Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2021 Aug 9;6(32):20887–20894. doi: 10.1021/acsomega.1c02300

Structural and Functional Characterization of Covalently Modified Proteins Formed By a Glycating Agent, Glyoxal

Gurumayum Suraj Sharma †, Reshmee Bhattacharya ‡, Snigdha Krishna ‡, Suliman Y Alomar §, Afrah F Alkhuriji ∥, Marina Warepam ⊥, Kritika Kumari ‡, Hamidur Rahaman ⊥, Laishram Rajendrakumar Singh ‡,*
PMCID: PMC8374913  PMID: 34423196

Abstract

graphic file with name ao1c02300_0008.jpg

Glycation, the main consequence of hyperglycemia, is one of the major perpetrators of diabetes and several other conditions, including coronary and neurodegenerative complications. Such a hyperglycemic condition is represented by a large increase in levels of various glycation end products including glyoxal, methylglyoxal, and carboxymethyl-lysine among others. These glycation end products are known to play a crucial role in diabetic complications due to their ability to covalently modify important proteins and enzymes, specifically at lysine residues (a process termed as glycation), making them non-functional. Previous studies have largely paid attention on characterization and identification of these reactive glycating agents. Structural and functional consequences of proteins affected by glycation have not yet been critically investigated. We have made a systematic investigation on the early conformational changes and functional alterations brought about by a glycating agent, glyoxal, on different proteins. We found that the early event in glycation includes an increase in hydrodynamic diameter, followed by minor structural alterations sufficient to impair enzyme activity. The study indicates the importance of glyoxal-induced early structural alteration of proteins toward the pathophysiology of hyperglycemia/diabetes and associated conditions.

Introduction

Hyperglycemia is known to be the main perpetrator of diabetes and other related complications, such as chronic renal diseases, cataracts, heart diseases, rheumatoid arthritis, atherosclerosis, peripheral vascular diseases, and neurodegenerative diseases (NDs).1−3 Hyperglycemia is characterized by the accumulation of highly reactive reducing sugars such as glucose, fructose, and ribose-5-phosphate among others. Under such conditions, several proteins undergo non-enzymatic covalent modifications via a process called glycation by reducing sugars, specifically at lysine or arginine residues. Such a modification (via Maillard reaction) is a complex set of reactions initiated by a nucleophilic addition between a free amino group of protein and electrophilic carbonyl groups of glucose, forming an unstable Schiff’s base, aldimine. Schiff’s base undergoes Amadori rearrangement to form a stable ketoamine called an Amadori product.4 These Amadori products further undergo enolization reactions to form 1,2-dicarbonyl compounds (glyoxal, methylglyoxal) or 2,3-dicarbonyl compounds (1-deoxyglucosone). Finally, the advanced glycation end products (AGEs) are formed by following two different pathways: the irreversible rearrangement of the Amadori products through both oxidative and non-oxidative pathways and the subsequent condensation reaction between the dicarbonyls and the side chain of lysine, cysteine, and arginine residues (see Scheme 1).

Scheme 1. Sequential Events of AGE Formation.

Scheme 1

Open in a new tab

The amino group of proteins reacts with the carbonyl group of reducing sugars to form the Schiff base which undergoes rearrangement forming Amadori products. Finally, AGEs are formed via two different pathways: by irreversible breakdown of Amadori products via an oxidative/non-oxidative pathway or by reaction between dicarbonyl (e.g., glyoxal, methylglyoxal, 1-deoxyglucosone; 1-DG) and Lys, Arg, and Cys residues.

In spite of the fact that sugars are the main precursors of AGE formation, these intermediate dicarbonyls are much more reactive than other reducing sugars (like ribosyl or d-glucose) and act as propagators of the non-enzymatic glycation and cross-linking of several proteins. Accumulation of such AGEs and consequent glycation of proteins in tissues and serum are considered to be the major causes of pathologies associated with hyperglycemia. Consequently, glycation index of hemoglobin has been used to determine the magnitude of hyperglycemia in diabetes.5,6

The complexity and heterogeneous nature of AGEs have made their studies one of the most challenging research areas. As a result, large focus has been paid to characterize different AGE products and identification of the nature of protein oligomers induced by different AGEs.7−11 Although there is existence of a few literature studies, the structural and functional characterization of proteins upon glycation have not been properly understood. In this communication, we have made a systematic investigation on the early and late conformational changes and functional alteration brought about by the glycating agent, glyoxal, on different proteins (alpha lactalbumin, α-LA; lysozyme, Lyz; carbonic anhydrase, CA; and myoglobin, Myo). These proteins were chosen as they have different physico-chemical properties and variable functions and the functional impairment of some of these proteins is linked with different pathophysiology in humans. Furthermore, Lyz, CA, etc. have been reported to undergo modification by glyoxals in vitro and in vivo.12,13 We found that all proteins undergo minor structural changes in the early time period sufficient to impair or exhibit unwanted enzyme activity. The study indicates the importance of early structural alteration on proteins induced by glyoxal toward the pathophysiology of hyperglycemia or diabetes.

