguously as a result of a fast intermolecular crosslinking event associated to the glycation. These intermolecular crosslinking AGEs, and therefore insoluble HEWL-GLA aggregates, could only be detected at HEWL concentrations above 2µM HEWL. It seems that below this HEWL concentration protein-protein contacts are limited and insufficient to produce crosslinking AGEs. Therefore glycation evolves towards a less favoured pathway ending with the formation of non-crosslinking AGEs capable to inhibit the formation of insoluble HEWL-GLA aggregates. Consequently, it becomes clear that the chemical nature of the glycating agent, as well as the protein concentration, strongly influence the aggregation mechanism, although with a little effect on the size and morphology of the final aggregates. Aggregates formed from HEWL glycated with ribose displayed a completely random molecular-oriented association while retaining the HEWL native structure.38 This was also described for insulin45 and cytochrome c.46 However, FT-IR data suggest a slight conformational rearrangement in the HEWL native structure when forming HEWL-GLA aggregates, which mainly display a random molecular orientation. This structural change could occur during the crosslinking/aggregation process of monomeric HEWL, or alternatively appear as a minor partially folded HEWL monomer also (or only) seeding the formation of HEWL-GLA aggregates (Figure 7). We could not detect the formation of unfolded glycated monomers, which would represent an off-pathway byproduct without a noteworthy role within the aggregation mechanism (Figure S17a). Although highly unlikely, the crosslinking process tying the HEWL-GLA aggregates could also be a later event occurring on pre-existing aggregates. However this would imply that hydrophobic interactions and/or hydrogen bonds are formed between noncrosslinking AGEs, a doubtful process given their chemical nature. Additionally, a conformational rearrangement on native HEWL-GLA aggregates after or concomitant with their crosslinking could also occur (Figure S17b).

15



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

The results assessed in this work represent a detailed mechanistic description on how glycation mediated by GLA induces rapid aggregation of HEWL. Besides their plausible extrapolation on different proteins (also glycated with GLA), and their relevance within the overall understanding of the molecular mechanism linking PG with aggregation, our work showcases both similarities and differences in the aggregation mechanism of a protein glycated by two different agents (i.e. ribose38 and GLA).

16


Page 16 of 32

Page 17 of 32

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


METHODS Detailed methods are included in the Supporting Information. ASSOCIATED CONTENT Supporting Information Figures S1-S17, Tables S1-S2, materials and methods, and supplementary references. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Miquel Adrover: 0000-0002-4211-9013 Funding The overall work has been supported in part by the MINECO/FEDER, UE (Project CTQ-2014-55835-R). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors are grateful for the excellent technical assistance from the Serveis Cientificotècnics at the UIB, especially to Dr. G. Martorell for his generous assistance with NMR measurement, to Dr. J. Cifre for his help with AFM and X-ray powder diffraction measurements, and to Dr. R. Gomila for her aid with the MALDI-TOF set up and analysis. In addition, L. Mariño wants to thank MINECO for the FPU PhD grant FPU14/01131. C. Maya-Aguirre wishes to acknowledge the “Fundación Carolina” for his fellowship (2011). Dr. K. Pauwels is supported by a FWO long-term postdoctoral fellowship.

