International Journal of Biological Macromolecules 72 (2015) 875–882

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Preferential recognition of peroxynitrite-modified human serum albumin by circulating autoantibodies in cancer Parvez Ahmad a , Badar ul Islam a , Shaziya Allarakha a , Gulam Rabbani b , Kiran Dixit a , Moinuddin a , Rizwan Hasan Khan b , Shahid Ali Siddiqui c , Asif Ali a,∗ a

Department of Biochemistry, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India c Department of Radiotherapy, Faculty of Medicine, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India b

a r t i c l e

i n f o

Article history: Received 12 September 2014 Received in revised form 27 September 2014 Accepted 1 October 2014 Available online 13 October 2014 Keywords: Cancer Peroxynitrite Nitration Human serum albumin ELISA.

a b s t r a c t Peroxynitrite is a potent oxidizing and nitrating agent and has in vivo existence. Several studies have shown the damaging role of this molecule in biological system. Human serum albumin (HSA), being most abundant plasma protein, is easily targeted by different oxidizing and nitrating agents. Free radicals increase the onset of different cancers as evident by several researchers. In the present study, structural perturbations in HSA by peroxynitrite were observed by MALDI-MS, DSC and DLS. Immunological studies showed enhanced binding of peroxynitrite-modified HSA with cancer autoantibodies, compared to the native protein. A decline in the antioxidant property of peroxynitrite-modified HSA was also observed. Therefore, we may conclude that peroxynitrite exposure results in structural alteration and hence generation of neo-epitopes in HSA molecule along with the decrease in its antioxidant property. The possible role of peroxynitrite-modified HSA in carcinogenesis has been discussed. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cancer is ranked as the second cause of death, led only by heart disease [1]. Cancer appears to be a significant public health problem, with an estimated 1.19 million cases in 2011 and 1.87 million cases in 2026, in India [2]. There are different causes of cancer but free radicals have emerged as one of the most prominent reasons that can derive carcinogenesis by altering targets and pathways that are crucial to normal tissue homeostasis. Free radicals increase cancer risk through mutations in cancer-related genes; post-translational modifications in proteins, like oxidation, nitration, acetylation, phosphorylation; lipid peroxydation, etc. [3]. Sometimes the generation of reactive oxygen species (ROS)/reactive nitrogen species (RNS) exceeds the ability of antioxidant defense system and this imbalance leads to the oxidative/nitrosative stress causing damage to cellular constituents (DNA, proteins, lipids and sugars) [4,5]. Among ROS/RNS, peroxynitrite has got more attention from different research groups as it is a compelling oxidizing and nitrating agent. It is an extremely reactive molecule [6] which is produced irreversibly as a result of the reaction between nitric oxide (NO• ) and superoxide (O2 •− )

∗ Corresponding author. Tel.: +91 941 227 3580; fax: +91 571 272 0030. E-mail address: asifali [email protected] (A. Ali). http://dx.doi.org/10.1016/j.ijbiomac.2014.10.001 0141-8130/© 2014 Elsevier B.V. All rights reserved.

and has a physiological half-life of just about 1 s [7]. Irrespective of its short half-life at physiological pH, it is able to influence surrounding target cells within one to two cell diameters (5–20 ␮m) due to its ability to cross plasma membranes [8,9]. A high affinity reaction of peroxynitrite is with free and protein bound tyrosine residues resulting in the formation of 3-nitrotyrosine which is a covalent modification, results in attachment of a nitro (–NO2 ) group on the aromatic ring of tyrosine residues [10] and is considered as a specific marker of peroxynitrite-mediated damage to proteins [11]. The reaction between peroxynitrite and tyrosine is an irreversible process and can be detected as long as the nitrated protein is in circulation [12]. Researchers have seen the presence of 3-nitrotyrosine in vivo in a number of inflammatory conditions [13]. In addition to nitration, peroxynitrite also causes oxidation of proteins, resulting in increase in the carbonyl content of proteins which is the outcome of oxidative modifications of the side chains of lysine, proline, arginine and threonine [14]. Increase in protein carbonyl content has been observed in various human diseases including cancer [15]. Despite other clearly defined functions of HSA, like maintenance of colloidal osmotic pressure, binding and transport of important solutes, the protein has got a significant antioxidant activity and may stand for the key and leading circulating antioxidant in plasma. Due to its abundance in plasma, HSA is continuously exposed to oxidative stress, which can alter the conformation and function of