Results

Sensitivity toward Glycation Varies for Different Proteins

We have taken four proteins having different physico-chemical properties, as summarized in Table 1.

Table 1. Physico-Chemical Properties of Proteins Under Study.

protein molecular weight no. of lysine residue pI
α-LA 14.2 12 4.5
Myo 17.6 18 6.97
Lyz 14.3 6 11.3
CA 29 18 5.9
Open in a new tab

Protein adduct formation was assessed by analyzing the increase in free carbonyl content upon treatment with glyoxal. It is seen in Table 2 that there is an increase in the free carbonyl content upon treatment with glyoxal. However, the increases at 1.0 and 2.5 mM concentrations were not significant in some proteins; hence, we have chosen 5.0 mM as the standard concentration for further analyses.

Table 2. Protein Carbonyl Content upon Treatment with Different Concentrations of Glyoxals.

carbonyl content (in μM/μM of protein)
protein (mM) alpha-LA lysozyme CA myoglobin
0 0.0071 0.0168 0.0249 0.0119
1 0.0593 0.0408 0.0696 0.0415
2.5 0.1902 0.0584 0.2088 0.0616
5 0.2581 0.1052 0.3465 0.4767
Open in a new tab

First, the time-dependent structural changes were investigated by measuring the increase in hydrodynamic diameter of the proteins (Table 3, also see Figure S1 for size distribution by volume for each protein recorded at different time frames).

Table 3. Changes in Hydrodynamic Diameters of α-LA, Myo, Lyz, and CA upon Modification by Glyoxal.

time (h) α-LA (nm) Myo (nm) Lyz (nm) CA (nm)
0 3.67 ± 0.22 3.94 ± 0.24 3.60 ± 0.18 4.34 ± 0.30
1 3.62 ± 0.24 4.09 ± 0.26 3.41 ± 0.19 4.42 ± 0.33
2 3.70 ± 0.23 4.26 ± 0.25 3.32 ± 0.20 4.56 ± 0.34
3 3.84 ± 0.25 4.23 ± 0.29 3.39 ± 0.20 4.63 ± 0.32
4 4.68 ± 0.30 3.99 ± 0.25 3.36 ± 0.21 4.47 ± 0.39
5 95.89 ± 7.09, 335 ± 24.89 4.01 ± 0.26 3.35 ± 0.17 4.59 ± 0.36
6   4.42 ± 0.30 3.47 ± 0.22 4.78 ± 0.36
7   4.83 ± 0.32 3.26 ± 0.24 4.80 ± 0.35
8   200 ± 11, 930 ± 58 3.42 ± 0.19 4.81 ± 0.37
9   238 ± 16, 1358 ± 99 3.49 ± 0.20 141 ± 10.50, 749 ± 52.43
10     3.65 ± 0.22  
12     3.55 ± 0.18  
14     3.53 ± 0.26  
16     3.49 ± 0.21  
18     3.62 ± 0.30  
20     3.64 ± 0.21  
22     3.68 ± 0.23  
24     3.79 ± 0.25  
26     287 ± 14.35, 1115 ± 74.70  
Open in a new tab

A time-dependent plot of the change in hydrodynamic diameter illustrates at least two regimes of minor initial changes, followed by gross oligomerization (see Figure S2). The monomers of all proteins undergo a maximum increase in hydrodynamic diameter, beyond which the formation of multimers/oligomers appeared. Importantly, all proteins have different time frames to reach their maximum hydrodynamic diameter. For instance, the tentative time for the maximum increase in hydrodynamic diameter was 4 h for α-LA, 7 h for Myo, 8 h for CA, and 24 h for Lyz. The maximum change in hydrodynamic diameter ranges from 25 to 30% in the case of α-LA and Myo but only 5–10% in the case of Lyz and CA, as can be seen in Figure 1. The results indicate that the time frame and the increase in hydrodynamic diameter are different for different proteins.

Figure 1.

Figure 1

Open in a new tab

Alteration in hydrodynamic diameter of the monomeric state of proteins upon glycation. Percent increase in hydrodynamic diameter of monomers of α-LA, Myo, Lyz, and CA upon modification with 5 mM glyoxal.

Enzyme Activity Is Altered at the Maximum Increase in Hydrodynamic Diameter

Our next curiosity is to investigate if these alterations in structures bring about changes in functional activity of the glycated proteins. We have performed activity assays for Lyz, CA, and Myo using appropriate substrates, and the results are presented as percent change (Figure 2, also see Figure S3 for the activity curve of each enzyme).

Figure 2.

Figure 2

Open in a new tab

Effect of glycation on functional activity of proteins.

It is seen that there was a 30–40% decrease in activity in the cases of Lyz and CA. We also observed an increase in peroxidase function of Myo upon glycation. Indeed, native Myo is devoid of peroxidase activity since heme is buried in the protein matrix and remains in the hexa-coordinated state.22 Such gain in peroxidase activity suggests conformational alteration upon modification. We also recorded absorption spectra of heme of modified Myo. We observed a decrease in the absorbance at 409 nm, suggesting alteration in the heme microenvironment (see Figure S4).

