Opinion - (2023) Volume 14, Issue 4
Aims: Both in vivo and in vitro non-enzymatic glycation of DNA produces free radicals, or glycoxidation. Neo-antigenic epitopes, which play a role in autoimmune diseases like diabetes mellitus, are produced as a result of DNA structural alterations caused by glycolysis. In this study, autoantibody binding to human placental DNA was probed in Type 1 diabetes patients after it was glycated with methylglyoxal (MG) and lysine (Lys) in the presence of Cu2+.
Methods: DNA was lysed by incubating it for 24 hours at 37 °C with MG, Lys, and Cu2+. LC-MS and ESI-MS techniques were used to investigate the pathway for Amadori formation and the carboxyethyl deoxyguanosine (CEdG) formation during the glycation reaction. In addition, a direct binding, competitive ELISA, and band shift assay were used to evaluate the autoantibodies' binding properties in diabetics.
Results: CEdG, a marker of DNA glycation, is produced when DNA is glycated with MG, Lys, and Cu2+, as demonstrated by LC-MS. The ESI-MS method confirmed the intermediate glycation stages. When compared to the native form of DNA, serum from diabetic patients exhibited enhanced binding and specificity for glycated DNA.
Conclusions: DNA glycation has altered the structure, resulting in the generation of neo-antigenic epitopes that recognize diabetes-associated autoantibodies.
DNA; MethylGlyoxal; Glycation; Auto-antibody; Diabetes mellitus
Diabetes mellitus is a typical endocrine problem portrayed by hyperglycemia because of the lack of insulin or insulin obstruction. By increasing glycation intermediates and the gradual accumulation of advanced glycation endproducts (AGEs) in body tissues, hyperglycemia contributes significantly to the pathogenesis of diabetes complications. In diabetes, the biomolecular damage caused by glycoxidation and AGEs is accompanied by an increase in the activity of free radicals [1]. The receptors for AGEs (RAGE) that can be found on a variety of cell types-particularly those that are affected by diabetesare the subject of much research interest. Recent research suggests that the pathogenesis of secondary complications is influenced by the interaction of AGEs with RAGE, which alters gene expression, intracellular signalling [2], and the release of pro-inflammatory molecules and free radicals. Long-term complications are often caused by hyperglycemia, and diabetics with poor blood glucose control are especially vulnerable. In addition, complications appear to affect organs, such as the nervous system, heart, kidneys, and small blood vessels, where cells do not require insulin for glucose uptake. Consequently, during hyperglycemia, these cells have high intracellular glucose concentrations. It is still unclear what exact role hyperglycemia plays in the development of long-term complications. However, it has been extensively reported that the level of methylglyoxal (MG) in diabetic patients increases 5- 6 fold under hyperglycemic conditions [3]. Additionally, the concentration of MG in human lenses typically exceeds that of plasma by about 20 times. In mononuclear cells, physiological levels of MG have been shown to decrease cellular adhesion and induce DNA cleavage and ROS production. As a result, the glycation reaction's initiation and progression are directly influenced by MG.
The nonenzymatic addition of reducing sugars and sugar-related compounds like ascorbic acid, MG, glyoxal, and 3-deoxyglucosone, among others, is known as glycation. into macromolecules in biology like DNA. The Maillard reaction is a series of chemical reactions in which the free carbonyl groups of the sugar and related moieties interact with the free amino residues of the macromolecules [4]. The formation of acid-labile Schiff base adducts that undergo Amadori rearrangements into more stable products initiates glycation. The irreversible AGEs are produced by slow transformation of the early glycation products. Due to their connection to free radicals, which are involved in the development of cancer, diabetes, heart disease, cataract, atherosclerosis, and neurodegenerative disorders, these reactions have recently received a lot of attention. The addition of sugars to DNA alters its structure and function, resulting in harmful modifications and mutations, as demonstrated by previous biochemical and molecular biology studies [5].
As previously mentioned, commercially available human placental DNA was glycated in this study. A lot of research has been done on the structural changes that MG and Lys in the presence of Cu2+ cause in the DNA macromolecule. The adduct shaped by MG-Lys-Cu2+ framework to human DNA was examined by LC-MS strategy. In addition, the ESI-MS method is used to observe the DNA glycation intermediates that are formed. In addition, the band shift assay, direct binding, and competitive inhibition ELISA utilized the glycated DNA as an antigen to detect anti-DNA antibodies in the sera of individuals with type 1 diabetes [6].
Materials
Tween 20, Protein A-agarose (2.5 ml pre-pack column), p-nitrophenyl phosphate, sodium dodecyl sulfate, methylglyoxal (MG), dialysis tubing, and anti-human IgG alkaline phosphatase conjugates were all purchased from Sigma Chemical Company in the United States. Dihydroxy acetone (DHA) was purchased from Merck in Germany. Lysine was a product of the Sisco laboratory. Hi–Media supplied the Triton X-100. The base for Trizma came from Spectrochem in Mumbai, India. 96-well ELISA plates were obtained from NUNC, Denmark. Bio-Rad Laboratories, in the United States, supplied acrylamide, bisacrylamide, ammonium persulfate, and N,N,N′,N′- tetramethylethylenediamine (TEMED). Qualigens, in India, supplied EDTA (disodium salt), silver nitrate, sodium carbonate, and sodium nitrite. The highest analytical grade was used for all of the other chemicals and reagents. After receiving informed consent, blood samples from type 1 diabetes patients who were fasting were collected from J.N. Medical College Hospital, A.M.U., Aligarh. None of the diabetics had any other autoimmune conditions. Specifically, lupus and other connective tissue disorders were not present. Healthy individuals provided normal human sera. In a glass test tube, the samples were collected and allowed to clot for 30 minutes at 37 °C. Ten minutes of centrifugation at 3000 rpm separated the serum. After being heated for 30 minutes at 56 °C to inactivate complement proteins, serum samples were stored in aliquots at 20 °C with 1% sodium azide as a preservative.
Purification of human placental DNA
As previously mentioned, commercially available human placental DNA was purified free of proteins and single-stranded regions.
According to Ahmad et al.'s findings, methylglyoxal (MG) and lysine were able to modify human placental DNA in the presence and absence of Cu2+. 2011). In 10 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl, 37.8 M of human DNA was thoroughly mixed with MG (40 mM), lysine (40 mM), and Cu2+ (300 M). After incubating for 24 hours at 37 °C, extensive dialysis against PBS was performed to remove any unbound components.
Combination of the norm, carboxyethyl deoxyguanosine (CEdG)
The combination of CEdG was done as portrayed by Ashraf et al. (2012), with minor adjustments. In brief, a shaking water bath at 70 °C was used to incubate 50 mg of deoxy-guanosine suspended in 1 ml of 100 mM phosphate buffer (pH 7.4) with 100 mg of dihydroxyacetone. CEdG was isolated by preparative HPLC using 50 mM ammonium acetate buffer solution and methanol as eluents. It was dissolved at 70 °C during the reaction after 24 hours.
An Agilent 1100 capillary HPLC System with a Synergi C18 analytical column was used for the HPLC analysis of both the native and modified analogs of human placental DNA. This allowed for the LC-MS detection of a glycated product known as CEdG in the modified form of the DNA. The following were the general chromatographic conditions: C18 segment (2 mm × 150 mm with 4 μm molecule size); eluant A, a pH 7 solution of 5 mM aqueous ammonium acetate buffer; In the first five minutes of eluant B, a gradient solution containing acetonitrile (CH3N), the CH3N concentration increased from 0 to 4.0%; from 4.0 to 6.5 percent in 30 minutes; held at 6.5 percent for five minutes before being increased to 90 percent to wash any remaining material off the column at a constant flow rate of 500 L/min. The diode array detector (DAD) detected DNA bases at their maximum absorption wavelength of 254 nm. A Micromass Quattro Ultima Triple Quadrupole Mass Spectrometer interfaced with an Agilent 1100 capillary HPLC system was used for the LC-MS analysis of the CEdG standard.
Electrospray ionization mass spectrometry was used to characterize DNAAGEs. An orthogonal time of flight (TOF) mass spectrometer with a standard electrospray ionization source (Applied Biosystems Mariner Atmospheric Pressure Ionization TOF Workstation, Framingham, MA, USA) was used. Positive ion polarity was used to collect the mass spectral data. The nebulizer, heater, and collision gas were all made of nitrogen. For direct infusion onto the mass spectrometer, the instrument was outfitted with an integrated syringe pump and a dual syringe rack. The sciex heater was set to 350 °C and the spray tip potential was set to 4000 V. Full scan mode (m/z 100–1000) was used by the mass spectrometry system. Ten spectra were gathered by performing spectral acquisition every two seconds. An average of 4–6 scans are depicted in the final spectrum.
As previously mentioned, enzyme-linked immunosorbent assay (ELISA) was performed on flat-bottom polystyrene plates. In a nutshell, microtitre wells were coated with one microliter of 2.5 g/ml of DNA (in TBS, pH 7.4), incubated for two hours at 37 °C, and then kept at 4 °C for an overnight period. Each sample was coated twice, and the antigen-free half of the plate served as the control. To get rid of the unbound antigen, the test plate wells were emptied and washed three times with TBS-T. In direct binding ELISA, antibodies were directly added to antigen-coated wells and incubated for 2 h at 37 °C and overnight at 4 °C, respectively. Unoccupied sites were blocked with 150 l of 1.5% non-fat dry milk (in TBS, pH 7.4) for 4–5 h at 4 °C, followed by a single wash with TBS-T. After the wells were emptied and thoroughly washed with TBS-T, anti-immunoglobulin G alkaline phosphatase conjugate was added to each well, and the plates were incubated for two hours at 37 °C. The plates were then washed once with distilled water and three times with TBS-T. After adding para-nitrophenyl phosphate, a microplate reader read the developed color at 410 nm. The mean of the absorbance differences between the test and control wells (Atest Acontrol) were used to represent the findings.
Competition ELISA The competitive binding assay was used to determine the specific antibody binding properties. A constant amount of antiserum or IgG was mixed with varying amounts of inhibitors (0–20 g/ml), and the mixture was incubated for two hours at room temperature and overnight at 4 °C. Instead of the serum, the immune complex that had formed was coated in the wells. The leftover advances were equivalent to in coordinate restricting ELISA.
The following formula was used to calculate percent inhibition: Gel retardation assay The gel retardation assay provided additional evidence of the specificity of the antibody against the target antigen. Mixing varying amounts of IgG with a constant amount of DNA antigen (0.5 g) was done for two hours at 37 °C and overnight at 4 °C. After the incubation period had ended, the antigen–antibody complex was electrophoresed for two hours at 30 mA current on 1% agarose gel in TAE buffer (pH 7.8). The gels were photographed and visualized under ultraviolet light after being stained with ethidium bromide (0.5 mg/ml) [7].
Statistics show data as mean minus standard deviation. Student's t-test (Statgraphics, Origin 6.1) was used to determine the data's statistical significance. The study was deemed statistically significant when p 0.05.
Glycation of human DNA Human DNA (37.8 M) was glycated for 24 hours at 37 °C using 40 mM MG, 40 mM lysine, and 150 M Cu2+. The glycation reaction's structural perturbation of the DNA macromolecule's structure has been confirmed in the published literature.
N2-(1-carboxyethyl)-2-deoxyguanosine (CEdG) was synthesized and characterized in the same way that it was previously described. CEdG was isolated by preparative HPLC using 50 mM ammonium acetate buffer solution and methanol as eluents following final preparation. When a UV detector was used for the experiment, the elution of CEdG was obtained at a retention time of 14.399 minutes. Deoxyguanosine (dG), on the other hand, eluted with a retention time of 9.1 minutes.
LC-MS detection of N2-(1-carboxyethyl)-2-deoxyguanosine (CEdG) formed in modified human DNA The mass spectrometer determined that the CEdG synthesized from dG had a mass (m/z) that was 338 times greater than the 266 mass of dG. They demonstrated mass value matching with CEdG when analyzed in the same conditions as modified DNA. The MG-Lys-Cu2+ glycated DNA suggests CEdG formation, and modified human DNA has an m/z value of 338. 1b). However, analysis of native DNA indicates that CEdG formation is not present. 1c)
Electrospray ionization mass spectrometry (ESI-MS) characterization of DNA-AGEs The hydrolyzed glycated human DNA was analyzed with mass spectrometry in an effort to confirm the formation of the Schiff base and the Amadori product in glycated DNA. The condensation reaction of dG (Mr 285.26) with methylglyoxal (Mr 70.06) in a dehydration reaction that results in the loss of a water molecule produces an ion at m/z 341 that is consistent with a [Schiff base + H]+ molecule. The ion at m/z 679 suggests that a [Schiff base + H]+ dimer product was formed [8]. The ion with m/z 385 is thought to be a fragment formed by the degradation of MG reacting with the Schiff base product or its enaminol or Amadori intermediates, and the ion at 268 is thought to be the result of the loss of a hydroxyl group from dG.
Immunogenicity of modified DNA
Glycated DNA has been shown to be a potent immunogen that induces highly specific, non-precipitating, high titers (>1:12800) antibodies in laboratory animals.
Autoantibodies' adherence to native and MG-Lys-Cu2+-modified human DNA in diabetics The objective of the pilot study was to eliminate positive sera samples—sera that had a higher affinity for the immunogen—from diabetics with type 1 diabetes. After receiving informed consent, the sera were obtained from patients attending J.N. Medical College and Hospital, A.M.U., Aligarh [9]. 40 serum samples from patients with type 1 diabetes were used in our study. Twenty healthy, normal people provided control serum samples from individuals whose sexes and age were matched.
All sera were weakened to 1:100 in TBS-T and exposed to coordinate restricting ELISA on strong stage independently covered with equivalent measures of local and MG-Lys-Cu2+ changed human DNA [10]. Out of 40 sera from type 1 diabetes, 27 examples (67.5%) showed higher restricting with the glycated DNA when contrasted with the local structure. While samples whose absorbance was less than or equal to that of the control were excluded, those serum samples that demonstrated enhanced binding (double or more than double binding) were taken into consideration for future research.
Competition ELISA was used to examine the specific binding of circulating autoantibodies in the sera of Type 1 diabetes patients to native and MGLys- Cu2+ modified human DNA. Immuno-cross reactivity of autoantibodies from diabetes type 1 patients [11]. The maximum inhibition with MG-Lys- Cu2+ modified human DNA was 46.9–63.1% in the 27 sera chosen from Type 1 diabetes patients that showed enhanced binding, whereas with native human DNA, it was 20.2–33%. With native human DNA, the mean inhibition for the entire sample was 26.98 3.8%, while with MG-Lys-Cu2+ modified human DNA, it was 54.95 5.4%. However, MG-Lys-Cu2+ modified human DNA inhibited normal human subjects by a mean of 32.5 2.2 percent under similar experimental conditions, whereas native human DNA inhibited by 24.4 2.3 percent.
Purification of IgG from type 1 diabetes
Patients' Serum IgG was isolated using a protein A-agarose column from a few high-binding sera. On the affinity column, the purified IgG eluted as a symmetrical single peak. Under non-reducing conditions, a single homogenous band on SDS-PAGE confirmed IgG purity [12].
Glycation adducts of DNA may have potential as biomarkers since all nucleated cells contain the same DNA content and should reflect the relative level of MG in the target tissue. Reaction of double-stranded DNA with MG or glucose in vitro produces primarily N2-carboxyethyl- 2′-deoxyguanosine (CEdG), suggesting to be the likely major adduct formed in vivo. This implies that CEdG might be a useful biomarker for monitoring oxoaldehyde-induced stress in response to enhanced glycolytic flux or environmental exposure to MG [13]. The preparative HPLC was employed for the synthesis of the standard, carboxyethyl deoxyguanosine (CEdG). The LC-MS was performed to detect the glycated adduct, CEdG formed with the double stranded human DNA. The acid hydrolyzate of MGLys- Cu2+ glycated human DNA showed an m/z value of 338 in the negative ion mode, which is in conformity with the m/z value for standard CEdG [14]. This finding is consistent with an earlier study from our group. Since CEDG has been reported as the major DNA adduct formed as a result of glycation, it could serve as an effective biomarker for the detection of glycation events taking place in our body. It has been reported that the reaction of deoxy-guanosine (dG) with MG proceeds via Amadori pathway. In our case the ESI-MS, mass-spectroscopic data has shown similar results, i.e., the reaction of human DNA with MG-Lys-Cu2+ proceeds via the classic Amadori pathway and yields glycation-like products similar to those generated between a nucleoside and a carbohydrate. This is in conformity with the results we obtained for ESI-MS [15]. The ion at m/z 341, 679, 268 and 385 is consistent with a [Schiff base + H]+, [Schiff base + H]+ dimer product, dG-H2O and a fragment formed by the degradation of MG reacting with the Schiff base product, or its enaminol or Amadori intermediate respectively.
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Citation: Musraf Uddin. The Autoimmune Response to AGE-Modified Human DNA and Its Implications for Type 1 Diabetic Mellitus. J Diabetes Metab, 2023, 14(4): 989.
Received: 27-Mar-2023, Manuscript No. dm-23-22193; Editor assigned: 30-Mar-2023, Pre QC No. dm-23-22193(PQ); Reviewed: 13-Apr-2023, QC No. dm-23-22193; Revised: 20-Apr-2023, Manuscript No. dm-23-22193(R); Published: 28-Apr-2023, DOI: 10.35248/2155-6156.1000989
Copyright: © 2023 Uddin M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
