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Antibodies and Fab fragments protect Cu,Zn-SOD against methylglyoxal-induced inactivation
Biochimica et Biophysica Acta 1760 (2006) 1167 – 1174 http://www.elsevier.com/locate/bba
Antibodies and Fab fragments protect Cu,Zn-SOD against methylglyoxal-induced inactivation Rukhsana Jabeen a,b , Amin A. Mohammad a , Elizabeth C. Elefano a , John R. Petersen a , Mohammed Saleemuddin b,⁎ b
a Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0551, USA Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India
Received 2 December 2005; received in revised form 10 April 2006; accepted 11 April 2006 Available online 19 April 2006
Abstract Methyl glyoxal (MG) is a highly reactive α-oxoaldehyde that plays an important role in non-enzymatic glycosylation reactions, formation of Advanced Glycation End products (AGEs) and other complications associated with hyperglycemia and related disorders. Unlike sugars, glycation by MG is predominantly arginine directed, which is particularly more damaging since arginine residues have a high-frequency occurrence in ligand and substrate recognition sites in receptor and enzyme active sites. Using bovine erythrocyte Cu,Zn-superoxide dismutase (SOD) as model enzyme, the potential of anti-enzyme antibodies in imparting protection against MG-induced inactivation was investigated. A concentration- and time-dependent inactivation of SOD was observed when the enzyme was incubated with MG. The enzyme lost over 80% activity on incubation with 5 mM MG for 5 days. More marked inactivation was observed in 24 h when the MG concentration was raised up to 30 mM. The SOD inactivation was accompanied by the formation of high molecular weight aggregates as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI/TOF mass spectrometry). Inclusion of specific anti-SOD antibodies raised in rabbits or monomeric Fab fragments derived thereof offered remarkable protection against MGinduced loss in enzyme activity. The protection, however, decreased with increase in the concentration of MG. SELDI/TOF mass spectrometry also revealed that the antibodies restricted the formation of high molecular weight aggregates. The results emphasize the potential of antibody based therapy in combating glycation and related complications. Published by Elsevier B.V. Keywords: Methyl glyoxal; Glycation; SOD; Polyclonal antibodies
1. Introduction Non-enzymatic glycosylation or glycation comprises a complex series of reactions between reducing sugars and amino groups of proteins, lipids and nucleic acids [1]. During glycation, the carbonyl groups of sugars react slowly with the free amino groups yielding Schiff base. The Schiff bases undergo Amadori rearrangement and through a series of further rearrangements, cyclizations, dehydrations, etc. form a variety of diverse compounds, collectively described as Advanced Glycation End products (AGEs) [2,3]. AGE formation is accompanied by the formation, among others of a number of reactive oxygen species (ROS), α-oxoaldehydes including ⁎ Corresponding author. Tel.: +91 5712720449; fax: +91 5712721776. E-mail address: [email protected] (M. Saleemuddin). 0304-4165/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.bbagen.2006.04.002
methyl glyoxal (MG), that further react and damage the proteins and other important biological molecules [4]. MG formation in vivo can also take place from glycolytic intermediates, ketone bodies, as a result of threonine catabolism, oxidative decomposition of polyunsaturated fatty acids and during autoxidation of sugars by transition metal ions [5– 7]. The glyoxalase system converts MG to D-lactate and ensures the maintenance of low in vivo levels of MG [8]. Increased levels of MG have been however reported in the blood of diabetic patients and in the lens of streptozotosin-induced diabetic rats [9,10]. MG has been shown to be highly reactive both towards proteins and nucleic acids [11–13]. Glycation of proteins by MG, unlike that by reducing sugars, is mainly arginine-directed while other amino acids such as lysine, and to a lesser extent histidine and cysteine are also modified during the reactions of proteins with MG [14–17].