Glycation Induces Different Structural Alterations at the Early Time Frame

To further characterize the structural consequences in the proteins due to glycation, we monitored the conformational changes using multiple spectroscopic probes. We use similar time frames obtained from the dynamic light scattering (DLS) studies, that is, 4 h for α-LA, 7 h for Myo, 8 h for CA, and 24 h for Lyz. Far-UV CD spectra indicate a subtle increase in the secondary structure of the proteins (Figure 3A). Indeed, analysis of secondary structural components revealed no alterations in α-helix or β-sheet contents (see Figure S5).

Figure 3.

Figure 3

Open in a new tab

Glyoxal inducing structural alteration of proteins. (A) Far-UV CD spectra of native and glycated proteins. (B) Near-UV CD spectra of native and glycated proteins. (C) ANS fluorescence spectra of native and glycated proteins. Solid and dashed lines represent native and glycated proteins, respectively.

Near-UV CD measurements were carried out to evaluate gross changes in tertiary contacts (Figure 3B). A decrease in ellipticity at θ270 in α-LA and Myo revealed certain alterations in tertiary contacts, while a slight increase was observed in the case of Lyz. However, there was complete absence of any observable effect in the case of CA. In addition to these observations, ANS binding assays also provided indications for certain variations in the conformational status of the glycated proteins (Figure 3C). ANS binding was prominent with concomitant blue shift in α-LA and Myo. However, there was an unappreciable increase in ANS intensity with no shift in λmax, indicating the absence of dye binding in Lyz and CA. ANS is a hydrophobic dye that specifically binds to the exposed hydrophobic clusters and therefore considered to be a signature of the exposition of the hydrophobic groups to the solvent. Thus, our results indicate loosening of tertiary contacts in α-LA and Myo, accompanied by exposition of hydrophobic clusters to the solvent. No binding of ANS in the cases of Lyz and CA is expected since no significant changes were observed in the tertiary structural level in both proteins.

Glycation Induces the Formation of Oligomers of Different Morphologies

The maximum increase in hydrodynamic diameter in the monomeric state is followed by the formation of multimers with different sizes, as can be seen in Table 3.

We have further examined the aggregates formed after prolonged incubation. TEM images (Figure 4) revealed that the glycated proteins exist as clusters of large spherical structures (in Myo), while α-LA and CA aggregates exist as a mixture of fibrillar and spherical structures. The TEM image of glycated Lyz shows the presence of amorphous fibrillar structures. The results indicate different influences of the glycation event on the aggregation profile of different proteins.

Figure 4.

Figure 4

Open in a new tab

Transmission electron micrographs of glycated proteins. TEM images of glycated α-LA, Myo, Lyz, and CA incubated with 5 mM Glyoxal.

Glycation Exhibits Different Magnitudes of Cytotoxicity

Figure 5 shows the level of toxicity of different protein oligomers induced by glycation on HeLa cells.

Figure 5.

Figure 5

Open in a new tab

MTT reduction assay. Effect of glyoxal-induced protein oligomers on viability of HeLa cells.

Cells were treated for 24 h with modified protein oligomers (obtained after incubation with glyoxal for 24 h). All oligomers were found to show cytotoxicity, with Myo oligomers exhibiting the highest level of cytotoxicity. The results indicate that oligomers of different proteins have different morphologies and their effects on HeLa cells viability are variable.

Discussion

Our results on DLS measurements provide at least two important observations; (i) glycation induces certain structural changes in all proteins (except lysozyme) as evident from the increase in hydrodynamic diameter, followed by appearance of high-order oligomers, (ii) susceptibility to structural change is different for all proteins as the time frame for appearance of oligomers or the maximum change in the hydrodynamic diameter are different. Importantly, the structural susceptibility of the proteins toward the glycating agent does not depend strictly on the number of lysine contents or the extent of glycation. For instance, α-LA (with 12 lysine residues) and Lyz (with 6 lysine residues) are the most and least sensitive to structural change, respectively. Additionally, CA and Myo (with the maximum lysine residues, 18) exhibit less structural susceptibility toward glycation relative to α-LA. Alternatively, proteins with similar lysine contents (CA and Myo) showed a 3-fold difference in the extent of glycation (Table 2). The results led us to believe that certain lysine residues may stay protected from glycation (inaccessible) or their reactivity toward the glycating agent is very poor (perhaps because of large alteration in pKa of lysine due to the surrounding charge environment).

We were further interested to discern the conformational alterations due to the increase in hydrodynamic diameter. It is evident from our conformational measurements that there is no significant change in secondary structural elements due to glycation. Similarly, there is no change in tertiary interactions in the case of CA, but the tertiary interactions were different for the other proteins. α-LA and Myo exhibit destabilization or opening of tertiary contacts, while Lyz shows a slight increase in tertiary contacts. The opening of tertiary contacts (in the case of α-LA and Myo) might have influenced the packing pattern of some of the hydrophobic groups making them exposed to the solvent. The ANS binding assay indicates that some of the hydrophobic groups are exposed to the solvent in both α-LA and Myo, whereas there is no significant change in the packing pattern of hydrophobic groups in the case of Lyz and CA. Thus, it is clear that changes in the tertiary level conformational changes are not enough to bring about significant change in secondary structures of the proteins. Taken together, the results indicate that glycation induces conversion of the native proteins into conformationally altered species.

Structural transitions from native to a conformationally altered state might have affected the enzyme’s active sites and hence disrupted their catalytic efficiency. As evident in Figure 2, there is a decrease in percent activity in the case of Lyz and CA and an increase in peroxidase activity in Myo. It may be noted that all the three enzymes do not contain lysine residues as functional groups in their catalytic site. The results indicate that the observed alterations in enzyme activity are due to structural alterations brought about by covalent modification (not by covalent modification of residues in the active site) by glyoxal. Perhaps the structural alterations are sufficient to perturb the catalytic site of the enzymes, thus impairing their functional efficiency. It may be noted that such alterations in enzyme functions would bring changes in cellular machinery. For instance, CA is present in serum, and our results indicate that it is prone to glycation. A decrease in CA activity might lead to imbalance in pH homeostasis and might result in acidosis.23,24 Increased peroxidase activity (due to glycation) could also bring about unwanted gain of functions, thereby affecting the cellular H2O2 pool and hence overall cell signaling.25 Previously, our laboratory reported that early structural alteration in cytochrome c by the glycating agent converts into its apoptotically competent conformation.26 Taken together, we conclude that early structural changes brought about by glyoxal impair functional activity of various proteins and enzymes and therefore contribute to pathophysiology of hyperglycemia and associated diseases. Our results add novel insight that early structural changes (due to glycation) associated with important driver proteins linked with each disease might play an important role in pathophysiology. Specifically, AGEs are known to interfere with cellular functions via multiple processes including impairment of normal cell signaling, damaging the functions of various extracellular receptors, and causing oxidative stress among others. The present results indicate that the early structural changes in the cell signaling proteins or extracellular receptors might be the major consequence for the impaired functions.

Glycation is known to induce the formation of cross-links or high-order oligomers, and different oligomers appear to exhibit different magnitudes of cytotoxicity.11,27,28 DLS studies revealed that there are formations of very large oligomers for all the proteins. Lyz, even though it does not undergo significant structural transitions upon glycation, exhibited multimer formation. TEM images further revealed that Myo forms spherical aggregates, while α-LA and CA form a mixture of spherical and fibrillar aggregates. Interestingly, Lyz appears to form fibrillar species. Since all proteins have different conformations due to the modification, the variations in the nature of oligomers indicate that structural changes might be responsible for the different types of oligomers formed. Table 4 summarizes the consequences of all proteins studied to date for their oligomeric nature and cytotoxicities. There are several important observations arising out of this table. They are (i) morphologies of the oligomers are highly variable (amyloid, native-like oligomers, molten globule-like oligomers, amorphous, globular, etc.) depending on the protein and/or glycating agent used on a case-by-case basis.

Table 4. Consequences of Glycation on Various Proteins and Their Cytotoxicity.

protein glycating agent aggregate morphology cytotoxicity cell lines used refs
Aβ-peptide glucose amyloid aggregation (accelerated) non-cytotoxic neuroblastoma cells (29)(30),
β2-microglobulin d-ribose granular morphology to amyloid aggregation cytotoxic human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells (31)(32),
  d-glucose amyloid fibril formation (inhibition)      
insulin methylglyoxal native-like aggregate not determined   (33)(34),
  glucose (reducing conditions) amyloid formation (accelerate)      
  glucose (non-reducing conditions) amyloid formation (inhibition)      
cytochrome c methylglyoxal native-like aggregates non-cytotoxic   (35)
α-synuclein methylglyoxal amyloid fibrils (inhibition) non-cytotoxic HeLa and SH-SY5Y cells (36)
  d-ribose molten globule-like aggregates high cytotoxicity SH-SY5Y cells (37)
Lyz d-glucose, d-fructose, d-ribose (most effective) cross-linked β-sheet-rich, amorphous and globular oligomers not determined   (39)
albumin d-ribose β-rich aggregates evolve to amyloid fibrils highly cytotoxic neurotypic SH-SY5Y and MCF-7 cells (40−)
W7FW14F apomyoglobin d-ribose amyloid fibrils non-cytotoxic NIH-3T3 cells (44)(45),
α-LA glyoxal mixture of globular and fibrillar aggregates mildly cytotoxic HeLa cells this study
Lyz glyoxal amyloid fibrils mildly cytotoxic HeLa cells this study
CA glyoxal globular aggregates mildly cytotoxic HeLa cells this study
Myo glyoxal globular aggregates cytotoxic HeLa cells this study
Open in a new tab

The results indicate that there may be several variations on the late conformational changes induced by covalent modification depending on the glycating agent and proteins, leading to different oligomerization/aggregation pathways. (ii) Not all oligomers are cytotoxic (at least on the cell lines tested), indicating that some of the oligomers might not be a crucial factor toward pathophysiology of the glycation event. (iii) It is also evident that some of the agents enhance aggregation/amyloid fibril formation, while some agents exhibit inhibitory effects, suggesting that some of the modifications might be beneficial. Thus, all modified intracellular and extracellular proteins will ultimately end up in the formation of oligomers that may exhibit cytotoxic effects. Taken together, for a given modified protein, in addition to the early functional loss, the oligomers may again add up in the overall cytotoxicities under hyperglycemic conditions.

Methods

Materials

Alpha lactalbumin (α-LA) from bovine milk, myoglobin (Myo) from equine skeleton muscles, lysozyme (Lyz) from chicken egg white, carbonic anhydrase (CA) from bovine erythrocyte, glyoxal, 2,4-dinitrophenylhydrazine and trichloroacetic acid, the Micrococcus lysodeikticus cell wall, p-nitrophenylacetate, guaiacol, and 1-anilinonaphthalene-8-sulfonate (ANS) were purchased from Sigma-Aldrich.

Preparation of Protein Stocks and Determination of Concentrations

Protein stock solutions were dialyzed extensively against 0.1 M KCl solution at 4 °C and filtered using a Millipore syringe filter (0.22 μm). Concentrations of protein solutions were determined using the molar absorption coefficient, ε (M–1 cm–1), value of 29,210 at 280 nm for α-LA,14 3.9 × 104 at 280 nm for Lyz,15 57,000 at 280 nm for CA,16 and 0.188 at 408 nm for Myo.17 All solutions for optical measurements were prepared in degassed 0.05 M phosphate buffer, pH 7.0, containing 0.1 M KCl.

Protein Modification

Proteins were incubated with varying concentrations of glyoxal (0–5 mM) in phosphate buffer, pH 7.0 at 37 °C. These glyoxal-treated and untreated samples were further used for subsequent analysis.

Determination of Protein Carbonyl Content

Total protein carbonyl content was determined following the method described by Levine et al.(18) Briefly, aliquots of overnight glyoxal-treated proteins were incubated for 1 h at room temperature with 2,4 di-nitrophenyl hydrazine (DNPH) (0.1% w/v in 2 N HCL). DNPH reacts with the aldehydes/ketones generated on adducted proteins to form the colored hydrazone product (via nucleophilic reduction reaction). The reaction was stopped by addition of equal volumes of 20% trichloroacetic acid and centrifuged to obtain a pellet. DNPH was removed by extracting the pellets two times using 1 mL of ethyl acetate/ethanol (1:1 v/v) solution. Pellets were dried and dissolved in 6.0 M GdmCl (pH 7.0). Solubilized hydrazones were measured at 370 nm, and the concentration of DNPH-derivatized proteins was determined using a molar extinction coefficient of 22,000 M–1 cm–1.

DLS Measurements

DLS measurements were carried out in a Malvern Zetasizer MicroV to obtain the hydrodynamic diameter of unmodified and modified proteins at 37 ± 0.1 °C. Protein samples were filtered through a 0.22 μm filter. Measurements were made at a fixed angle of 90° using an incident laser beam of 689 nm. A total of 15 measurements were made for each sample with an acquisition time of 30 s at a sensitivity of 10%. Data were analyzed using Zetasizer software to get the hydrodynamic diameter, which is a measure of standard deviation of the size of the particle. The protein concentration used was 2 mg/mL.

Enzyme Activity Assay

Activity of modified proteins was analyzed using the M. lysodeikticus cell wall (0.14 mg/mL) as a substrate for Lyz (0.015 mg/mL), p-nitrophenylacetate (0.5 mM) for CA (0.03 mg/mL), and guaiacol (1 mM) for Myo (1 μM). Glyoxal treatments were carried out for 7 h in the case of Myo, 8 h for CA, and 24 h for Lyz. Lyz activity was followed by measuring the decrease in absorbance due to lysis of the bacterial cell wall at 450 nm for 1 h in a Jasco V-660 UV/Vis spectrophotometer with constant stirring.19 Activity of CA was assayed by monitoring hydrolysis of p-nitrophenylacetate at 400 nm for 1 h.20 Peroxidase activity of Myo was analyzed by measuring absorption at 470 nm for tetraguaiacol formed as a product.21

UV–Visible Spectrophotometry

Absorption spectra of the unmodified/modified Myo were recorded with a Jasco V-660 spectrophotometer equipped with a Peltier-type temperature controller at 37 °C. The protein concentration used was 5 μM for Soret band measurements. Cells of a 1.0 cm path length were used for all measurements.

Structural Measurements

Far- and near-UV CD spectra of glycated and non-glycated proteins were recorded in a Jasco J-810 Spectropolarimeter equipped with a Peltier-type temperature controller (Jasco PTC-424S). The concentration of proteins used was 0.5 mg/mL. The path lengths for far- and near-UV CD measurements were 1.0 and 10 mm, respectively. ANS was used to assess surface hydrophobicity of proteins upon treatment with glyoxal. The protein concentration used was 5 μM, and ANS was kept 16-fold that of the protein concentration for the assay. ANS was excited at 360 nm, and emission was obtained from 400 to 600 nm. Each spectrum was corrected for contribution of its blank in the entire wavelength range.

TEM Imaging

Pelleted samples were placed on copper grids and air-dried. Negative staining was done with 1.0% uranyl acetate, and samples were allowed to air-dry. The samples were examined under an FEI Tecnai G2-200 kV HRTA transmission electron microscope operating at 200 kV.

Cell Viability Assay

Treatment of cell lines with glyoxal-induced protein oligomers was carried out in a 96-well plate. Viability of HeLa cells was measured using the 3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide (MTT) reduction assay. MTT reduction was quantified spectrophotometrically using an ELISA reader.

Acknowledgments

This work was supported partly by grants from SERB, Department of Science and Technology [PDF/2015/001090], provided to M.W., SRF-ICMR [45/06/2019-BIO/BMS] provided to R.B., and UGC-SAP and DST-PURSE grant provided to L.R.S. The authors thank the All India Institute of Medical Science (AIIMS), New Delhi, for the TEM facility. The authors are grateful to the Deanship of Scientific research, King Saud University, for funding through the Vice Deanship of Scientific research Chairs.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c02300.

  • Size distribution by volume graph of DLS data; time-dependent plot of hydrodynamic diameter; time-dependent kinetics plot; visible absorption spectra; secondary structural components analysis; size distribution by volume of α-LA, Myo, Lyz, and CA upon treatment with 5 mM glyoxal obtained at different time frames; time-dependent plot of hydrodynamic diameter of α-LA, Myo, Lyz, and CA upon treatment with 5 mM glyoxal; activity assays of native and glycated enzymes; Time-dependent kinetics for hydrolysis of substrates by Myo, Lyz, and CA; heme absorbance measurement of Myo upon glycation; visible absorption spectra of unmodified and modified Myo at 408 nm; and secondary structural components of native and glycated proteins (PDF)

Author Contributions

# G.S.S. and R.B. contributed equally to this manuscript. G.S.S., R.B., M.W., S.K., and K.K. performed the experiments. G.S.S., M.W., and L.R.S. analyzed the data. R.B., S.K., and L.R.S. wrote the manuscript. S.Y.A., A.F.A., and H.R. reviewed and commented on the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao1c02300_si_001.pdf (460.6KB, pdf)

References

  1. Ahmed N. Advanced glycation endproducts-role in pathology of diabetic complications. Diabetes Res. Clin. Pract. 2005, 67, 3–21. 10.1016/j.diabres.2004.09.004. [DOI] [PubMed] [Google Scholar]
  2. Brownlee M.; Cerami A. Nonenzymatic glycosylation and the pathogenesis of diabetic complications. Ann. Intern. Med. 1984, 101, 527–537. 10.7326/0003-4819-101-4-527. [DOI] [PubMed] [Google Scholar]
  3. Younus H.; Anwar S. Prevention of non-enzymatic glycosylation (glycation): Implication in the treatment of diabetic complication. Int. J. Health Sci. 2016, 10, 247–263. 10.12816/0048818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Thornalley P. J.; Langborg A.; Minhas H. S. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem. J. 1999, 344, 109–116. 10.1042/0264-6021:3440109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Rahbar S.; Blumenfeld O.; Ranney H. M. Studies of an unusual hemoglobin in patients with diabetes mellitus. Biochem. Biophys. Res. Commun. 1969, 36, 838–843. 10.1016/0006-291x(69)90685-8. [DOI] [PubMed] [Google Scholar]
  6. Soros A. A.; Chalew S. A.; McCarter R. J.; Shepard R.; Hempe J. M. Hemoglobin glycation index: a robust measure of hemoglobin A1c bias in pediatric type 1 diabetes patients. Pediatr. Diabetes 2010, 11, 455–461. 10.1111/j.1399-5448.2009.00630.x. [DOI] [PubMed] [Google Scholar]
  7. Singh R.; Barden A.; Mori T.; Beilin L. Advanced glycation end-products: a review. Diabetologia 2001, 44, 129–146. 10.1007/s001250051591. [DOI] [PubMed] [Google Scholar]
  8. Schmitt A.; Schmitt J.; Münch G.; Gasic-Milencovic J. Characterization of advanced glycation end products for biochemical studies: side chain modifications and fluorescence characteristics. Anal. Biochem. 2005, 338, 201–215. 10.1016/j.ab.2004.12.003. [DOI] [PubMed] [Google Scholar]
  9. Ghosh S.; Pandey N. K. Prolonged glycation of hen egg white lysozyme generates non amyloidal structures. PloS One 2013, 8, e74336 10.1371/journal.pone.0074336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bouma B.; Kroon-Batenburg L. M. J.; Wu Y.-P.; Brünjes B.; Posthuma G.; Kranenburg O.; de Groot P. G.; Voest E. E.; Gebbink M. F. B. G. Glycation induces formation of amyloid cross-β structure in albumin. J. Biol. Chem. 2003, 278, 41810–41819. 10.1074/jbc.m303925200. [DOI] [PubMed] [Google Scholar]
  11. Wei Y.; Chen L.; Lin G.; Qiao R. Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells. BMC Mol. Cell Biol. 2009, 10, 10. 10.1186/1471-2121-10-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fazili N. A.; Naeem A. In vitro hyperglycemic condition facilitated the aggregation of lysozyme via the passage through a molten globule state. Cell Biochem. Biophys. 2013, 66, 265–275. 10.1007/s12013-012-9479-2. [DOI] [PubMed] [Google Scholar]
  13. Kondo T.; Murakami K.; Ohtsuka Y.; Tsuji M.; Gasa S.; Taniguchi N.; Kawakami Y. Estimation and characterization of glycosylated carbonic anhydrase I in erythrocytes from patients with diabetes mellitus. Clin. Chim. Acta 1987, 166, 227–236. 10.1016/0009-8981(87)90425-6. [DOI] [PubMed] [Google Scholar]
  14. Sugai S.; Yashiro H.; Nitta K. Equilibrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride. Biochim. Biophys. Acta 1973, 328, 35–41. 10.1016/0005-2795(73)90327-9. [DOI] [PubMed] [Google Scholar]
  15. Hamaguchi K.; Kurono A. Structure of Muramidase (Lysozyme). I. The Effect of Guanidine Hydrochloride on Muramidase. J. Biochem. 1963, 54, 111–122. [PubMed] [Google Scholar]
  16. Yazdanparast R.; Khodarahmi R.; Soori E. Comparative studies of the artificial chaperone-assisted refolding of thermally denatured bovine carbonic anhydrase using different capturing ionic detergents and beta-cyclodextrin. Arch. Biochem. Biophys. 2005, 437, 178–185. 10.1016/j.abb.2005.03.003. [DOI] [PubMed] [Google Scholar]
  17. Antonini E.; Brunori M.. Hemoglobin and myoglobin in their reactions with ligands; North-Holland Pub. Co.: Amsterdam, 1971. Series: Frontiers of biology, v. 21. [Google Scholar]
  18. Levine R. L.; Garland D.; Oliver C. N.; Amici A.; Climent I.; Lenz A.-G.; Ahn B.-W.; Shaltiel S.; Stadtman E. R. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990, 186, 464–478. 10.1016/0076-6879(90)86141-h. [DOI] [PubMed] [Google Scholar]
  19. Maurel P.; Douzou P. Catalytic implications of electrostatic potential: The lytic activity of lysozyme as a model. J. Mol. Biol. 1976, 102, 253–264. 10.1016/s0022-2836(76)80052-6. [DOI] [PubMed] [Google Scholar]
  20. Wells J. W.; Kandel M.; Gornall A. G. The esterase activity of Bovine carbonic anhydrase B above pH 9. J. Biol. Chem. 1975, 250, 3522–3530. 10.1016/s0021-9258(19)41546-9. [DOI] [PubMed] [Google Scholar]
  21. Hayashi T.; Hitomi Y.; Ando T.; Mizutani T.; Hisaeda Y.; Kitagawa S.; Ogoshi H. Peroxidase activity of Myoglobin is enhanced by chemical mutation of heme-propionates. J. Am. Chem. Soc. 1999, 121, 7747–7750. 10.1021/ja9841005. [DOI] [Google Scholar]
  22. Wan L.; et al. In vitro evolution of horse heart myoglobin to increase peroxidase activity. Proc. Natl. Acad. Sci. 1998, 95, 12825–12831. 10.1073/pnas.95.22.12825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sly W. S.; Hewett-Emmett D.; Whyte M. P.; Yu Y. S.; Tashian R. E. Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc. Natl. Acad. Sci. 1983, 80, 2752–2756. 10.1073/pnas.80.9.2752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Diez-Fernandez C.; et al. Defective hepatic bicarbonate production due to carbonic anhydrase VA deficiency leads to early-onset life threatening metabolic crisis. Genet. Med. 2016, 18, 991–1000. 10.1038/gim.2015.201. [DOI] [PubMed] [Google Scholar]
  25. Rezaeinezhad A.; Pegah E.; Hossein M.; Hamid G. The effect of cold atmospheric plasma on diabetes-induced enzyme glycation, oxidative stress, and inflammation; in vitro and in vivo. Sci. Rep. 2019, 9, 19958. 10.1038/s41598-019-56459-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sharma G. S.; Warepam M.; Bhattacharya R.; Singh L. R. Covalent modification by glyoxals converts cytochrome c into its apoptotically competent state. Sci. Rep. 2019, 9, 4781. 10.1038/s41598-019-41282-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sajithlal G. B.; Chithra P.; Chandrakasan G. Advanced glycation end products induce crosslinking of collagen in vitro. Biochim. Biophys. Acta (BBA) - Mol. Basis Dis. 1998, 1407, 215–224. 10.1016/s0925-4439(98)00043-x. [DOI] [PubMed] [Google Scholar]
  28. Iannuzzi C.; Carafa V.; Altucci L.; Irace G.; Borriello M.; Vinciguerra R.; Sirangelo I. Glycation of Wild-Type apomyoglobin induces formation of highly cytotoxic oligomeric species. J. Cell. Physiol. 2015, 230, 2807–2820. 10.1002/jcp.25011. [DOI] [PubMed] [Google Scholar]
  29. Vitek M. P.; Bhattacharya K.; Glendening J. M.; Stopa E.; Vlassara H.; Bucala R.; Manogue K.; Cerami A. Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 4766–4770. 10.1073/pnas.91.11.4766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Fernandez-Busquets X.; Ponce J.; Bravo R.; Arimon M.; Martianez T.; Gella A.; et al. Modulation of amylois β peptide 1-42 cytotoxicity and aggregation in vitro by glucose and chondroitin sulfate. Curr. Alzheimer Res. 2010, 7, 428–438. 10.2174/156720510791383787. [DOI] [PubMed] [Google Scholar]
  31. Hashimoto N.; Naiki H.; Gejyo F. Modification of beta 2-microglobulin with D-glucose or 3-deoxyglucosone inhibits A beta 2M amyloid fibril extension in vitro. Amyloid 1999, 6, 256–264. 10.3109/13506129909007337. [DOI] [PubMed] [Google Scholar]
  32. Kong F.-L.; Cheng W.; Chen J.; Liang Y. D-Ribose glycates b2-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway. Chem. Biol. Interact. 2011, 194, 69–78. 10.1016/j.cbi.2011.08.003. [DOI] [PubMed] [Google Scholar]
  33. Oliveira L. M.; Lages A.; Gomes R. A.; Neves H.; Família C.; Coelho A. V.; Quintas A. Insulin glycation by methylglyoxal results in native-like aggregation and inhibition of fibril formation. BMC Biochem. 2011, 12, 41. 10.1186/1471-2091-12-41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Alavi P.; Yousefi R.; Amirghofran S.; Karbalaei-Heidari H. R.; Moosavi-Movahedi A. A. Structural analysis and aggregation propensity of reduced and nonreduced glycated insulin adducts. Appl. Biochem. Biotechnol. 2013, 170, 623–638. 10.1007/s12010-013-0207-1. [DOI] [PubMed] [Google Scholar]
  35. Oliveira L. M. A.; Gomes R. A.; Yang D.; Dennison S. R.; Família C.; Lages A.; Coelho A. V.; Murphy R. M.; Phoenix D. A.; Quintas A. Insights into the molecular mechanism of protein native-like aggregation upon glycation. Biochim. Biophys. Acta 2013, 1834, 1010–1022. 10.1016/j.bbapap.2012.12.001. [DOI] [PubMed] [Google Scholar]
  36. Lee D.; Park C. W.; Paik S. R.; Choi K. Y. The modification of alpha-synuclein by dicarbonyl compounds inhibits its fibril-forming process. Biochim. Biophys. Acta 2009, 1794, 421–430. 10.1016/j.bbapap.2008.11.016. [DOI] [PubMed] [Google Scholar]
  37. Chen L.; Wei Y.; Wang X.; He R. Ribosylation rapidly induces alpha-synuclein to form highly cytotoxic molten globules of advanced glycation end products. PLoS One 2010, 5, e9052 10.1371/journal.pone.0009052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ghosh S.; Pandey N. K.; Singha Roy A.; Tripathy D. R.; Dinda A. K.; Dasgupta S. Prolonged glycation of hen egg white lysozyme generates non amyloidal structures. PLoS One 2013, 8, e74336 10.1371/journal.pone.0074336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sattarahmady N.; Moosavimovahedi A.; Ahmad F.; Hakimelahi G.; Habibirezaei M.; Saboury A.; Sheibani N. Formation of the molten globule-like state during prolonged glycation of human serum albumin. Biochim. Biophys. Acta 2007, 1770, 933–942. 10.1016/j.bbagen.2007.02.001. [DOI] [PubMed] [Google Scholar]
  40. Wei Y.; Chen L.; Chen J.; Ge L.; He R. Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells. BMC Cell Biol. 2009, 10, 10. 10.1186/1471-2121-10-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Khan M. S.; Dwivedi S.; Priyadarshini M.; Tabrez S.; Siddiqui M. A.; Jagirdar H.; Al-Senaidy A. M.; Al-Khedhairy A. A.; Musarrat J. Ribosylation of bovine serum albumin induces ROS accumulation and cell death in cancer line (MCF-7). Eur. Biophys. J. 2013, 42, 811–818. 10.1007/s00249-013-0929-6. [DOI] [PubMed] [Google Scholar]
  42. Iannuzzi C.; Maritato R.; Irace G.; Sirangelo I. Misfolding and amyloid aggregation of apomyoglobin. Int. J. Mol. Sci. 2013, 14, 14287–14300. 10.3390/ijms140714287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Iannuzzi C.; Maritato R.; Irace G.; Sirangelo I. Glycation accelerates fibrillization of the amyloidogenic W7FW14F apomyoglobin. PLoS One 2013, 8, e80768 10.1371/journal.pone.0080768. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao1c02300_si_001.pdf (460.6KB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES