https://doi.org/10.1007/s11010-018-3403-z
Structural effects of methylglyoxal glycation, a study on the model
protein MNEI
Serena Leone1 · Jole Fonderico1 · Chiara Melchiorre1 · Andrea Carpentieri1 · Delia Picone1
Received: 17 February 2018 / Accepted: 12 July 2018 / Published online: 16 July 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
The reaction of free amino groups in proteins with reactive carbonyl species, known as glycation, leads to the formation of mixtures of products, collectively referred to as advanced glycation endproducts (AGEs). These compounds have been implicated in several important diseases, but their role in pathogenesis and clinical symptoms’ development is still debated. Particularly, AGEs are often associated to the formation of amyloid deposits in conformational diseases, such as Alzheimer’s and Parkinson’s disease, and it has been suggested that they might influence the mechanisms and kinetics of protein aggrega-tion. We here present the characterization of the products of glycation of the model protein MNEI with methylglyoxal and their effect on the protein structure. We demonstrate that, despite being an uncontrolled process, glycation occurs only at specific residues of the protein. Moreover, while not affecting the protein fold, it alters its shape and hydrodynamic proper-ties and increases its tendency to fibrillar aggregation. Our study opens the way to in deep structural investigations to shed light on the complex link between protein post-translational modifications, structure, and stability.
Keywords Protein glycation · Advanced glycation endproducts · Protein aggregation · Protein hydrodynamics ·
Post-translational modifications
Introduction
The term “glycation” indicates the reaction of the free amino groups of lysines and arginines in proteins with reactive carbonyl compounds and reducing sugars. Differently from glycosylation, this process is not mediated by enzymes and depends only on the reaction conditions. The early steps of glycation follow the Maillard reaction and produce Schiff bases, which then evolve over time to yield mixtures of prod-ucts, collectively known as advanced glycation endproducts (AGEs) [1]. In biological systems, AGEs are due to circulat-ing sugars and are sensed by specific receptors (RAGEs),
determining increased oxidative stress and inflammation [2,
3]. Elevated AGE levels are typically associated to aging and certain pathologies, among which hyperglycaemia/diabetes, rheumatoid arthritis, and Alzheimer’s and Parkinson’s dis-eases, but their role in pathogenesis and symptoms’ develop-ment is still unclear [1, 4–6]. Glycation products generally form at the expenses of long-lived proteins, such as collagen, elastin, myelin, fibrinogen, or hemoglobin, whose structure, function, and clearance by the proteasome are consequently impaired [7, 8]. Moreover, AGEs are often found on amy-loid deposits, typical of neurodegenerative diseases, which is why the correlation between AGEs and protein aggregation has currently become a hot topic of investigation [5, 9, 10]. Studies to clarify this link for different proteins and peptides, in both pathogenesis-related and model systems, have been undertaken and have recently been reviewed by Iannuzzi et al. [9]. From the analysis of the literature available so far, no general trend or feature has emerged yet: the outcome of the glycation reaction in terms of products distribution, structure, toxicity, and aggregation propensity still seems strongly dependent on the glycating agent and the reaction conditions [9]. For instance, in terms of consequences on aggregation, glycation promotes the formation of fibrils in
Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1101 0-018-3403-z) contains supplementary material, which is available to authorized users. * Serena Leone
serena.leone@unina.it * Delia Picone delia.picone@unina.it
1 Department of Chemical Sciences, University of Naples
Federico II, Complesso Universitario di Monte Sant’Angelo, Via Cintia, 80126 Naples, Italy
Aβ peptide [10, 11], but inhibits it in α-synuclein, insulin, lysozyme, and cytochrome c [12–15]. For other proteins, such as β2-microglobulin or BSA, a variable outcome has
emerged, with fibrils promoted or disfavored according to the incubation conditions [16–19]. Among several possible glycating agents in vivo, one of the most relevant is methyl-glyoxal (MGO), a very reactive dicarbonyl compound, ubiq-uitous byproduct of glycolysis. MGO quickly reacts with proteins to give different MGO-derived AGEs (MAGEs) and, in some cases, this reaction has shown a certain degree of specificity: for instance, both insulin and cytochrome c react at a single arginine residue [12, 13], whereas Aβ pep-tides are glycated only at Arg5 and Lys16 [20]. Aggregation prone proteins with abundant available structural data are ideal models to study glycation, in the attempt to highlight correlations between structure/stability alterations and fibril formation. MNEI, a small, globular protein able to produce fibrillar aggregates [21–23], has recently emerged as a reli-able model for folding and aggregation studies, alongside with structural and computational investigations [24–31]. MNEI possesses a β-grasp fold and its sequence contains 16 potential glycation sites, 9 lysines, and 7 arginines [32]. We have characterized the products of MGO glycation of MNEI (gMNEI) and analyzed the effect of their formation on the protein’s structure, stability, hydrodynamic properties, and aggregation propensity. Our data confirm that MNEI repre-sents a good model to study also this important biological process, in the attempt of defining a correlation between structural features, AGEs formation, and their effects.
Materials and methods
Protein glycation
MNEI was produced and purified as previously described [33]. Glycation was performed by incubating 0.5 mg/mL (44 µM) protein with 5 mM MGO in 50 mM sodium phos-phate buffer, pH 7.4, at 37 °C for 21 days. Negative con-trol samples of protein and MGO alone were also prepared. MAGEs formation was monitored by fluorescence spectros-copy as described in [34] and by native gel electrophore-sis according to [35]. The glycated protein is indicated as gMNEI.
FPLC analysis
Size exclusion chromatography (SEC) was performed on a Superdex75 HR 10/300 column equilibrated with 50 mM sodium phosphate buffer, pH 7.4, containing 200 mM NaCl. The flow rate was at 0.5 mL/min on an AKTA pure FPLC system (GE Life Sciences). The column calibration was based on the elution volumes of BSA, Ovalbumin, Ribonuclease A
and Vitamin B, whereas void volume was determined by the injection of a solution of blue dextran.
Mass spectrometry
Mass spectra were recorded on a 5800 MALDI-TOF/TOF (Sciex, Framingham, MA). Intact proteins were analyzed in positive linear mode using a matrix of 50 mg/mL α-sinapinic acid in acetonitrile/50 mM citric acid 70/30. MAGEs were characterized after chymotrypsin digestion of the proteins according to [12]. Mass spectra were recorded in positive reflectron mode using a matrix of 10 mg/mL α-cyano-4-hydroxy-cinnamic acid (α-CHCA) in acetonitrile/50 mM citric acid 70/30. The peptides having masses consistent with modi-fications by MGO were selected for MS/MS fragmentation experiments, using Collision Induced Dissociation (CID) with 1 kV collision energy and air as collision gas. Only peaks with confirmed MS/MS sequences were considered.
Circular dichroism
CD spectra were recorded on a Jasco J-715 spectropolarimeter. Protein concentration was 0.2 or 1.0 mg/mL for far- and near-UV spectra, respectively. Spectra were acquired as previously described [33]. Near-UV CD spectra and thermal denaturation experiments prior to aggregation measures were recorded in 20 mM sodium phosphate, pH 2.5, far-UV in 50 mM sodium phosphate, pH 7.4. Denaturation spectra were converted to fraction of unfolded protein (fu) and analyzed as previously
described [31]. NMR spectroscopy
Diffusion Ordered Spectroscopy (DOSY) experiments were performed on a Bruker Avance III 400 MHz spectrometer at 25 °C. Samples were prepared by mixing 400 µL of MNEI and gMNEI after 21 days incubation with 100 µL D2O. The
spectra were recorded with the Bruker stebpgp1s19 sequence, with bipolar gradient pulses for diffusion and watergate water suppression. A δ of 2 ms and Δ of 100 and 200 ms were used. Signal attenuation was measured for the integral of the regions 0.60–1.00 and 6.37 and 7.31 ppm, where no MGO peak was present, and used to calculate the diffusion coefficients (Dcoeff)
according to the method by Stejskal and Tanner [36]. For each species, Dcoeff resulted from the average over the 2 spectral
regions and the 2 Δs. Hydrodynamic radiuses were calculated using the Stokes–Einstein equation:
where RH is the hydrodynamic radius, k is the Boltzmann constant and η is the viscosity of the solution, which was R
H= kT 6𝜋𝜂Dcoeff
assumed equal to that of pure water (8.904 × 10−4 kg m−1 s−1
at 25 °C).
Aggregation kinetics
Aggregation kinetics of MNEI and gMNEI were moni-tored in the conditions of [21]. Incubation mixtures with or without MGO after 21 days were buffer exchanged to 20 mM sodium phosphate buffer, pH 2.5, on a Sephadex G25 column (40 mL, GE lifesciences) and concentrated to 2.5 mg/mL using a centrifugal concentrator with 3000 Da MWCO. The samples were incubated at 70 °C for 7 days. At different time intervals, aliquots were assayed by Thi-oflavin T (ThT) assay [37]. Experimental points were fit-ted as described in [12].
Results
Characterization of the glycation products
The reaction between MNEI and MGO was carried out for 21 days and the formation of MAGEs was monitored by flu-orescence spectroscopy, which reflects the formation of the fluorescent MAGE Argpyrimidine (ArgPyr), until the signal at 385 nm reached a plateau (Fig. S1) [34]. The analysis of the mixture by native PAGE showed that unmodified protein had disappeared from the reaction mixture after as little as 1 day, in favor of the appearance of slow migrating spe-cies (Fig. 1a). This reduced mobility could reflect either the formation of MAGE-induced soluble oligomers, or merely the reduction of the protein charge accompanying glycation, which neutralizes the positive side chains of arginines and lysines. To clarify this point, we checked the reaction mix-ture by analytical size exclusion chromatography (SEC) after 7, 15, and 21 days of incubation. Elution profiles (Fig. 1b)
a
b
c
Fig. 1 Time course of the glycation reaction: Native PAGE (a) and SEC elution profiles (b) of samples taken at different time points dur-ing the incubation of MNEI with MGO. c MALDI-ToF analysis on
the intact protein sample of MNEI (pink) and gMNEI (green). The signal for gMNEI appears broader, due to the heterogeneity of the MAGEs product mixture. (Color figure online)
confirmed that after 7 days MNEI has quantitatively reacted with MGO. Moreover, they clarify that the slow migrating species observed with native PAGE are not oligomers, but monomeric glycated MNEI (gMNEI). Based on the column calibration curve for standard globular proteins, we calcu-lated a MW of 11 kDa for MNEI, in perfect accordance with the theoretical value of 11.4 kDa. By comparison, the estimated MW for gMNEI is ~ 17 kDa. The apparent mass increase of ~ 6 kDa seems too high to be uniquely due to covalent binding of MGO, and it is too low to indicate dimerization. Most likely, it is symptomatic of changes in the hydrodynamic properties of gMNEI, in accordance with what previously observed for MGO-glycated insulin [12]. MALDI-ToF analysis of intact MNEI and gMNEI indeed confirmed that the actual increase in protein mass due to glycation is only ~ 600 Da (Fig. 1c). The signal of intact gMNEI in the MALDI-ToF spectrum appeared broader than the one relative to the control protein, due to the formation of a MAGEs mixture, and exhibited a remarkable intensity decrease. This latter effect is ascribable to a loss of ioniza-tion efficiency deriving from the reacioniza-tion of the free amino groups of the basic residues. To identify the major glycation products, MNEI and gMNEI samples were digested with chymotrypsin and the resulting peptides were also analyzed by MALDI-ToF (Fig. S2). In the absence of protein oligom-ers, which would derive from the formation of the 1,3-di(N ε-lysino)-4-methyl-imidazolium adduct (MOLD), four main MAGEs are expected upon reaction with MGO: the lysine specific carboxyethyl-lysine (CEL) and the arginine specific Hydroimidazolone (MG-H1), Tetrahydropyrimidine (THP) and ArgPyr (Fig. S3). The formation of each of these prod-ucts is accompanied by a characteristic mass increase, which translates in a corresponding Δm/z of the glycated peptide. To identify glycation sites, potential matching glycated pep-tides were subjected to MS/MS fragmentation experiments. Only peptides with confirmed identities were considered. The results, summarized in Table 1, indicate that out of 16 potential glycation sites in MNEI (9 lysines and 7 arginines), only 4 arginines react with MGO, i.e., R31, R72, R53, and R88. Moreover, only two adducts, MG-H1 and ArgPyr, are formed. Other arginine residues are never found modified, independently on their position on the protein surface. More-over, none of the lysines, nor the N-terminus of the protein,
reacted with MGO, despite the long incubation time. Since glycated peptides could suffer the same loss of ionization efficiency already observed for the intact protein, no quan-titation was attempted for the different MAGEs produced. Structural effects of MAGEs formation
The change, hinted by SEC, in hydrodynamic properties of gMNEI versus MNEI could indicate that, upon glycation, the protein begins to unfold. In order to assess the effects of glycation on secondary structure elements, we also moni-tored the evolution of MAGEs formation by CD spectros-copy. Figure 2 shows an overlay of the far-UV CD spectra recorded at different time points during the incubation with or without MGO. The slight intensity decrease in the control mixture depends on the minor collateral precipitation, which accompanies prolonged incubation of MNEI at pH 7.4, and is expected based on the sensitivity of MNEI to neutral pH [21, 38]. This effect was not observed for gMNEI, suggest-ing that MAGEs formation increases the protein solubility at neutral pH. The appearance of the CD spectra is globally unaffected by glycation, indicating that secondary struc-ture elements are not modified by MAGEs formation. The hydrodynamic effects observed in SEC experiments must then indicate that the protein shape changes. To confirm this hypothesis, we recorded the near-UV CD spectra of MNEI and gMNEI at the incubation end point, after removal of the MGO (Fig. 3). The differences visible in the near UV CD spectrum may be dependent on a local modification of the aromatic side chains environments and are hardly interpret-able in this case because of the heterogeneity of the product. However, the remarkable difference registered within the two species suggests that glycation induces a conformational change in the molecule. To quantify this effect, we used Diffusion Ordered Spectroscopy (DOSY) to estimate the hydrodynamic radius of MNEI and gMNEI. The calculated Dcoeff of 1.37 and 1.07 × 10−10 m2/s corresponds to
hydrody-namic radiuses of 17.9 and 23.0 Å for MNEI and gMNEI, respectively. While the value found for MNEI is consist-ent with a typical globular shape, the radius of gMNEI increases by roughly 33% upon glycation; nonetheless, it is still below what would be expected upon unfolding for a protein with the same size of MNEI [39]. Together with
Table 1 Summary of MS/MS data for the identification of MAGEs
m/z Fragment Sequence MAGE location MAGE
647.29 [30–34] (Y)GRLTF(N) R31 MG-H1 673.02 [30–34] (Y)GRLTF(N) R31 ArgPyr 1022.35 [72–79] (F)RADISEDY(K) R72 MG-H1 1034.44 [88–96] (L)RFNGPVPPP(–) R88 MG-H1 1060.46 [88–96] (L)RFNGPVPPP(–) R88 ArgPyr 1395.53 [48–58] (Y)ENEGFREIKGY(E) R53 MG-H1
the altered chromatographic mobility and the near-UV CD spectra, these data suggest that gMNEI deviates from the ideal globular behavior, without losing structural integrity. Instead, upon introduction of the hydrophobic MAGEs on its surface, the protein undergoes a deformation, with a con-sequent alteration of its hydrodynamic radius.
Effects of MAGEs formation on MNEI aggregation MNEI has become a well-established model for protein folding and aggregation [21–28]. It is known that, upon incubation at acidic pH, MNEI forms fibrils, whereas at neutral to alkaline pH it produces amorphous aggregates [21–23, 38]. MNEI fibrils have been observed after 1-week incubation at a temperature close to the protein’s Tm. Before analyzing the aggregation kinetics, we first evalu-ated whether MAGEs formation altered the protein stabil-ity by following its thermal unfolding by CD spectroscopy. As visible in Fig. 4, the Tms of MNEI and gMNEI at pH 2.5 are quite close, namely 72.6 and 74.6 °C, respectively. More than on stability, MAGEs formation seems to affect unfolding cooperativity, as suggested by the different slopes of the two curves. Nonetheless, as both proteins begin to unfold around 70 °C, we chose this temperature to monitor the aggregation kinetics. Figure 5 shows the evolution of the ThT fluorescence signal upon 1-week incubation of MNEI and gMNEI in the fibrillization condi-tions. Glycation strongly promotes the formation of fibril-lar aggregates, as suggested by the drastic increase in the ThT fluorescence signal after only few hours of incuba-tion. While for MNEI the ThT fluorescence signal reaches a plateau after 1 week, fibrils of gMNEI are completely formed after as little as 24 h.
a
b
Fig. 2 Effect of MAGEs formation on MNEI secondary structure: overlay of the far-UV CD spectra recorded on MNEI (a) and gMNEI (b) at t0 (black) and after 7 (red), 15 (green) and 21 (blue) days of incubation. (Color figure online)
CD (mdeg) −3 −2.5 −2 −1.5 −1 −0.5 0 0.5 nm 260 270 280 290 300 310 320 MNEI gMNEI
Fig. 3 Effect of MAGEs formation on the tertiary structure of MNEI: overlay of the near-UV CD spectra of MNEI (green) and gMNEI (pink) after 21 days of incubation. (Color figure online)
Fig. 4 Effect of MAGEs formation on MNEI stability: CD thermal denaturation curves for MNEI (green) and gMNEI (pink)
Discussion
Glycation has been associated to several pathologies and seems to have an important role in directing protein aggrega-tion in conformaaggrega-tional diseases, characterized by the pres-ence of amyloid deposits. Outside living organism, glyca-tion can also occur at the expenses of food proteins during processing and, also in this case, the mechanisms directing the reaction outcome are still poorly understood [40]. Thus, from different perspectives, AGEs formation assumes great biological relevance, making it mandatory to develop models for characterizing the various aspects of this complex reac-tion. We have employed several instrumental techniques to study the products of the glycation of MNEI with MGO. In analogy with previous studies on insulin, cytochrome c and Aβ peptides [12, 13, 20], we also found that glycation does not occur at all possible positions: only four out of seven arginines are modified to form MAGEs, independently on their location on the protein surface, and no lysine residues are involved in the process. The formation of these adducts does not alter significantly the protein secondary structure nor its stability, while modifies instead its three-dimensional arrangement, as suggested by near-UV CD spectroscopy. Diffusion measures and SEC show in fact that the hydrody-namic radius of the protein is enlarged, indicating the loss of globular arrangement. This could in turn be related to the fact that, in the case of MNEI, MAGEs formation is a trigger for amyloid aggregation, which, in comparable conditions, occurs with much faster kinetics than for the native protein. This is in contrast with previous findings on other model proteins, such as cytochrome c and insulin [12, 13], for which MAGEs formation had been associated to decreased amyloidogenicity. In both proteins, MAGEs formation did not alter the secondary structure distribution, leading to the
formation of small, native-like oligomers and preventing the α to β transitions required for the formation of amyloid fibrils. Similar oligomeric species were not detectable for gMNEI, which instead rapidly evolved to form fibrils. Such a different behavior could derive from the intrinsic structural differences between MNEI, which has an elevated content of β structure, and model proteins like insulin and cytochrome c, displaying instead an elevated helical content. A further clarification of this point would require deeper structural investigation and comparative analyses on the morphol-ogy of the aggregates. Nonetheless, our studies suggest that MNEI could indeed become a useful model protein to char-acterize the mechanistic details of AGEs formation. In fact, the available data on MNEI structure, stability and folding, and the possibility to stabilize aggregates of different sizes, which makes the protein amenable for solution and solid state studies performed with high-resolution techniques [41], will provide the background to elucidate the determinants which govern the outcomes of glycation resulting upon exploring various experimental conditions and to figure out the physiological effects of its products.
Funding This work was supported by “Fondazione con il SUD,” Grant 2011-PDR-19.
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