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MG reacts irreversibly with amino groups in proteins, forming AGEs [18,19]. Nϵ-(carboxyethyl)lysine (CEL) and MG-derived lysine dimer (MOLD) are the main adducts of the reaction of MG with lysine residues, while with arginine it forms mainly arginine-derived hydroimidazolone AGE, Nδ-(5methyl-imidazolone-2-yl)-ornithine (MG-H1) and Nδ-(5-hydroxy-4,6-dimethyl pyrimidine-2-yl)-L-ornithine commonly known as argpyrimidine [20–25]. Argpyrimidine is considered as a specific marker of protein glycation by MG and has been detected in renal tissues, lens proteins from diabetic patients, in kidney mesangial cells from diabetic rats, human carcinoma cells and in neurodegenerative disorders [15,20,26,27]. A number of proteins including BSA, RNase A, collagen and lysozyme have been shown to react with MG, accompanying alterations in conformation, biological activity and modification in interaction with other molecules [28]. MG is also toxic to cultured cells and it is believed that ROS-mediated apoptosis is responsible for the observed cytotoxic effects [29–31]. It is now well recognized that ROS cause widespread damage to biological macromolecules leading to lipid peroxidation, protein oxidation, enzyme inactivation, DNA base modifications and strand breaks in the DNA [32]. The damage caused by oxidative stress is expected to be exacerbated if the antioxidant enzymes themselves are inactivated by such events. Elevated levels of MG have been reported to adversely effect SODs which constitute the first and the most important line of anti-oxidant defense, particularly against superoxide radicals [33]. Exposure of Cu,Zn-SOD to MG has been shown to cause its covalent crosslinking associated with loss of enzymatic activity and release of copper ions [34]. Our recent work revealed that anti-SOD antibodies provide remarkable protection against inactivation of SOD by various sugars [35]. The objective of the present study was to evaluate the possible protective role of anti-enzyme antibodies and the Fab fragments derived thereof against MG-induced inactivation of Cu,ZnSOD. The results suggest that the anti-enzyme antibodies and the Fab fragments provide appreciable protection against MGinduced inactivation of the enzyme. 2. Materials and methods 2.1. Materials Bovine erythrocyte SOD (EC 1.15.1.1.), Methylglyoxal (MG 40% aqueous solution), Phenazine methosulfate (PMS), NADH and Nitro Blue Tetrazolium (NBT) were purchased from Sigma (St. Louis, MO, USA). Fab preparation kit was purchased from Pierce, Rockford, USA. Colloidal Blue Staining Kit (catalog no. LC6025) was purchased from Invitrogen Corporation, CA, USA. All other chemicals used were of analytical grade.
preserved at −20 °C. Formation of SOD specific antibodies was monitored by Ouchterlony double immunodiffusion, ELISA and Western blotting.
2.3. Purification of IgG The specific antibodies were purified by affinity chromatography following the published procedure [36]. The anti-SOD antiserum was applied to SODSepharose 4B packed column and allowed to bind at pH 7.8 in the presence of 20 mM sodium phosphate buffered saline. Unbound protein was removed by thorough washings and bound IgGs were eluted with 0.1 M glycine/HCl buffer, pH 3.0. The eluate was neutralized immediately with 1 M Tris buffer, pH 8.8. IgG from the sera of non-immunized rabbits was isolated by applying serum to Protein-A Sepharose packed column.
2.4. Incubation of SOD with MG Cu,Zn-SOD (0.25 mg/ml) was incubated with different concentrations of MG in 20 mM sodium phosphate buffer, pH 7.4 in dark, under sterile conditions for various time periods. At the end of incubation period, incubation mixtures were placed in Microcon filters (molecular mass cut off 3 kDa) and centrifuged to remove unbound MG. Protection by specific polyclonal antibodies was measured by incubating the enzyme with MG in presence of antibodies/Fab. Protein concentration was determined using Bio- Rad DC protein assay kit (Hercules, CA, USA).
2.5. Activity measurement and Protein crosslinking After incubation with various concentrations of MG in presence and absence of specific polyclonal antibodies for various time intervals, activity of Cu,ZnSOD was measured spectrophotometrically by employing PMS–NADH–NBT system where one unit of activity is defined as the quantity of enzyme that inhibits the NBT reduction by 50% [37]. Cu,Zn-SOD samples incubated with 30 mM MG for varying time periods were separated by SDS/PAGE in a Miniprotean 3 (Bio-Rad), using a 15% polyacrylamide separation gel and 5% stacking gel, as described by Laemmli [38]. Unstained precision plus protein standards (Bio-Rad) were also loaded on the gel. The gels were stained using Colloidal Blue Staining Kit (Invitrogen).