17



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

REFERENCES (1) Thorpe, S. R., Baynes, J. W. (2003) Maillard reaction products in tissue proteins: new products and new perspectives. Amino Acids 25, 275-281. (2) Ulrich, P., Cerami, A. (2001) Protein glycation, diabetes, and aging. Recent. Prog. Horm. Res. 56, 1–22. (3) Thornalley, P.J., Langborg, A., Minhas, H. S. (1999) Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem. J. 344, 109-116. (4) Wolff, S. P., Dean, R.T. (1987) Glucose autoxidation and protein modification. The potential role of 'autoxidative glycosylation' in diabetes. Biochem. J. 245, 243-250. (5) Odani, H., Shinzato, T., Matsumoto, Y., Usami, J., Maeda, K. (1999) Increase in three alpha,beta-dicarbonyl compound levels in human uremic plasma: specific in vivo determination of intermediates in advanced Maillard reaction. Biochem. Biophys. Res. Commun. 256, 89-93. (6) Lorenzi, R., Andrades, M. E., Bortolin, R. C., Nagai, R., Dal-Pizzol, F., Moreira, J. C. (2010) Glycolaldehyde induces oxidative stress in the heart: a clue to diabetic cardiomyopathy? Cardiovasc. Toxicol. 10, 244-249. (7) Ledru, F., Ducimetière, P., Battaglia, S., Courbon, D., Beverelli, F., Guize, L., Guermonprez, J. L., Diébold, B. (2001) New diagnostic criteria for diabetes and coronary artery disease: insights from an angiographic study. J. Am. Coll. Cardiol. 37, 1543-1550. (8) Thornalley, P. J. (2005) Dicarbonyl intermediates in the Maillard reaction. Ann. N.Y. Acad. Sci. 1043, 111-117. (9) Thornalley, P. J. (2008) Protein and nucleotide damage by glyoxal and methylglyoxal in physiological systems--role in ageing and disease. Drug Metabol. Drug Interact. 23, 125-150. (10) Niwa, T. (1999) 3-Deoxyglucosone: metabolism, analysis, biological activity, and clinical implication. J. Chromatogr. B Biomed. Sci. Appl. 731, 23-36. (11) Vistoli, G., De Maddis, D., Cipak, A., Zarkovic, N., Carini, M., Aldini, G. (2013) Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation. Free Radical Res. 47 (suppl 1), 3-27. (12) Anderson, M. M., Requena, J. R., Crowley, J. R., Thorpe, S. R., Heinecke, J. W. (1999) The myeloperoxidase system of human phagocytes generates N-epsilon(carboxymethyl)lysine on proteins: a mechanism for producing advanced glycation end products at sites of inflammation. J. Clin. Invest. 104, 103-113. (13) Hayashi, T., Namiki, M. (1980) Formation of two-carbon sugar fragments at an early stage of the browning reaction of sugar and amine. Agric. Biol. Chem. 44, 25752580. (14) Al-Enezi, K. S., Alkhalaf, M., Benov, L. T. (2006) Glycolaldehyde induces growth inhibition and oxidative stress in human breast cancer cells. Free Radic. Biol. Med. 40, 1144-1151. (15) Acharya, A. S., Manning, J. M. (1983) Reaction of glycolaldehyde with proteins: latent crosslinking potential of alpha-hydroxyaldehydes. Proc. Natl. Acad. Sci. USA 80, 3590-3594. (16) Glomb, M. A., Monnier, V. M. (1995) Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction. J. Biol. Chem. 270, 10017-10026.