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this molecule, resulting in modification of its biological properties [16]. The presence of oxidized albumin has been investigated in various diseases [17]. It has been investigated that structural alteration in HSA molecule may result in impairment of its biological properties [18]. In the present study, structural changes in HSA molecule, after peroxynitrite treatment, was evaluated and the autoantibodies in cancer patients were analyzed for specific recognition of peroxynitrite-modified epitopes on HSA. We have discussed the role of peroxynitrite-modified HSA in the etiopathogenesis of cancer of different tissue origins. 2. Materials and methods Human serum albumin, Protein A-agarose affinity column, pnitrophenyl phosphate, anti-rabbit and anti-human IgG alkaline phosphatase were obtained from Sigma-Aldrich (St. Louis, MO). Flat bottom polysorp ELISA modules (NUNC, Denmark). Unless otherwise indicated, all other reagents and solvents were of high analytical grade and all solutions were prepared in deionized and distilled water.

2.4. Dynamic light scattering (DLS) measurements DLS measurements were carried out at 830 nm by using DynaPro-TC–04 dynamic light scattering equipment (Protein Solutions, Wyatt Technology, Santa Barbara, CA) equipped with a temperature-controlled microsampler. Before measurement, all the solutions were spun at 10,000 rpm for 10 min and filtered through a microfilter (Whatman International, Maidstone, UK) with an average pore size of 0.22 ␮m directly into a 12 ␮l black quartz cell and the protein concentration was 18 ␮M. Measured size was presented as the average value of 20 runs. All data were analyzed by using Dynamics 6.10.0.10 software at optimized resolution. The mean hydrodynamic radius (Rh ) and polydispersity were estimated on the basis of an autocorrelation analysis of scattered light intensity data based on translational diffusion coefficient (D) by the Stokes–Einstein equation: Rh =

kT 25 6Dw

◦C

where Rh is the hydrodynamic radius, k is the Boltzman’s constant, 25 ◦ C T is the absolute temperature,  is the viscosity of water and Dw ◦ is translational diffusion coefficient of water at 25 C.

2.1. Collection of serum samples Informed consents from patients as well as healthy subjects were obtained before taking serum samples. Sera of pre-treated cancer patients (n = 98) having cancer of different tissue origin (lung, n = 31; head and neck, n = 30; breast, n = 22; liver, n = 15) with proven histopathological diagnosis attending the medicine outdoor clinic or those admitted in the medicine ward of the Jawaharlal Nehru Medical College and Hospital (JNMCH), Aligarh Muslim University (AMU), Aligarh, India, were collected. Total numbers of male and female patients were 60 and 38, respectively, with a mean age of 40 ± 20 years. Sera from healthy individuals (n = 50), with a mean age of 35 ± 15 years, served as negative control. All the serum samples were heated at 56 ◦ C for 30 min to inactivate complement proteins and stored at −20 ◦ C with 0.2% sodium azide. The present research work was awarded institutional ethical clearance certificate.

2.5. Protein 3-nitrotyrosine determination by MALDI mass spectrometry MALDI measurements were performed on 4800 plus mass spectrometer (AB Sciex, MA, USA) operated in the positive linear mode. Ions formed by a pulsed UV laser beam were accelerated at 30 keV. The matrix was sinapinic acid, dissolved in acetonitrile/water (50:50, v/v) at a concentration of about 50 mM. Samples were diluted five times in 0.1% trifluoroacetic acid aqueous solution. Equal volumes of sample and matrix solutions were added, and approximately 1–2 ␮l of the mixture was deposited on the stainless steel multiprobe and allowed to dry before introduction into the mass spectrometer. Mass spectra were obtained and averaged over 10 shots; three independent MALDI measurements were made for each sample to evaluate reproducibility. 2.6. Free radical-induced hemolysis test

2.2. Peroxynitrite-modification of HSA Peroxynitrite was synthesized by rapid quenched flow process [19] and stored in 1.2 M NaOH at −20 ◦ C. Concentration of stored peroxynitrite was determined from absorbance at 302 nm using molar extinction coefficient of 1670 M−1 cm−1 . HSA, at a concentration of 5 ␮M in phosphate buffered saline (PBS), pH 7.4, was incubated with 500 ␮M peroxynitrite at 37 ◦ C for 3 h. After incubation, the solutions were extensively dialyzed against PBS to remove excess peroxynitrite. To maintain the same condition for the control, unmodified HSA was also dialyzed in the same manner. 2.3. Differential scanning calorimetry (DSC) For a thermodynamic evaluation of the structural properties of native and peroxynitrite-modified HSA, DSC was carried out on a VP-DSC microcalorimeter (MicroCal, Northampton, MA). The DSC scans were run between 20 and 90 ◦ C at a rate of 1.0 ◦ C/min. The experiments were performed using 18 ␮M HSA. The respective reference scan was run under identical DSC set up conditions and was subtracted from each sample scan. The heat capacity curves (CP ), thermal denaturation temperature (Tm ), calorimetric enthalpy (Hcal ) and van’t Hoff enthalpy (HvH ) were analyzed using Origin 7.0 scientific plotting software.

This test was performed to check the antioxidant properties of native as well as peroxynitrite-modified HSA and HSA isolated from healthy human serum as well as from lung cancer patient using the standard procedure with slight modification [20]. Pellets of erythrocytes were used to prepare 10% (v/v) erythrocyte suspension in phosphate buffer saline (pH 7.4). Oxidative hemolysis was started by adding 100 mM of peroxyl radical initiator AAPH [2,2 azobis (2-amidinopropane) dihydrochloride] in 500 ␮l of RBC suspension mixed with 0.5 mg/ml of native or peroxynitrite-modified HSA. A control sample, having AAPH only, was taken for reference. The free radical generation by AAPH was started by thermal shock by heating the samples to 37 ◦ C. The final volume of the reaction mixtures was 2 ml. Reading was taken after every 30 min by taking 0.02 ml of the sample in 1.0 ml of PBS. After 10–60 s centrifugation at 3000 × g, an aliquot (0.2 ml) of the supernatant was taken in microtitre plate and read at 540 nm. 2.7. Protein A-agarose affinity chromatography IgG from cancer sera was isolated by affinity chromatography on Protein A-agarose affinity column as described previously [21]. The IgG concentration was determined considering 1.4 OD278 = 1.0 mg IgG/ml. The homogeneity of isolated IgG was checked on 10%

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SDS–PAGE. The isolated IgG was dialyzed against PBS and stored at −20 ◦ C with 0.1% sodium azide. 2.8. Enzyme-linked immunosorbent assay (ELISA) ELISA was performed on flat bottom polysorp ELISA plate. The plates were coated with 100 ␮l of native or peroxynitrite-modified HSA (10 ␮g/ml) in antigen coating buffer. Each sample was assayed in duplicate and half of the plate, devoid of antigen coating, served as control. The remaining steps were the same as described earlier [22]. Results were expressed as mean of Atest − Acontrol . 2.9. Competition ELISA The antigenic specificity of antibodies was determined by competition ELISA as described earlier [23]. Percent inhibition was calculated using the equation: Percent inhibition = 1 −

 A  inhibited Auninhibited

× 100

2.10. Band shift assay Band shift assay was performed for the visual detection of antigen–antibody interaction and immune complex formation [24]. In assay, a constant amount (10 ␮g) of native or peroxynitritemodified HSA was incubated with increasing concentrations of affinity purified lung cancer IgG (0–50 ␮g) in PBS at 37 ◦ C for 2 h and overnight at 4 ◦ C. Immune complexes (ICs) thus formed were electrophoresed on 10% SDS–PAGE for 4 h at 80 V and the bands were visualized by silver staining. 2.11. Isolation of HSA The isolation of HSA from the sera of normal healthy individuals and cancer patients was done with slight modifications according to the published procedure [25]. A column of Sephacryl S-200 HR (60 × 1.5 cm) was used for gel chromatography. Sodium phosphate buffer (0.06 M, pH, 7.0) was used to equilibrate it. The determination of the void volume of the column was done by passing blue dextran (5 mg/ml). Each dialyzed sample was loaded on top of the column and eluted with 0.06 M sodium phosphate buffer pH 7.4 at a flow rate of 15–20 ml/h in 3 ml fractions. To determine the concentration of HSA, Lowry et al. method was used [26]. 2.12. Statistical analysis Data are presented as mean ± standard deviation (±SD). Statistical significance of data was determined by Student’s t test and a p value of