2.6. Mass spectrometric analysis Cu,Zn-SOD samples incubated with various MG concentrations in presence and absence of anti-enzyme antibodies were analyzed by SELDI-TOF mass spectrometry using PBS-II mass reader (Ciphergen Biosystems, Fremont, CA). Samples were loaded on to NP20 Protein chip array (Ciphergen Biosystems). Prior to sample loading, chips were pre-rinsed with HPLC grade water. 2.0 μl sample was loaded per spot and the sample was allowed to air dry onto the surface. The chip was washed with binding buffer thrice with shaking in Micromix and the binding buffer was removed. Finally, the chips were air-dried and 0.5 μl of saturated sinapinic acid (Ciphergen Biosystem) solution in 50% acetonitrile in water containing 0.5% trifluoroacetic acid was applied twice with air-drying step in between. The protein bound chips were then transferred into the chip reader for analysis. Spectra were collected with a laser intensity of 170 and a detector sensitivity of 10. Mass accuracy was calibrated externally using the All-in-1 peptide and All-in-1 Protein molecular mass standards (Ciphergen Biosystems).
2.7. Preparation of Fab monomers 2.2. Immunization Antibodies were raised in the rabbits by injecting Cu,Zn-SOD emulsified in Freund's complete/incomplete adjuvant. Briefly, rabbits were immunized subcutaneously with SOD preparation (200 μg/0.4 ml) in 20 mM sodium phosphate buffered saline, pH 7.2 using Freund's complete adjuvant. The animals were rested for 21 days and then boosted weekly with antigen preparation (100 μg) in Freund's incomplete adjuvant. After the fourth booster, the rabbits were bled through a marginal ear vein and blood was allowed to clot at room temperature for 6–8 h. Serum was then collected by centrifugation and
Fab monomers were prepared using the kit supplied by Pierce Co following manufacturer's protocol. Briefly, the antibodies raised against Cu,Zn-SOD were extensively dialyzed against sodium phosphate buffer (20 mM, pH 7.0) and concentrated to approximately 20 mg/ml. Around 0.5 ml of the immobilized papain was taken in a glass tube and equilibrated with 4.0 ml digestion buffer (cysteine–HCl in phosphate buffer). Papain was separated from buffer using serum separator tubes and washed again with digestion buffer. After washings, immobilized papain was resuspended in 0.5 ml of digestion buffer. Then, 0.5 ml of IgG solution (∼10 mg) was mixed with 0.5 ml digestion buffer and finally
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added to the tube containing equilibrated immobilized papain solution. The mixture was incubated for 5 h in a shaking water bath at 37 °C. The solubilized Fab, Fc fragments and undigested IgG was recovered from immobilized papain using the separator tube and the crude digest was applied to a 1.0 ml protein A column. The immobilized protein A binds the Fc and undigested IgG, allowing the Fab fragments to flow through the column. Non-specific Fab were prepared from IgG, isolated from the sera of non-immunized rabbits following the above mentioned protocol.
3. Results 3.1. Effect of MG on the activity and aggregation of Cu,Zn-SOD The effect of MG concentration and exposure time on SOD activity was studied. The extent of enzyme inactivation was found to be both concentration- and time-dependent (Fig. 1). At the end of day 5, SOD retained 43, 25 and 12% activity upon incubation with 1, 3 and 5 mM MG, respectively (Fig. 1A). SOD inactivation was more rapid at higher MG concentrations and the enzyme retained less than 10% activity after incubation with 30 mM MG for 24 h (Fig. 1B). Fig. 1C and D show the protective effect of anti Cu,Zn-SOD antibodies or the Fab fragments derived thereof on the MG-induced inactivation of the enzyme. Protection was remarkable at low concentrations of MG and decreased with increasing concentration of the oxoaldehyde. It is also clear from the figures that comparable concentrations of non-specific antibodies/Fab monomers were ineffective in protecting the enzyme even at lower MG concentrations. Formation of high molecular weight cross-linked materials in the SOD is evident from Fig. 2. Higher molecular weight bands started appearing within 6 h of incubation and their intensity increased with time. The efficiency of methylglyoxal in crosslinking is thought to be due to its ability to form stable heterocyclic compounds that crosslink proteins. Fig. 2 provides strong evidence for the non-enzymatic crosslinking property of MG. 3.2. Mass spectral analysis The degradation and modification of Cu,Zn-SOD, on incubation with MG, was analyzed by SELDI-TOF-MS as a function of peak intensity and net gain in molecular weight of the enzyme upon its binding with MG. Fig. 3A represents the mass spectra of unmodified Cu,Zn-SOD (control). As conceived, the molecular ion of Cu,Zn-SOD produced a major peak at m/z ratio of 15,582.5 Da, which is indicative of the natural monomer. Another peak with lesser intensity at 31059.8 Da corresponds to the dimer. Minor peaks, corresponding to the masses of doubly charged trimers (46 kDa) and tetramers (64 kDa) of SOD are also present. The modified Cu,Zn-SOD, in contrast reveals the different mass profile. There is a clear broadening of peaks indicative of MG binding, accompanied by decreased peak intensity, reflecting the loss of the natural monomer by excessive crosslinking. There is a 3-fold decrease in the monomer peak intensity, compared to control, after 12 h incubation of SOD with 30 mM MG. The intensity of monomer peak continues to decrease in a time-dependent manner where as the dimer and
Fig. 1. Effect of MG on the activity of Cu,Zn-SOD. (A) Cu,Zn-SOD (0.25 mg/ ml) was incubated with indicated concentrations of MG in 20 mM phosphate buffer, pH 7.4 at 37 °C for 1, 3 and 5 days. Activity of untreated enzyme is expressed as 100%. Specific activity of the untreated enzyme (control) was 4371 units/mg protein. (B) Cu,Zn-SOD (0.25 mg/ml) was incubated with 5, 10, 20 or 30 mM MG and activity determined after incubation at 37 °C for 24 h. Values are the averages from three independent experiments ± S.D. (C) Cu,ZnSOD (0.25 mg/ml) was incubated with varying MG concentrations (0, 5, 10, 20 & 30 mM) for 24 h in presence of specific anti-SOD IgG and non-specific IgG. Specific activity of the control was 4371 units/mg protein whereas the specific activity of SOD incubated with 30 mM MG for 24 h in absence and presence of antibodies was 367.16 and 1424.94 units/mg protein, respectively. (D) Cu,Zn-SOD (0.25 mg/ml) was incubated indicated MG concentrations for 24 h in presence of specific and non-specific Fab monomers. Specific activity of the control was 4371 units/mg protein while the activities in Fab absence and presence were 367.164 and 3777 units/mg protein, respectively.
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The mass spectra of Cu,Zn-SOD incubated with 30 mM MG in presence of the antibodies showed little evidence of aggregation even after 24 h incubation (Fig. 3B). 4. Discussion
Fig. 2. SDS-PAGE of Cu,Zn-SOD incubated with MG. Cu,Zn-SOD (0.25 mg/ ml) was incubated with 30 mM MG in 20 mM phosphate buffer, pH 7.4 at 37 °C for various time intervals and aliquots were subjected to SDS-PAGE. 6 μg of protein was applied in each lane (Lane 1 contains molecular weight markers (kDa); Lanes 2–6 contain SOD samples incubated with 30 mM MG for 0, 3, 6, 12 and 24 h, respectively).
tetramer peak intensity increases with time thereby reflecting the formation of high molecular weight aggregates due to crosslinking. However, there was significant increase in m/z ratio with time and finally the m/z ratio reached as high as 16319.6 as compared to monomer peak of control after 24 h incubation with 30 mM MG implying modification of SOD by MG binding. 3.3. Effect of the anti-SOD antibodies on MG induced alteration in Cu,Zn-SOD Inclusion of specific polyclonal antibodies in the incubation mixtures containing MG resulted in remarkable protection of SOD as is evident from Fig. 4A–C. Cu,Zn-SOD retained an appreciable 93, 84 and 79% enzymatic activity after incubation with 5 mM MG for 1, 3 and 5 days, respectively (Fig. 4A–C). The antibodies exhibited significant protective effect even at higher MG concentrations. When Cu,Zn-SOD was treated with extremely high MG concentrations for 24 h (10, 20 and 30 mM MG), it still retained around 71, 50 and 33% activity, respectively, as compared to 35, 20 and 8% in absence of antibodies (Fig. 5A–D). 3.4. Effect of Fab fragments on MG induced modification/ inactivation As shown in Figs. 4 and 5, specific Fab monomers derived from polyclonal anti-SOD IgG protected SOD against MG induced inactivation. The Fig. 4 clearly depicts that in presence of Fab monomers, enzyme retained around 87, 80 and 72% of its initial activity after incubating with 5 mM MG for 1, 3 and 5 days. Fab monomers rendered significant protection to Cu,ZnSOD even at higher MG concentrations. The extent of protection is summarized in Fig. 5A–D. Mass spectral analysis of the incubation mixtures in presence of antibodies also revealed the striking protective effect of antibodies against aggregation/cross-linking. The molecular ion of SOD incubated with MG in presence of antibodies showed monomer major peak at m/z ratio close to that of the control.
Post-translational modifications of proteins due to glycation and oxidation play a significant role in the development of a variety of complications associated with diabetes and aging. The accumulation of glycation intermediates like MG, leads to modification of DNA causing mutagenesis and apoptosis [39,40]. Modification of proteins with MG may in turn result in protein degradation, enzyme inhibition and a cytokinemediated immune response [41]. In vitro reaction with MG has been shown to induce conformational changes in proteins that expose the hydrophobic core and facilitate the oxidation of newly exposed thiols resulting in protein crosslinking [42,43]. Currently, several strategies are being employed to control protein glycation. These include inhibition of AGE formation and conversion of post-Amadori intermediates to AGEs, use of crosslinking inhibitors and manipulation of AGE receptor pathways [44]. A number of AGE inhibitors have also been proposed. Inhibitors like aspirin or pyridoxal-5-phosphate act to modify the free amino groups, thereby preventing sugar attachment [45,46]. Inhibitors like aminoguanidine, on the other hand react with sugars, diverting them from Maillard reactions on proteins [47]. Penicillamine, DETAPAC, Vitamin C and E act as chelators of in vivo metal ions, while inhibitors like L-arginine trap dicarbonyl intermediates to form substituted triazenes. Amadorins, like pyridoxamine (vitamin B6 derivative), are the compounds with potential to scavenge postAmadori products, inhibiting the conversion of glycation intermediates to AGEs [48,49]. Crosslink breakers disrupt the crosslinks between the protein molecules [50]. Recently, researchers have been investigating chaperones for protection against glycation-induced inactivation and loss of antigenicity [51,52]. Molecular chaperones not only assist during protein folding but also stabilize proteins and prevent aggregation [53,54]. Substantiating the earlier observation [35], this work shows the potential of the antibodies and monomeric Fab fragments in protecting the inactivation of Cu,Zn-SOD by a far more reactive α-oxoaldehde MG. MG reacts with arginine, lysine and to a lesser extent to cysteine and histidine residues leading to crosslinking of the enzyme [55]. Cu,Zn-SOD contains copper and zinc ions at the active site, bridged by a common ligand histidine and treatment of SOD with MG leads to the release of the ions at the active site and hence enzyme inactivation [34,56]. Fig. 1 shows that inactivation of the Cu,Zn-SOD is rapid and is accompanied by marked aggregation, especially at higher MG concentrations (Figs. 2 and 3). Specific polyclonal antibodies/Fab monomers protected significantly Cu,Zn-SOD against MG induced inactivation (Figs. 4 and 5) and crosslink formation (Fig. 3B). When enzyme was incubated with lower doses of MG (1, 3 and 5 mM), the antibodies were found to be highly effective in preventing MGinduced inactivation of SOD. Antibodies and Fab monomer fragments protected the enzyme, although to a smaller extent,
R. Jabeen et al. / Biochimica et Biophysica Acta 1760 (2006) 1167–1174 Fig. 3. SELDI-TOF mass spectra of Bovine Cu,Zn-SOD from an SPA matrix. Cu,Zn-SOD (0.25 mg/ml) was incubated with 30 mM MG at 37 °C for 0, 3, 6, 12 and 24 h in 20 mM phosphate buffer pH 7.4 in absence (A) or presence of 5-fold molar excess of anti-SOD antibodies (B). 1171
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aggregation upon binding to the antigen molecules. Alternatively, binding of antibodies may shift equilibrium between unfolded and native conformations of the protein antigen towards the native form or lower the activation barrier through stabilization of the folded transition state [57]. We believe that binding of antibody restricts the unfolding of the protein and consequently the further
Fig. 4. Effect of inclusion of specific anti-SOD antibodies/monomeric Fab against the MG-induced Cu,Zn-SOD inactivation. After incubation of Cu,ZnSOD (0.25 mg/ml) with various indicated MG concentrations for 1, 3 or 5 days in presence and absence of 5-fold molar excess specific polyclonal antibodies/ Fab monomers at 37 °C in dark, under sterile conditions, its enzymatic activity was determined (A–C). Values are the averages from three independent experiments ± S.D.
against the MG-induced inactivation even at very high concentrations of the α-oxoaldehyde (up to 30 mM). This protection is specific, since non-specific IgG and Fab monomers derived thereof did not show any protection against the inactivation (Fig. 1C and D). The exact mechanism for the protective role of antibodies needs to be elucidated. It has been found that some antibodies act as chaperones under specific conditions and can facilitate folding and prevent aggregation of protein antigens [57]. α-crystallin, a molecular chaperone and lens structural protein, protects the membrane enzyme Na/K-ATPase and preserves its activity upon incubation with methylglyoxal, fructose and H2O2. It is believed that α-crystallin may act through dynamic interactions, such that the chaperone may prevent the further unfolding but not bind to the target protein [58]. Antibodies may sterically hinder the
Fig. 5. Effect of anti-SOD antibodies/monomeric Fab on the inactivation of SOD induced by higher MG concentrations. Cu,Zn-SOD (0.25 mg/ml) was incubated with indicated MG concentrations and activity was measured after 0, 3, 6, 12 and 24 h. Values are the averages from three independent experiments ± S.D.
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modification and inactivation of Cu,Zn-SOD. Since monomeric Fab also protected the enzyme, binding rather than crosslinking and aggregation by the bifunctional anti-enzyme IgG may play an important role in restricting the MG-induced damage. Inclusion of antibodies prevents dimer formation by MG, probably the antibodies inhibit the exposure of glycation susceptible functional groups by preventing enzyme unfolding (Fig. 3B). Ulbrich-Hoffman and co-workers proposed that enzymes may contain specific regions where unfolding of the molecule may begin and blocking of such regions may lead to restriction of unfolding and enhancement in the stabilization of enzymes [59]. It has been shown in earlier studies that antibodies recognizing the labile regions of RNase can impart stability to the enzyme on binding [60]. Binding of the antibody also improved the stability of a labile mutant RNase with variation of the labile region aminoacid sequence [61]. Polyclonal antibodies raised in rabbits have been used in this study and the preparation may contain the populations that bind to such crucial regions of SOD, preventing further unfolding of the enzyme. Since the protection by specific polyclonal antibodies and Fab monomers is remarkable, enzyme therapy may turn out to be a promising approach in the treatment of disorders associated with glycation. The Fab monomers can be advantageous owing to the fact that interaction of complement and other molecules with the Fc portion can be avoided. We have recently demonstrated that soluble yet stable enzyme preparations can be prepared by binding of bromelain with monomeric Fab fragment derived from anti-bromelain IgG [62]. Valency and halflife of the fragments can be tailored through protein engineering approaches to suit the desired mechanism of action. Furthermore, antibody fragments are better suited for expression in microbial systems, providing benefits in terms of scaling up and ease of production [63]. A few attempts of using antibodies to restrict AGE-mediated damage are also available. An antiglycated albumin antibody has been shown to prevent BM thickening in diabetic db/.db mice and a monoclonal antibody A 717 was also shown to be effective in retarding the development of diabetic retinopathy in diabetic animal models [64,65]. The present study has been carried out using affinity purified anti-enzyme polyclonal IgG. It is quite likely that few epitope specific monoclonal antibody populations may be far more protective and attempts are being made to isolate such antibodies. To conclude, this work suggests a remarkable potential of anti-enzyme antibodies and Fab fragments in the protection of SOD against inactivation induced by MG.
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Acknowledgments [19]
This work has been primarily done at Department of Pathology, University of Texas Medical Branch, Galveston, USA. Authors are highly thankful to Dr. M.A. Khan for his valuable suggestions.
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[43] J.N. Liang, M. Rossi, In vitro non-enzymatic glycation and formation of browning products in the bovine lens α-crystallin, Exp. Eye Res. 50 (1990) 367–371. [44] A.W. Stitt, The role of advanced glycation in the pathogenesis of diabetic retinopathy, Exp. