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Methylglyoxal mediated conformational changes in histone H2A—generation of carboxyethylated advanced glycation end products
International Journal of Biological Macromolecules 69 (2014) 260–266
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Methylglyoxal mediated conformational changes in histone H2A—generation of carboxyethylated advanced glycation end products Abdul Rouf Mir, Moin uddin ∗ , Khursheed Alam, Asif Ali Department of Biochemistry, Jawaharlal Nehru Medical College, Faculty of Medicine, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
a r t i c l e
i n f o
Article history: Received 1 March 2014 Received in revised form 15 May 2014 Accepted 16 May 2014 Available online 28 May 2014 Keywords: Methylglyoxal Histone H2A Advance glycation end products (AGEs) N -(carboxyethyl) lysine
a b s t r a c t Methylglyoxal, an oxo-aldehyde has been implicated as a potential precursor in non enzymatic glycation reactions. Its role in the modification of extra cellular proteins has been extensively reported, but little is known about its modification of nuclear proteins, like histones. Here, we report the methylglyoxal induced modification of histone H2A which forms an essential part of intact core nucleosome. In this study commercially available histone H2A was subjected to in vitro non-enzymatic glycation by methylglyoxal. The structural alterations in the histone were characterised by various biophysical and biochemical techniques. The modified histone showed hyperchromicity at 276 nm, loss in intrinsic tyrosine fluorescence intensity at 305 nm along with a red shift, cross linking and dimer formation in SDS PAGE, induction of ␣-helix in CD spectroscopy, reduced hydrophobicity in ANS binding studies, accumulation of AGE products, increased carbonyl content, and appearance of a novel peak showing carboxyethylation complemented by a shift in amide I and amide II bands in ATR-FTIR spectroscopy. The modified histone exhibited increased melting temperatures (Tm ) and enhanced heat capacities (Cp ) in differential scanning calorimetric analysis. The results suggest that methylglyoxal significantly altered the structure of the nuclear histone H2A by non enzymatic glycation reaction. The conformational changes in histone H2A may influence the chromatin integrity which may have implications in various pathological conditions. © 2014 Published by Elsevier B.V.
1. Introduction Methylglyoxal (MG) is a dicarbonyl compound produced physiologically during various metabolic pathways, including from dihydroxyacetone phosphate and d-glyceraldehyde-3-phosphate, during glycolysis [1]. It is a highly reactive by product of nonenzymatic glycation at an early stage of the Maillard reaction. It is the most potent dicarbonyl compound that irreversibly reacts with amino and sulfhydryl groups in lipids, nucleic acids and proteins, forming MG-derived advanced glycation end-products [2]. Reports have established that MG modifies a variety of proteins including BSA, RNase A, collagen, lysozyme, lens crystallins and insulin. It has been commonly implicated in endothelium dysfunction, diabetes and in neurodegenerative diseases [3–8]. Proteins susceptible to MG modification with related functional impairment are called the “dicarbonyl proteome” [9]. Previous studies
have mostly concentrated on extra and intra cellular proteins [5–8] or on DNA damage [10,11], with little work on nuclear proteins [12–15]. This study looks at the impact of MG on histone H2A, one of the essential eukaryotic proteins that package the genetic material (DNA) inside the cell nucleus [16]. Histone H2A protein with other core histones (H2B, H3 and H4) forms an octameric histone core around which 147 bp of DNA is wrapped to form a nucleosome, the fundamental repeating unit of chromatin having regulatory role in addition to maintaining the genomic integrity [17]. The integrity of genome has gross dependence on the three-dimensional structures of the different histones as modifications impact diverse biological processes such as gene regulation, DNA repair, chromosome condensation (mitosis) and spermatogenesis (meiosis) [18]. 2. Experimental procedures 2.1. Modification of H2A histone by MG
∗ Corresponding author. Tel.: +91 9412596816; fax: +91 571 2702758. E-mail address: moin [email protected] (M. uddin). http://dx.doi.org/10.1016/j.ijbiomac.2014.05.057 0141-8130/© 2014 Published by Elsevier B.V.
The modification was carried out by incubating 42 M of H2A, dissolved in phosphate buffer saline (10 mM sodium phosphate
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buffer, pH 7.4 containing 150 mM NaCl) with varying concentrations of MG (2.5, 5, 7.5 and 10 mM) for 24 h at 37 ◦ C. The unreacted low molecular weight constituents were removed by extensive dialysis against sodium phosphate buffer. 2.2. Absorbance spectroscopy The absorption profile of native and MG-modified H2A histones were recorded on Shimadzu Spectrophotometer (UV 1700 model) in the wavelength range of 250–500 nm. 2.3. Fluorescence studies
F.I. of native sample − F.I. of glycated sample % loss of F.I. = × 100 F.I. of native sample
2.4. Circular dichroism determination Far-UV CD studies (190–250 nm) were carried on J-815 JASCO spectropolarimeter. Data are reported as molar residual ellipticity [] (deg cm−2 dmol−1 ) at a wavelength , based on the following equation [19]
Binding of 1-anilinonaphthalene-8-sulfonic acid (ANS) with native H2A and its MG modified counterpart was evaluated by fluorescence spectroscopy. The molar ratio between H2A histone and ANS was 1:10 and emission spectra were recorded between 400 and 600 nm wavelengths after exciting at 370 nm. Increase in fluorescence intensity (F.I.) was calculated as given below: % decrease of F.I. =
F.I. of native H2A − F.I. of MG-H2A F.I. of native H2A
× 100
Carbonyl content of native and MG modified histones was analysed as per the published protocol [24]. The concentration of DNPH was determined by absorbance measurement at 360 nm against guanidinium chloride using the molar extinction coefficient of 22,000 M−1 cm−1 and the carbonyl content was expressed as nmol per mg of protein. 2.9. Differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC) is a powerful technique for obtaining data on the thermodynamics of protein unfolding. The impact of modifying agents on the protein unfolding is obtained through Tm and H studies. DSC was performed on VPDSC microcalorimeter (MicroCal, Northampton, MA). Scans were recorded from 25 ◦ C to 85 ◦ C at a heating rate of 2 ◦ C min−1 . The heat capacity curves, Tm and H were analyzed using Origin 7.0 software. 2.10. Fourier transform infrared spectroscopic analysis-attenuated total reflectance (FTIR-ATR)
obs (m deg) MRE = 10 × n × Cp × l where obs is the observed ellipticity in degrees, Cp is the molar fraction and l is the length of the light path in centimetre. The ␣helical content of different histone H2A samples were calculated from values at 222 nm (MRE222 ) using the following equation (MRE222 − 1340) 30300
FTIR-ATR spectroscopy of the protein samples was performed on Perkin Elmer FT-IR spectrophotometer. Infrared spectra were recorded between 1000 cm−1 and 4000 cm−1 . FTIR studies were carried out at 10 mg ml−1 of protein concentration. 2.11. Statistical analysis of results All measurements were done in duplicates. Results are expressed as mean ± S.D. A two tailed p value lower than 0.05 was considered to be significant.
2.5. Electrophoresis Protein samples (25 g each) were loaded into the wells of 10% non reducing SDS polyacrylamide gel [20]. Electrophoresis was performed at 80 V for 4 h at room temperature. The gels were stained with silver nitrate and photographed by Molecular Imager Gel Doc XR system from Bio-Rad Laboratories, U.S.A. All other experiments were carried out with 42 M of native histone H2A and its counterpart modified by 7.5 mM MG. 2.6. AGE Assay The formation of AGE aggregates, and cross linking is well demonstrated by characteristic florescence emission at 440 nm after excitation at 370 nm as a consequence of non enzymatic glycation [21,22]. AGE-specific fluorescence was recorded and increase in fluorescence intensity (F.I.) was computed by the following equation [23]: % increase of F.I. =
2.7. Determination of surface hydrophobicity (H0 )
2.8. Determination of reactive carbonyl content
Fluorescence spectra were recorded on Shimadzu (RF-5301-PC) spectrofluorophotometer at 25 ◦ C in a 1 cm path length cell at 10 nm slit width. Intrinsic fluorescence was measured by exciting the protein samples at 275 nm and emission spectra were recorded in 300–400 nm range. Loss in the fluorescence intensity (F.I.) was calculated using the following equation:
%˛ helix =
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F.I. of MG-H2A − F.I. of native H2A F.I. of MG-H2A
× 100
3. Results 3.1. Absorbance spectroscopy Histone H2A showed a peak at 276.5 nm. The hyperchromicities shown by 2.5, 5, 7.5 and 10 mM MG modified H2A histone, in comparison to that of native H2A histone, at 276.5 nm were 53.20%, 67.81%, 72.35% and 74.23%, respectively. Appearance of a hump like peak at 330 nm was observed with the elevating concentration of MG (Fig. 1a). 3.2. Intrinsic fluorescence Native histone H2A gave fluorescence maxima at 305 nm, characteristic of tyrosine emission. The 2.5, 5, 7.5 and 10 mM MG modified H2A exhibited a decrease of 52.91%, 68.4%, 72.21% and 74.28% in intrinsic fluorescence intensity when compared to native H2A histone, respectively. MG modified H2A also showed a red shift of 2, 3, 4 and 4 nm with four increasing concentration of
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Fig. 1. (a) UV profile of native histone H2A (--), and histone H2A modified with 2.5 mM (--), 5 mM (-䊉-), 7.5 mM (--) and 10 mM (- -), methylglyoxal. (b) Fluorescence profile of native histone H2A (--), and histone H2A modified with 2.5 mM (--), 5 mM (-䊉-),7.5 mM (--) and 10 mM (-+-) methylglyoxal. (c) CD spectra of native histone H2A (--), and histone H2A modified with 7.5 mM methylglyoxal (--) in far UV region (200–250 nm).
MG, respectively. Furthermore, hump like peaks also appeared at 440 nm (Fig. 1b). 3.3. Circular dichroism Compared to native histone H2A, the modified histone exhibited an increase in negative ellipticity in the 208–224 nm regions. Two enhanced negative bands appeared at 222 and 208 nm in the modified sample. CD () values for native histone and MG modified H2A at 222 nm were found to be −1.1768 and −4.6383 mdeg, respectively (Fig. 1c). 3.4. Electrophoretic analysis Compared to the native histone H2A, MG modified histones showed slightly increased electrophoretic mobility along with increase in band stretching in SDS PAGE studies. Extra bands appeared in the modified histone H2A at higher molecular weight positions. The brightness of the bands showed a gradual increase with higher concentrations of MG (Fig. 2).
Fig. 2. SDS PAGE: 25 g each of native histone H2A and 2.5, 5, 7.5 and 10 mM methylglyoxal modified H2A were loaded into the wells of 10% polyacrylamide gel under non-denaturing conditions (lane 1–5), lane 6 shows the molecular weight marker.
3.5. AGE assay The characteristic fluorescence properties of AGEs were detected by maximum excitation and emission wavelengths at 370 nm and 443 nm, respectively. Native histone H2A showed no appreciable AGE fluorescence. The histone H2A modified with 7.5 mM of MG showed an increase in fluorescence intensity by 57.19% at 443 nm (Fig. 3). 3.6. Surface hydrophobicity The histone H2A modified with 7.5 mM of MG showed a decrease in ANS fluorescence intensity by 59.39% at 477 nm as compared to native H2A. The blue shift of 2 nm is observed as the native histone showed emission maxima at 479 nm (Fig. 4). 3.7. Reactive carbonyl content The carbonyl content in native H2A was found to be 0.8 nmol per mg of protein. Upon modification with 7.5 mM of MG, the carbonyl content increased to 32.5 nmol per mg (Fig. 5).
Fig. 3. AGE fluorescence profile of native histone H2A (dotted line), and histone H2A modified with 7.5 mM methylglyoxal (thick line).
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and 62.4 ◦ C with an enthalpy change (H1 ) of 79.2 kcal mol−1 and (H2 ) of 76.2 kcal mol−1 . Native and MG modified histone showed the heat capacities (Cp ) of 1.36 kcal mol−1 ◦ C−1 and 1.97 kcal mole−1 ◦ C−1 at neutral pH in phosphate buffer with 150 mM NaCl (Fig. 6). 3.9. FTIR-ATR spectroscopic analysis In FTIR spectra, native histone exhibited characteristic amide I band (C O stretching) at 1639 cm−1 . This band got shifted to 1635 cm−1 upon modification with MG. In the amide II region, native histone showed less transmittance as compared to the modified analogue. We also observed a new stretching band in the modified histone at 1731 cm−1 in comparison to native histone. Furthermore, a large band appeared for modified histone at 1082 cm−1 with lesser percent transmittance than its native counterpart. Large bands in the region between 3000 cm−1 and 3500 cm−1 with broader band for MG-H2A were also seen (Figs. 7 and 8 and in Table 1). Fig. 4. ANS fluorescence profile of native histone H2A (Thick line), and histone H2A modified with 7.5 mM methylglyoxal (thin line).
4. Discussion
Fig. 5. Protein-bound carbonyl concentration in native and modified H2A.
3.8. DSC measurements The melting temperature for native histone was obtained at 48.6 ◦ C with an enthalpy change of 63.6 kcal mol−1 . MG Modified histone H2A showed two melting temperatures i.e. at 57.2 ◦ C
Reports suggest the role of MG as an AGE precursor in potential damage of nucleic acids and proteins. Many works have appeared on DNA damage but the research on damage to histone proteins has not been very comprehensive [10,25]. In eukaryotes, some works have been carried on post translational modifications of H1, H3 and H4 histones but little is known about histone H2A [13,26]. Thus in this study, we have focussed on the modification of histone H2A by MG. We report a significant damage to native conformation of histone H2A upon modification by MG. The significant hyperchromicities in the case of MG modified H2A histone, in comparison to that of native H2A points towards the structural damage in histone H2A upon modification. The appearance of hump like peak at 330 nm in the modified histone may be attributed to the formation of AGEs. The intrinsic (tyrosine) fluorescence spectrum is determined chiefly by the polarity of the environment of tyrosine residues and it provides a sensitive means of monitoring conformational changes in proteins and protein-protein as well as ligand-protein interactions [27]. The loss of intrinsic fluorescence intensity by 74.28% on increasing the concentration of MG points towards the destruction or modification of tyrosine microenvironment. It is in conformity with earlier reports that the fluorescence emission maximum (m ) suffers a red shift when the tyrosine chromophores become more exposed to solvent and the quantum yield of fluorescence decreases when the chromophores interact with quenching agents [28]. Also the appearance of rising hump like peaks at 440 nm indicate the
Fig. 6. DSC thermograms [the heat capacity change at constant pressure (Cp ) versus temperature (T)] of native histone H2A (a) and histone H2A modified with 7.5 mM methylglyoxal (b).
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Table 1 Characterization of native and histone H2A modified by 7.5 mM methylglyoxal under identical experimental conditions. Parameters UV absorbance Florescence intensity (at nm) (exc . 275 nm) AGE florescence (exc . 370 nm) ANS florescence Carbonyl content (nmol mg−1 ) Ellipticity (mdeg) at 222 nm DSC measurements Cp (kcal mol−1 ◦ C−1 ) Tm 1 (◦ C) Tm 2 (◦ C) H1 (kcal mol−1 ) H2 (kcal mol−1 ) FTIR amide I (cm−1 )
Native H2A
MG-H2A
Modification (%)
0.163 86.74 (305)
0.59 24.11/309
72.35 72.21
13.68
31.96
57.19
62.82 32.5
59.39
154.7 0.8 −1.17684 1.36 48.6 – 63.6 1639
−4.6382 1.97 57.2 62.4 79.2 76.2 1635
increasing amounts of AGEs in the modified histone molecule. Several other studies have also shown similar quenching of intrinsic fluorescence in proteins, like immunoglobin G [23] and HSA [29]. CD spectral analysis showed large conformational changes in histone H2A as a consequence of MG modification. The increase in the negative ellipticity around 222 nm and 208 nm reflects the formation of ordered secondary structure and increased compactness in MG modified histone as compared to the less organised native protein. The appearance of two enhanced negative bands at 222 and 208 nm correspond to induction of ␣ helix in the modified histone. Increase in negative ellipticity CD () values for modified H2A correspond to 9.83% and 16.03% of ␣ helical structure in the respective proteins, showing an increase of 6.2% ␣ helix in the modified
histone. Rest of the protein molecule has both  components and random coil forms. Our CD findings stand in full conformity with the results published in case of MG modified haemoglobin [30] and ␣-crystallin [31]. The increased electrophoretic mobility of MG modified histones as compared to native histone H2A in SDS PAGE shows a loss of positive charge upon glycation of the epsilon amino groups of lysine residues by MG. Increase in band stretching with increase in the concentration of MG is indicative of cross linking and aggregate formation upon modification. The new bands that appeared at higher molecular weight positions in the modified samples indicate the formation of dimers and tetramers. Higher molecular weight aggregates (molecular weight above 200 kDa) that did not enter the gel were also observed in the proteins incubated with MG. The
Fig. 7. FTIR-ATR spectroscopic analysis of native (A) and MG modified histone H2A (B) recorded between 1000 and 3500 cm−1 .
Fig. 8. FTIR-ATR spectroscopic analysis of native (A) and MG modified histone (B) recorded between 1600 cm−1 and 1750 cm−1 .
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intensity of the bands showed a gradual increase with higher concentrations of modifying agent showing encapsulation of positive charges of lysine. Previous reports on other proteins suggest similar results for proteins modified by MG and histone H1 modified by glycating agents like glucose and ribose [13,14]. Altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation has also been reported for non-enzymatic browning of bovine ␣-crystallin [31]. The increase in AGE fluorescence intensity of histone H2A modified MG shows generation of AGE aggregates, and cross linking. The increased formation of AGEs is a consequence of the presence of abundant lysine residues in histone H2A that are glycated by MG. This increase in the amount of AGEs in MG-H2A shows that nuclear proteins are also prone to glycation like the extra-cellular proteins [32]. The most expected AGE production in histones is carboxyethylation of lysine residues and the same has been confirmed by FTIR studies. Previous studies on AGE-specific fluorescence in serum and urine of diabetic subjects showed similar increase in AGE fluorescence intensity suggesting the accumulation of AGEs [33]. Methylglyoxal has been widely reported to change the protein secondary structure [34], and this change varies depending upon type of protein under investigation. Some earlier studies on glycation of histones H1 and H2B using mass spectrometry have pointed out 14 possible glycation sites in histone H1 and 6 possible glycation sites in histone H2B after incubation with glucose. However it has been suggested that the glycated lysines can be different from molecule to molecule [35]. In another study on glycation of histone H1 analyzed with MS/MS spectrometry, two CML-modified lysine residues were reported [36]. From these studies, we can say that methylglyoxal mediated modification of histone H2A would be occurring at multiple sites. The surface hydrophilicity/hydrophobicity balance is altered upon glycation, changing the surface properties of histone H2A. Histone H2A being lysine rich showed enhanced binding to ANS indicating plenty of hydrophobic clusters in native protein. The decrease in ANS florescence upon MG treatment reflects folding of the histone molecule upon modification, leading to masking of hydrophobic patches, the structurally important regions in histones with role in nucleosome formation. The masking of hydrophobic patches may, therefore, effect the proper nucleosome formation. Methylglyoxal has been previously attributed with a decrease in ANS fluorescence in ␣-crystallin as well [37]. The blue shift of 2 nm in m in histone MG-H2A may be due to the charge transfer during the interaction of the charged group of lysine and arginine with the sulfonate group of ANS [38]. The carbonyl content was 40 times higher in modified histone as compared to the native form, showing an aggressive protein oxidation by MG. The substantially higher carbonyl content in H2A upon MG treatment can be explained by the abundant presence of oxidation prone aminoacids like lysine and arginine. The higher oxidation potential of MG has been previously reported for calf lens proteins where MG was reported to generate 24 times more carbonyls than the native proteins [39]. Our results show that MG is a strong glycating agent capable of causing significant damage to positively charged proteins. Furthermore DSC, which is the most direct approach for determining the intrinsic stability of a protein in dilute solution, showed enhanced thermodynamic stability in case of modified histone. The increase in melting temperature (Tm ) and the enthalpy change (H) in DSC results may be attributed to the methylglyoxal-mediated cross-linking and increase in hydrogen bonding in the more organised protein structure in histone. The deconvulated thermogram shows the presence of single and double calorimetric domains in native (Fig. 6a) and modified histone H2A (Fig. 6b), respectively, thus inferring to the structural changes in histone H2A upon modification that lead to the generation of a intermediate stage before the
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complete denaturation of the protein after modification suggesting the perturbation during unfolding. This variation in thermogram may be attributed to the MG induced structural stability in the modified protein and the formation of dimers that lead to the increased Tm and appearance of an addition peak. Native histone undergoes a two-state, the native and the unfolded form, folding-unfolding transition. Since histone H2A is a part of a histone dimer with H2B in nucleosome, its glyoxidated form with changed thermodynamic characteristics may affect the association in nucleosome. Previous studies have shown similar thermal effects of in vitro glycosylation of lens capsules resulting in elevations of Tm upon modification with glucose. DSC studies earlier have also revealed an elevation of the heat flow per unit mass during denaturation of other glycated proteins in diabetic patients [40,41]. FTIR ATR spectra showing the shifting of amide I band from 1639 cm−1 to 1635 cm−1 points towards a transition within  structure. It appears that MG has marginally changed the  structure of histone H2A. Also the increased percent transmittance and a shift in amide II band reflect an altered secondary structure. A new stretching that appeared in the modified histone at 1731 cm−1 marks the introduction of a carboxyethyl group exhibiting a typical C O stretching. Similar stretching has been reported in carboxymethylated chitin [42]. Since histone H2A is lysine rich, MG generates a carboxyethylated lysine residue, which has been reported as an AGE product for many proteins under oxidative stress. A large band appears at 1074 cm−1 for modified histone H2A which is lacking in the native histone because there is no characteristic N C␣ vibration in polypeptides in this region [43]. This shows that MG has introduced N C␣ stretching in the polypeptide. This band in the IR absorption spectra is also a marker for protein glycosylation—a post translation modification, indicating that MG may have glycosylated histone H2A. The absorbance bands between 3000 cm−1 and 3500 cm−1 are attributed to the N H and O H stretching. The broadness of band for modified protein is due to accumulation of OH groups by sugar moiety [44]. 5. Conclusion In conclusion, our study shows that MG is a strong glycating agent inducing substantial modifications in secondary and tertiary structure of nucleosomal protein histone H2A. Since structural characteristics of nuclear proteins are important for the integrity and normal functioning of genome, the non enzymatic glycation of histone H2A by MG may have severe implications in various pathological conditions. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Methylglyoxal mediated conformational changes in histone H2A—generation of carboxyethylated advanced glycation end products Abdul Rouf Mir, Moin uddin ∗ , Khursheed Alam, Asif Ali Department of Biochemistry, Jawaharlal Nehru Medical College, Faculty of Medicine, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
a r t i c l e
i n f o
Article history: Received 1 March 2014 Received in revised form 15 May 2014 Accepted 16 May 2014 Available online 28 May 2014 Keywords: Methylglyoxal Histone H2A Advance glycation end products (AGEs) N -(carboxyethyl) lysine
a b s t r a c t Methylglyoxal, an oxo-aldehyde has been implicated as a potential precursor in non enzymatic glycation reactions. Its role in the modification of extra cellular proteins has been extensively reported, but little is known about its modification of nuclear proteins, like histones. Here, we report the methylglyoxal induced modification of histone H2A which forms an essential part of intact core nucleosome. In this study commercially available histone H2A was subjected to in vitro non-enzymatic glycation by methylglyoxal. The structural alterations in the histone were characterised by various biophysical and biochemical techniques. The modified histone showed hyperchromicity at 276 nm, loss in intrinsic tyrosine fluorescence intensity at 305 nm along with a red shift, cross linking and dimer formation in SDS PAGE, induction of ␣-helix in CD spectroscopy, reduced hydrophobicity in ANS binding studies, accumulation of AGE products, increased carbonyl content, and appearance of a novel peak showing carboxyethylation complemented by a shift in amide I and amide II bands in ATR-FTIR spectroscopy. The modified histone exhibited increased melting temperatures (Tm ) and enhanced heat capacities (Cp ) in differential scanning calorimetric analysis. The results suggest that methylglyoxal significantly altered the structure of the nuclear histone H2A by non enzymatic glycation reaction. The conformational changes in histone H2A may influence the chromatin integrity which may have implications in various pathological conditions. © 2014 Published by Elsevier B.V.
1. Introduction Methylglyoxal (MG) is a dicarbonyl compound produced physiologically during various metabolic pathways, including from dihydroxyacetone phosphate and d-glyceraldehyde-3-phosphate, during glycolysis [1]. It is a highly reactive by product of nonenzymatic glycation at an early stage of the Maillard reaction. It is the most potent dicarbonyl compound that irreversibly reacts with amino and sulfhydryl groups in lipids, nucleic acids and proteins, forming MG-derived advanced glycation end-products [2]. Reports have established that MG modifies a variety of proteins including BSA, RNase A, collagen, lysozyme, lens crystallins and insulin. It has been commonly implicated in endothelium dysfunction, diabetes and in neurodegenerative diseases [3–8]. Proteins susceptible to MG modification with related functional impairment are called the “dicarbonyl proteome” [9]. Previous studies
have mostly concentrated on extra and intra cellular proteins [5–8] or on DNA damage [10,11], with little work on nuclear proteins [12–15]. This study looks at the impact of MG on histone H2A, one of the essential eukaryotic proteins that package the genetic material (DNA) inside the cell nucleus [16]. Histone H2A protein with other core histones (H2B, H3 and H4) forms an octameric histone core around which 147 bp of DNA is wrapped to form a nucleosome, the fundamental repeating unit of chromatin having regulatory role in addition to maintaining the genomic integrity [17]. The integrity of genome has gross dependence on the three-dimensional structures of the different histones as modifications impact diverse biological processes such as gene regulation, DNA repair, chromosome condensation (mitosis) and spermatogenesis (meiosis) [18]. 2. Experimental procedures 2.1. Modification of H2A histone by MG
∗ Corresponding author. Tel.: +91 9412596816; fax: +91 571 2702758. E-mail address: moin [email protected] (M. uddin). http://dx.doi.org/10.1016/j.ijbiomac.2014.05.057 0141-8130/© 2014 Published by Elsevier B.V.
The modification was carried out by incubating 42 M of H2A, dissolved in phosphate buffer saline (10 mM sodium phosphate
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buffer, pH 7.4 containing 150 mM NaCl) with varying concentrations of MG (2.5, 5, 7.5 and 10 mM) for 24 h at 37 ◦ C. The unreacted low molecular weight constituents were removed by extensive dialysis against sodium phosphate buffer. 2.2. Absorbance spectroscopy The absorption profile of native and MG-modified H2A histones were recorded on Shimadzu Spectrophotometer (UV 1700 model) in the wavelength range of 250–500 nm. 2.3. Fluorescence studies
F.I. of native sample − F.I. of glycated sample % loss of F.I. = × 100 F.I. of native sample
2.4. Circular dichroism determination Far-UV CD studies (190–250 nm) were carried on J-815 JASCO spectropolarimeter. Data are reported as molar residual ellipticity [] (deg cm−2 dmol−1 ) at a wavelength , based on the following equation [19]
Binding of 1-anilinonaphthalene-8-sulfonic acid (ANS) with native H2A and its MG modified counterpart was evaluated by fluorescence spectroscopy. The molar ratio between H2A histone and ANS was 1:10 and emission spectra were recorded between 400 and 600 nm wavelengths after exciting at 370 nm. Increase in fluorescence intensity (F.I.) was calculated as given below: % decrease of F.I. =
F.I. of native H2A − F.I. of MG-H2A F.I. of native H2A
× 100
Carbonyl content of native and MG modified histones was analysed as per the published protocol [24]. The concentration of DNPH was determined by absorbance measurement at 360 nm against guanidinium chloride using the molar extinction coefficient of 22,000 M−1 cm−1 and the carbonyl content was expressed as nmol per mg of protein. 2.9. Differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC) is a powerful technique for obtaining data on the thermodynamics of protein unfolding. The impact of modifying agents on the protein unfolding is obtained through Tm and H studies. DSC was performed on VPDSC microcalorimeter (MicroCal, Northampton, MA). Scans were recorded from 25 ◦ C to 85 ◦ C at a heating rate of 2 ◦ C min−1 . The heat capacity curves, Tm and H were analyzed using Origin 7.0 software. 2.10. Fourier transform infrared spectroscopic analysis-attenuated total reflectance (FTIR-ATR)
obs (m deg) MRE = 10 × n × Cp × l where obs is the observed ellipticity in degrees, Cp is the molar fraction and l is the length of the light path in centimetre. The ␣helical content of different histone H2A samples were calculated from values at 222 nm (MRE222 ) using the following equation (MRE222 − 1340) 30300
FTIR-ATR spectroscopy of the protein samples was performed on Perkin Elmer FT-IR spectrophotometer. Infrared spectra were recorded between 1000 cm−1 and 4000 cm−1 . FTIR studies were carried out at 10 mg ml−1 of protein concentration. 2.11. Statistical analysis of results All measurements were done in duplicates. Results are expressed as mean ± S.D. A two tailed p value lower than 0.05 was considered to be significant.
2.5. Electrophoresis Protein samples (25 g each) were loaded into the wells of 10% non reducing SDS polyacrylamide gel [20]. Electrophoresis was performed at 80 V for 4 h at room temperature. The gels were stained with silver nitrate and photographed by Molecular Imager Gel Doc XR system from Bio-Rad Laboratories, U.S.A. All other experiments were carried out with 42 M of native histone H2A and its counterpart modified by 7.5 mM MG. 2.6. AGE Assay The formation of AGE aggregates, and cross linking is well demonstrated by characteristic florescence emission at 440 nm after excitation at 370 nm as a consequence of non enzymatic glycation [21,22]. AGE-specific fluorescence was recorded and increase in fluorescence intensity (F.I.) was computed by the following equation [23]: % increase of F.I. =
2.7. Determination of surface hydrophobicity (H0 )
2.8. Determination of reactive carbonyl content
Fluorescence spectra were recorded on Shimadzu (RF-5301-PC) spectrofluorophotometer at 25 ◦ C in a 1 cm path length cell at 10 nm slit width. Intrinsic fluorescence was measured by exciting the protein samples at 275 nm and emission spectra were recorded in 300–400 nm range. Loss in the fluorescence intensity (F.I.) was calculated using the following equation:
%˛ helix =
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F.I. of MG-H2A − F.I. of native H2A F.I. of MG-H2A
× 100
3. Results 3.1. Absorbance spectroscopy Histone H2A showed a peak at 276.5 nm. The hyperchromicities shown by 2.5, 5, 7.5 and 10 mM MG modified H2A histone, in comparison to that of native H2A histone, at 276.5 nm were 53.20%, 67.81%, 72.35% and 74.23%, respectively. Appearance of a hump like peak at 330 nm was observed with the elevating concentration of MG (Fig. 1a). 3.2. Intrinsic fluorescence Native histone H2A gave fluorescence maxima at 305 nm, characteristic of tyrosine emission. The 2.5, 5, 7.5 and 10 mM MG modified H2A exhibited a decrease of 52.91%, 68.4%, 72.21% and 74.28% in intrinsic fluorescence intensity when compared to native H2A histone, respectively. MG modified H2A also showed a red shift of 2, 3, 4 and 4 nm with four increasing concentration of
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Fig. 1. (a) UV profile of native histone H2A (--), and histone H2A modified with 2.5 mM (--), 5 mM (-䊉-), 7.5 mM (--) and 10 mM (- -), methylglyoxal. (b) Fluorescence profile of native histone H2A (--), and histone H2A modified with 2.5 mM (--), 5 mM (-䊉-),7.5 mM (--) and 10 mM (-+-) methylglyoxal. (c) CD spectra of native histone H2A (--), and histone H2A modified with 7.5 mM methylglyoxal (--) in far UV region (200–250 nm).
MG, respectively. Furthermore, hump like peaks also appeared at 440 nm (Fig. 1b). 3.3. Circular dichroism Compared to native histone H2A, the modified histone exhibited an increase in negative ellipticity in the 208–224 nm regions. Two enhanced negative bands appeared at 222 and 208 nm in the modified sample. CD () values for native histone and MG modified H2A at 222 nm were found to be −1.1768 and −4.6383 mdeg, respectively (Fig. 1c). 3.4. Electrophoretic analysis Compared to the native histone H2A, MG modified histones showed slightly increased electrophoretic mobility along with increase in band stretching in SDS PAGE studies. Extra bands appeared in the modified histone H2A at higher molecular weight positions. The brightness of the bands showed a gradual increase with higher concentrations of MG (Fig. 2).
Fig. 2. SDS PAGE: 25 g each of native histone H2A and 2.5, 5, 7.5 and 10 mM methylglyoxal modified H2A were loaded into the wells of 10% polyacrylamide gel under non-denaturing conditions (lane 1–5), lane 6 shows the molecular weight marker.
3.5. AGE assay The characteristic fluorescence properties of AGEs were detected by maximum excitation and emission wavelengths at 370 nm and 443 nm, respectively. Native histone H2A showed no appreciable AGE fluorescence. The histone H2A modified with 7.5 mM of MG showed an increase in fluorescence intensity by 57.19% at 443 nm (Fig. 3). 3.6. Surface hydrophobicity The histone H2A modified with 7.5 mM of MG showed a decrease in ANS fluorescence intensity by 59.39% at 477 nm as compared to native H2A. The blue shift of 2 nm is observed as the native histone showed emission maxima at 479 nm (Fig. 4). 3.7. Reactive carbonyl content The carbonyl content in native H2A was found to be 0.8 nmol per mg of protein. Upon modification with 7.5 mM of MG, the carbonyl content increased to 32.5 nmol per mg (Fig. 5).
Fig. 3. AGE fluorescence profile of native histone H2A (dotted line), and histone H2A modified with 7.5 mM methylglyoxal (thick line).
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and 62.4 ◦ C with an enthalpy change (H1 ) of 79.2 kcal mol−1 and (H2 ) of 76.2 kcal mol−1 . Native and MG modified histone showed the heat capacities (Cp ) of 1.36 kcal mol−1 ◦ C−1 and 1.97 kcal mole−1 ◦ C−1 at neutral pH in phosphate buffer with 150 mM NaCl (Fig. 6). 3.9. FTIR-ATR spectroscopic analysis In FTIR spectra, native histone exhibited characteristic amide I band (C O stretching) at 1639 cm−1 . This band got shifted to 1635 cm−1 upon modification with MG. In the amide II region, native histone showed less transmittance as compared to the modified analogue. We also observed a new stretching band in the modified histone at 1731 cm−1 in comparison to native histone. Furthermore, a large band appeared for modified histone at 1082 cm−1 with lesser percent transmittance than its native counterpart. Large bands in the region between 3000 cm−1 and 3500 cm−1 with broader band for MG-H2A were also seen (Figs. 7 and 8 and in Table 1). Fig. 4. ANS fluorescence profile of native histone H2A (Thick line), and histone H2A modified with 7.5 mM methylglyoxal (thin line).
4. Discussion
Fig. 5. Protein-bound carbonyl concentration in native and modified H2A.
3.8. DSC measurements The melting temperature for native histone was obtained at 48.6 ◦ C with an enthalpy change of 63.6 kcal mol−1 . MG Modified histone H2A showed two melting temperatures i.e. at 57.2 ◦ C
Reports suggest the role of MG as an AGE precursor in potential damage of nucleic acids and proteins. Many works have appeared on DNA damage but the research on damage to histone proteins has not been very comprehensive [10,25]. In eukaryotes, some works have been carried on post translational modifications of H1, H3 and H4 histones but little is known about histone H2A [13,26]. Thus in this study, we have focussed on the modification of histone H2A by MG. We report a significant damage to native conformation of histone H2A upon modification by MG. The significant hyperchromicities in the case of MG modified H2A histone, in comparison to that of native H2A points towards the structural damage in histone H2A upon modification. The appearance of hump like peak at 330 nm in the modified histone may be attributed to the formation of AGEs. The intrinsic (tyrosine) fluorescence spectrum is determined chiefly by the polarity of the environment of tyrosine residues and it provides a sensitive means of monitoring conformational changes in proteins and protein-protein as well as ligand-protein interactions [27]. The loss of intrinsic fluorescence intensity by 74.28% on increasing the concentration of MG points towards the destruction or modification of tyrosine microenvironment. It is in conformity with earlier reports that the fluorescence emission maximum (m ) suffers a red shift when the tyrosine chromophores become more exposed to solvent and the quantum yield of fluorescence decreases when the chromophores interact with quenching agents [28]. Also the appearance of rising hump like peaks at 440 nm indicate the
Fig. 6. DSC thermograms [the heat capacity change at constant pressure (Cp ) versus temperature (T)] of native histone H2A (a) and histone H2A modified with 7.5 mM methylglyoxal (b).
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Table 1 Characterization of native and histone H2A modified by 7.5 mM methylglyoxal under identical experimental conditions. Parameters UV absorbance Florescence intensity (at nm) (exc . 275 nm) AGE florescence (exc . 370 nm) ANS florescence Carbonyl content (nmol mg−1 ) Ellipticity (mdeg) at 222 nm DSC measurements Cp (kcal mol−1 ◦ C−1 ) Tm 1 (◦ C) Tm 2 (◦ C) H1 (kcal mol−1 ) H2 (kcal mol−1 ) FTIR amide I (cm−1 )
Native H2A
MG-H2A
Modification (%)
0.163 86.74 (305)
0.59 24.11/309
72.35 72.21
13.68
31.96
57.19
62.82 32.5
59.39
154.7 0.8 −1.17684 1.36 48.6 – 63.6 1639
−4.6382 1.97 57.2 62.4 79.2 76.2 1635
increasing amounts of AGEs in the modified histone molecule. Several other studies have also shown similar quenching of intrinsic fluorescence in proteins, like immunoglobin G [23] and HSA [29]. CD spectral analysis showed large conformational changes in histone H2A as a consequence of MG modification. The increase in the negative ellipticity around 222 nm and 208 nm reflects the formation of ordered secondary structure and increased compactness in MG modified histone as compared to the less organised native protein. The appearance of two enhanced negative bands at 222 and 208 nm correspond to induction of ␣ helix in the modified histone. Increase in negative ellipticity CD () values for modified H2A correspond to 9.83% and 16.03% of ␣ helical structure in the respective proteins, showing an increase of 6.2% ␣ helix in the modified
histone. Rest of the protein molecule has both  components and random coil forms. Our CD findings stand in full conformity with the results published in case of MG modified haemoglobin [30] and ␣-crystallin [31]. The increased electrophoretic mobility of MG modified histones as compared to native histone H2A in SDS PAGE shows a loss of positive charge upon glycation of the epsilon amino groups of lysine residues by MG. Increase in band stretching with increase in the concentration of MG is indicative of cross linking and aggregate formation upon modification. The new bands that appeared at higher molecular weight positions in the modified samples indicate the formation of dimers and tetramers. Higher molecular weight aggregates (molecular weight above 200 kDa) that did not enter the gel were also observed in the proteins incubated with MG. The
Fig. 7. FTIR-ATR spectroscopic analysis of native (A) and MG modified histone H2A (B) recorded between 1000 and 3500 cm−1 .
Fig. 8. FTIR-ATR spectroscopic analysis of native (A) and MG modified histone (B) recorded between 1600 cm−1 and 1750 cm−1 .
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intensity of the bands showed a gradual increase with higher concentrations of modifying agent showing encapsulation of positive charges of lysine. Previous reports on other proteins suggest similar results for proteins modified by MG and histone H1 modified by glycating agents like glucose and ribose [13,14]. Altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation has also been reported for non-enzymatic browning of bovine ␣-crystallin [31]. The increase in AGE fluorescence intensity of histone H2A modified MG shows generation of AGE aggregates, and cross linking. The increased formation of AGEs is a consequence of the presence of abundant lysine residues in histone H2A that are glycated by MG. This increase in the amount of AGEs in MG-H2A shows that nuclear proteins are also prone to glycation like the extra-cellular proteins [32]. The most expected AGE production in histones is carboxyethylation of lysine residues and the same has been confirmed by FTIR studies. Previous studies on AGE-specific fluorescence in serum and urine of diabetic subjects showed similar increase in AGE fluorescence intensity suggesting the accumulation of AGEs [33]. Methylglyoxal has been widely reported to change the protein secondary structure [34], and this change varies depending upon type of protein under investigation. Some earlier studies on glycation of histones H1 and H2B using mass spectrometry have pointed out 14 possible glycation sites in histone H1 and 6 possible glycation sites in histone H2B after incubation with glucose. However it has been suggested that the glycated lysines can be different from molecule to molecule [35]. In another study on glycation of histone H1 analyzed with MS/MS spectrometry, two CML-modified lysine residues were reported [36]. From these studies, we can say that methylglyoxal mediated modification of histone H2A would be occurring at multiple sites. The surface hydrophilicity/hydrophobicity balance is altered upon glycation, changing the surface properties of histone H2A. Histone H2A being lysine rich showed enhanced binding to ANS indicating plenty of hydrophobic clusters in native protein. The decrease in ANS florescence upon MG treatment reflects folding of the histone molecule upon modification, leading to masking of hydrophobic patches, the structurally important regions in histones with role in nucleosome formation. The masking of hydrophobic patches may, therefore, effect the proper nucleosome formation. Methylglyoxal has been previously attributed with a decrease in ANS fluorescence in ␣-crystallin as well [37]. The blue shift of 2 nm in m in histone MG-H2A may be due to the charge transfer during the interaction of the charged group of lysine and arginine with the sulfonate group of ANS [38]. The carbonyl content was 40 times higher in modified histone as compared to the native form, showing an aggressive protein oxidation by MG. The substantially higher carbonyl content in H2A upon MG treatment can be explained by the abundant presence of oxidation prone aminoacids like lysine and arginine. The higher oxidation potential of MG has been previously reported for calf lens proteins where MG was reported to generate 24 times more carbonyls than the native proteins [39]. Our results show that MG is a strong glycating agent capable of causing significant damage to positively charged proteins. Furthermore DSC, which is the most direct approach for determining the intrinsic stability of a protein in dilute solution, showed enhanced thermodynamic stability in case of modified histone. The increase in melting temperature (Tm ) and the enthalpy change (H) in DSC results may be attributed to the methylglyoxal-mediated cross-linking and increase in hydrogen bonding in the more organised protein structure in histone. The deconvulated thermogram shows the presence of single and double calorimetric domains in native (Fig. 6a) and modified histone H2A (Fig. 6b), respectively, thus inferring to the structural changes in histone H2A upon modification that lead to the generation of a intermediate stage before the
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complete denaturation of the protein after modification suggesting the perturbation during unfolding. This variation in thermogram may be attributed to the MG induced structural stability in the modified protein and the formation of dimers that lead to the increased Tm and appearance of an addition peak. Native histone undergoes a two-state, the native and the unfolded form, folding-unfolding transition. Since histone H2A is a part of a histone dimer with H2B in nucleosome, its glyoxidated form with changed thermodynamic characteristics may affect the association in nucleosome. Previous studies have shown similar thermal effects of in vitro glycosylation of lens capsules resulting in elevations of Tm upon modification with glucose. DSC studies earlier have also revealed an elevation of the heat flow per unit mass during denaturation of other glycated proteins in diabetic patients [40,41]. FTIR ATR spectra showing the shifting of amide I band from 1639 cm−1 to 1635 cm−1 points towards a transition within  structure. It appears that MG has marginally changed the  structure of histone H2A. Also the increased percent transmittance and a shift in amide II band reflect an altered secondary structure. A new stretching that appeared in the modified histone at 1731 cm−1 marks the introduction of a carboxyethyl group exhibiting a typical C O stretching. Similar stretching has been reported in carboxymethylated chitin [42]. Since histone H2A is lysine rich, MG generates a carboxyethylated lysine residue, which has been reported as an AGE product for many proteins under oxidative stress. A large band appears at 1074 cm−1 for modified histone H2A which is lacking in the native histone because there is no characteristic N C␣ vibration in polypeptides in this region [43]. This shows that MG has introduced N C␣ stretching in the polypeptide. This band in the IR absorption spectra is also a marker for protein glycosylation—a post translation modification, indicating that MG may have glycosylated histone H2A. The absorbance bands between 3000 cm−1 and 3500 cm−1 are attributed to the N H and O H stretching. The broadness of band for modified protein is due to accumulation of OH groups by sugar moiety [44]. 5. Conclusion In conclusion, our study shows that MG is a strong glycating agent inducing substantial modifications in secondary and tertiary structure of nucleosomal protein histone H2A. Since structural characteristics of nuclear proteins are important for the integrity and normal functioning of genome, the non enzymatic glycation of histone H2A by MG may have severe implications in various pathological conditions. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
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