18


Page 18 of 32

Page 19 of 32

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


(17) Lorenzi, R., Andrades, M. E., Bortolin, R. C., Nagai, R., Dal-Pizzol, F., Moreira, J. C. (2011) Oxidative damage in the liver of rats treated with glycolaldehyde. Int. J. Toxicol. 30, 253-258. (18) Pedchenko, V. K., Chetyrkin, S. V., Chuang, P., Ham, A. J., Saleem, M. A., Mathieson, P. W., Hudson, B. G., Voziyan, P. A. (2005) Mechanism of perturbation of integrin-mediated cell-matrix interactions by reactive carbonyl compounds and its implication for pathogenesis of diabetic nephropathy. Diabetes 54, 2952-2960. (19) Fong, Y., Edelstein, D., Wang, E. A., Brownlee, M. (1993) Inhibition of matrixinduced bone differentiation by advanced glycation end-products in rats. Diabetologia 36, 802-807. (20) Indurthi, V. S., Leclerc, E., Vetter, S. W. (2014) Calorimetric investigation of diclofenac drug binding to a panel of moderately glycated serum albumins. Eur. J. Pharm. Sci. 59, 58-68. (21) Brown, B. E., Dean, R. T., Davies, M. J. (2005) Glycation of low-density lipoproteins by methylglyoxal and glycolaldehyde gives rise to the in vitro formation of lipid-laden cells. Diabetologia 48, 361-369. (22) Costal, F., Oliveira, E., Raposo, A., Machado-Lima, A., Peixoto, E., Roma, L., Santos, L., Lopes Faria, J. B., Carpinelli, A. R., Giannella-Neto, D., Passarelli, M., Correa-Giannella, M. L. (2013) Dual effect of advanced glycation end products in pancreatic islet apoptosis. Diabetes Metab. Res. Rev. 29, 296-307. (23) Moore, T. C., Moore, J. E., Kaji, Y., Frizzell, N., Usui, T., Poulaki, V., Campbell, I. L., Stitt, A. W., Gardiner, T. A., Archer, D. B., Adamis, A. P. (2003) The role of advanced glycation end products in retinal microvascular leukostasis. Invest. Ophthalmol. Vis. Sci. 44, 4457-4464. (24) Unno, Y., Sakai, M., Sakamoto, Y., Kuniyasu, A., Nagai, R., Nakayama, H., Horiuchi, S. (2005) Glycolaldehyde-modified bovine serum albumin downregulates leptin expression in mouse adipocytes via a CD36-mediated pathway. Ann. N. Y. Acad. Sci. 1043, 696-701. (25) Adrover, M., Vilanova, B., Muñoz, F., Donoso, J. (2008) Kinetic study of the reaction of glycolaldehyde with two glycation target models. Ann. N. Y. Acad. Sci. 1126, 235-240. (26) Nagai, R., Matsumoto, K., Ling, X., Suzuki, H., Araki, T., Horiuchi, S. (2000) Glycolaldehyde, a reactive intermediate for advanced glycation end products, plays an important role in the generation of an active ligand for the macrophage scavenger receptor. Diabetes 49, 1714-1723. (27) Wondrak, G. T., Varadarajan, S., Butterfield, D. A., Jacobson, M. K. (2000) Formation of a protein-bound pyrazinium free radical cation during glycation of histone H1. Free Radic. Biol. Med. 29, 557-567. (28) Argirov, O. K., Lin, B., Ortwerth, B. J. (2004) 2-ammonio-6-(3-oxidopyridinium-1yl)hexanoate (OP-lysine) is a newly identified advanced glycation end product in cataractous and aged human lenses. J. Biol. Chem. 279, 6487-6495. (29) Nagai, R., Hayashi, C. M., Xia, L., Takeya, M., Horiuchi, S. (2002) Identification in human atherosclerotic lesions of GA-pyridine, a novel structure derived from glycolaldehyde-modified proteins. J. Biol. Chem. 277, 48905-48912. (30) Tessier, F., Obrenovich, M., Monnier, V. M. (1999) Structure and mechanism of formation of human lens fluorophore LM-1. Relationship to vesperlysine A and the advanced Maillard reaction in aging, diabetes, and cataractogenesis. J. Biol. Chem. 274, 20796-20804. (31) Singh, V. P., Bali, A., Singh, N., Jaggi, A. S. (2014) Advanced glycation end products and diabetic complications. Korean J. Physiol. Pharmacol. 18, 1-14.

19



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

(32) Li, J., Liu, D., Sun, L., Lu, Y., Zhang, Z. (2012) Advanced glycation end products and neurodegenerative diseases: mechanisms and perspective. J. Neurol. Sci. 317, 1–5. (33) Miranda, H. V., Outeiro, T. F. (2010) The sour side of neurodegenerative disorders: the effects of protein glycation. J. Pathol. 221, 13–25. (34) da Costa, G., Gomes, R. A., Guerreiro, A., Mateus, É., Monteiro, E., Barroso, E., Coelho, A. V., Freire, A. P., Cordeiro, C. (2011) Beyond genetic factors in familial amyloidotic polyneuropathy: protein glycation and the loss of fibrinogen's chaperone activity. PLoS One 6, e24850. (35) Wei, Y., Chen, L., Chen, J., Ge, L., He, R. Q. (2009) Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells. BMC Cell Biol. 10, 10. (36) Kong, F. L., Cheng, W., Chen, J., Liang, Y. (2011) D-Ribose glycates β(2)microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway. Chem. Biol. Interact. 194, 69-78. (37) Khan, T. A., Amani, S., Naeem, A. (2012) Glycation promotes the formation of genotoxic aggregates in glucose oxidase. Amino Acids 43, 1311-1322. (38) Adrover, M., Mariño, L., Sanchis, P., Pauwels, K., Kraan, Y., Lebrun, P., Vilanova, B., Muñoz, F., Broersen, K., Donoso, J. (2014) Mechanistic insights in glycationinduced protein aggregation. Biomacromolecules 15, 3449-3462. (39) Roy, A., Sil, R., Chakraborti, A. S. (2010) Non-enzymatic glycation induces structural modifications of myoglobin. Mol. Cell. Biochem. 338, 105-114. (40) Fazili, N. A., Naeem, A. (2013) In vitro hyperglycemic condition facilitated the aggregation of lysozyme via the passage through a molten globule state. Cell Biochem. Biophys. 66, 265-275. (41) Gomes, R. A., Oliveira, L. M., Silva, M., Ascenso, C., Quintas, A., Costa, G., Coelho, A. V., Sousa Silva, M., Ferreira, A. E., Ponces Freire, A., Cordeiro, C. (2008) Protein glycation in vivo: functional and structural effects on yeast enolase. Biochem. J. 416, 317-326. (42) Bouma, B., Kroon-Batenburg, L. M., Wu, Y. P., Brünjes, B., Posthuma, G., Kranenburg, O., de Groot, P. G., Voest, E. E., Gebbink, M. F. (2003) Glycation induces formation of amyloid cross-beta structure in albumin. J. Biol. Chem. 278, 4181041819. (43) Obrenovich, M. E., Monnier, V. M. (2004) Glycation stimulates amyloid formation. Sci. Aging Knowledge Environ. 2004, pe3. (44) Iram, A., Alam, T., Khan, J. M., Khan, T. A., Khan, R. H., Naeem, A. (2013) Molten globule of hemoglobin proceeds into aggregates and advanced glycated end products. PLoS One 8, e72075. (45) Oliveira, L. M., Lages, A., Gomes, R. A., Neves, H., Família, C., Coelho, A. V., Quintas, A. (2011) Insulin glycation by methylglyoxal results in native-like aggregation and inhibition of fibril formation. BMC Biochem. 12, 41. (46) Oliveira, L. M., Gomes, R. A., Yang, D., Dennison, S. R., Família, C., Lages, A., Coelho, A. V., Murphy, R. M., Phoenix, D. A., Quintas, A. (2013) Insights into the molecular mechanism of protein native-like aggregation upon glycation. Biochim. Biophys. Acta 1834, 1010-1022. (47) Gu, L., Liu, C., Guo, Z. (2013) Structural insights into Aβ42 oligomers using sitedirected spin labeling. J. Biol. Chem. 288, 18673-18683. (48) Balbirnie, M., Grothe, R., Eisenberg, D. S. (2001) An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid. Proc. Natl. Acad. Sci. USA. 98, 2375-2380.

20


Page 20 of 32

Page 21 of 32

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


(49) Frare, E., Mossuto, M. F., de Laureto, P. P., Tolin, S., Menzer, L., Dumoulin, M., Dobson, C. M., Fontana, A. (2009) Characterization of oligomeric species on the aggregation pathway of human lysozyme. J. Mol. Biol. 387, 17-27. (50) Broersen, K., Jonckheere, W., Rozenski, J., Vandersteen, A., Pauwels, K., Pastore, A., Rousseau, F., Schymkowitz, J. (2011) A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer's disease. Protein Eng. Des. Sel. 24, 743-50. (51) Riley, M. L., Harding, J. J. (1995) The reaction of methylglyoxal with human and bovine lens proteins. Biochim. Biophys. Acta 1270, 36-43. (52) Gadad, B. S., Britton, G. B., Rao, K. S. (2011) Targeting oligomers in neurodegenerative disorders: lessons from α-synuclein, tau, and amyloid-β peptide. J. Alzheimers. Dis. 24 (Suppl 2), 223-232. (53) Johansen, M. B., Kiemer, L., Brunak, S. (2006) Analysis and prediction of mammalian protein glycation. Glycobiology 16, 844-853. (54) Borch, R. F., Bernstein, M. D., Durst, H. D. (1971) The cyanohydridoborate anion as a selective reducing agent. J. Am. Chem. Soc. 93, 2897-2904. (55) Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy, 3rd ed., pp 282286, Kluwer Academic, New York. (56) Masuda, T., Ide, N., Kitabatake, N. (2005) Structure-sweetness relationship in egg white lysozyme: role of lysine and arginine residues on the elicitation of lysozyme sweetness. Chem Senses. 30, 667-681. (57) Kharroubi, A. T., Darwish, H. M. (2015) Diabetes mellitus: the epidemic of the century. World J. Diabetes 6, 850-867. (58) Vitek, M. P., Bhattacharya, K., Glendening, J. M., Stopa, E., Vlassara, H., Bucala, R., Manogue, K., Cerami A. (1994) Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 91, 4766-4770. (59) Lee, D., Park, C. W., Paik, S. R., Choi, K. Y. (2009) The modification of alphasynuclein by dicarbonyl compounds inhibits its fibril-forming process. Biochim. Biophys. Acta 1794, 421-430. (60) Xu, Y. J., Qiang, M., Zhang, J. L., Liu, Y., He, R. Q. (2012) Reactive carbonyl compounds (RCCs) cause aggregation and dysfunction of fibrinogen. Protein Cell 3, 627-640. (61) Ghosh, S., Pandey, N. K., Singha Roy, A., Tripathy, D. R., Dinda, A. K., Dasgupta, S. (2013) Prolonged glycation of hen egg white lysozyme generates non amyloidal structures. PLoS One 8, e74336. (62) Amarnath, V., Amarnath, K., Avance, J., Stec, D. F., Voziyan, P. (2105) 5'-Oalkylpyridoxamines: lipophilic analogues of pyridoxamine are potent scavengers of 1,2dicarbonyls. Chem. Res. Toxicol. 28, 1469-1475. (63) Usha, R., Jaimohan, S. M., Rajaram, A., Mandal, A. B. (2010) Aggregation and self assembly of non-enzymatic glycation of collagen in the presence of amino guanidine and aspirin: an in vitro study. Int. J. Biol. Macromol. 47, 402-409. (64) Chen, L., Wei, Y., Wang, X., He, R. (2010) Ribosylation rapidly induces alphasynuclein to form highly cytotoxic molten globules of advanced glycation end products. PLoS One 5, e9052. (65) Chen, L., Wei, Y., Wang, X., He, R. (2009) D-Ribosylated Tau forms globular aggregates with high cytotoxicity. Cell. Mol. Life Sci. 66, 2559-2571.

21



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

Page 22 of 32

FIGURE LEGENDS Figure 1. Chemical structures of GLA-derived AGEs: Nε-(carboxymethyl)lysine (CML),19,29 1,4-disubstituted pyrazinium free radical cation (GLA-pyr•+),27 vesperlysine A, S-(carboxymethyl)cysteine (CMC)1 and the 3-hydroxypyridinium derivatives AGEs (OP-Lys28 and GLA-pyridine29). The formation of OP-Lys requires the simultaneous presence of GLA and glyceraldehyde, whereas vesperlysine A can be formed from ribose coexistent with GLA. The main pathways leading to their formation are depicted in the references, with the exception of verperlysine A, which chemical mechanism of formation has not yet been proposed. Figure 2. Formation of the HEWL-GLA aggregates through a concentration-dependent mechanism. (a) Turbidity curves collected for several reaction mixtures containing HEWL at different concentrations and GLA (50mM) incubated under quiescent conditions at 37ºC in phosphate buffer at pH 7.4. The insert graph shows a zoom of the data acquired during the first 400min. (b) Ratio between measured absorbance at different incubation time (At) and the initial absorbance (A0) plotted against the incubation time.

Absorbance

measurements were

carried at

281nm on the

supernatant collected after centrifugation –to eliminate the HEWL-GLA aggregates- for different HEWL/GLA (50mM) reaction mixtures at different incubation times. (c) Morphological characterization of the HEWL-GLA aggregates. AFM micrographs of a reaction mixture containing HEWL (5µM) and GLA (50mM) at different incubation times (37ºC and pH 7.4). The height scale bar is inset, while the length scale bar corresponds to 0.25µm. Figure 3. Structural features of the insoluble HEWL-GLA aggregates. (a) X-ray powder diffraction profiles of HEWL amyloid fibrils, native HEWL and HEW-GLA aggregates. The insert represents a zoom of the data acquired between scattering angles of 18 and 37. (b) Calculated FT-IR second derivative spectra of the amide I band of HEWL fibrils (blue line), native HEWL (red line) and HEWL-GLA aggregates (yellow line). The wavenumber of the different bands was assigned according literature (Table S1) using the following color code: β-sheet (red), turn/disordered (blue), α-helix (black) and turn (green). Dotted arrows indicate the increase (upper arrows) or decrease (lower arrows) in the content of each secondary structural element in the HEWL-GLA aggregates when compare with native HEWL, whereas single dotted lines indicate that there is not a remarkable change in the band intensity. The maximum intensity of the peaks corresponding to the asymmetric bending modes and to the symmetric stretching modes of the methyl groups of HEWL (~1457cm-1 and ~2874cm-1 respectively) were normalized to better compare the intensity of the different bands composing the amide 22


Page 23 of 32

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


I band, as it is shown in Figure S2b. (c) Tubes containing 5mg of HEWL-GLA aggregates in presence of 200µl of a TFA:HFIP solution (1:1). Pictures were taken after sonication during 10min at room temperature (left) and after centrifugation for 4min at room temperature of the sonicated suspension (right). (d) Scheme illustrating the formation of native HEWL amyloid fibrils (1), their glycation (2) and their solubilisation in an organic chaotropic mixture (3). AFM micrograph evidences the formation of unbranched amyloid fibrils (1) which become glycated, as evidence the yellowish colour acquired upon incubation with GLA. While a TFA:HFIP solution can solubilize native HEWL amyloid fibrils, it cannot dissolve glycated fibrils (3). Figure

4.

Effect

of

GLA

on

the

structure

of

monomeric

HEWL.

(a)

SEC

chromatographic profiles (from a Superdex-75 HR 10/300 column) of a HEWL (2µM)/GLA (50mM) reaction mixture incubated at different times (pH 7.4; 37ºC). (b) Temporal variation of the CD spectrum profile of HEWL (2µM) in the presence of GLA (50mM) before and after incubation at 37ºC and pH 7.4. (c) Temporal variation of the fluorescence spectra of HEWL (λexc 280nm; pH 7.4 and 37ºC) upon incubation in the presence of GLA. (d) Stern-Volmer plots for quenching of native (blue) and glycated HEWL (red) after 24h of incubation at 37ºC and at pH 7.4. F and F0 are the fluorescent intensities in the presence and absence of acrylamide, respectively (λexc 280nm; λem was 345nm for native HEWL and 398nm for HEWL glycated with GLA). (e) 1H-NMR spectra of the ring shifted resonances of HEWL at different incubation times (37ºC). Spectra were collected from a reaction mixture containing HEWL (2µM) and GLA (50mM) in presence of 5mM phosphate buffer at pH 7.4. Figure 5. Characterization of GLA-derived AGEs formed on HEWL. (a) MALDI-TOF spectra of a reaction mixture containing HEWL (2µM) and GLA (50mM) that was incubated during 0h (black line) and 20h (red line) in 5mM phosphate buffer (pH 7.4) at 37ºC. (b) Fluorescence spectra of a reaction mixture containing HEWL (2µM) and GLA (50mM) incubated at pH 7.4 and 37ºC (λexc 325nm). Spectra were collected each 0.5h during the first 20h of incubation. (c) MALDI-TOF spectrum of the 240-350Da region of a reaction mixture containing Ac-Lys (2mM) and GLA (50mM) that was previously incubated during 24h in 0.2M phosphate buffer at pH 7.4 and 37ºC (left). Peaks labelled in red are those assigned to Ac-GLA-pyridine (right). Peaks labelled in black correspond to relevant but unassigned peaks. Figure 6. The AGEs effect on the HEWL intrinsic fluorescence and on its enzymatic activity. (a) Changes in the intrinsic fluorescence spectra of a native HEWL solution (2µM; λexc 280 nm) upon addition of different aliquots of an Ac-Lys (0.1M)/GLA (0.5M) reaction mixture, which was previously incubated at 37 °C during 6h. (b) SternVolmer plot for a solution containing HEWL (2µM). F and F0 are the fluorescence intensities (λexc 280 nm; λem 342nm) in the absence or presence of increasing concentrations of an Ac-Lys (0.1M)/GLA (0.5M) reaction mixture previously incubated 23



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

at 37 °C during 6h. (c) Variation in HEWL enzymatic activity upon incubation in the absence (triangles) or in the presence of GLA (circles) in phosphate buffer (0.2M) at pH 7.4 and at 37ºC. Aliquots were taken at different incubation times and assayed by measuring the rate of lysis of Micrococcus lysodeikticus cells as described in the methods. Figure 7. Schematic representation of the proposed mechanism for the formation of HEWL-GLA insoluble aggregates. The process starts with the formation of noncrosslinking AGEs on HEWL, which do not alter the HEWL native structure. However, the fact that HEWL-GLA insoluble aggregates do not fully retain the HEWL native structure let us to hypothesise that glycated monomeric native-like HEWL could also display equilibrium with a minor misfolded form. Glycated monomeric HEWL does not evolve towards the formation of insoluble aggregates when the protein concentration is lower than 2µM. Above this protein concentration the formation of crosslinking AGEs (only or in combination with non-crosslinking AGEs) induce the formation of insoluble HEWL-GLA aggregates that could either arise from the glycated native-like monomeric HEWL concomitant with a conformational rearrangement, or from the minor misfolded glycated monomeric HEWL.

24


Page 24 of 32

Page 25 of 32

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


Figure 1 61x26mm (300 x 300 DPI)



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

Figure 2 104x78mm (300 x 300 DPI)


Page 26 of 32

Page 27 of 32

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


Figure 3 104x78mm (300 x 300 DPI)



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



Page 28 of 32

Page 29 of 32

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


Figure 5 104x78mm (300 x 300 DPI)



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

Figure 6 104x78mm (300 x 300 DPI)


Page 30 of 32

Page 31 of 32

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





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

Graphical Table of Contents 39x20mm (300 x 300 DPI)


Page 32 of 32

Glycation of Lysozyme by Glycolaldehyde Provides ... - ACS Publications

Recommend Documents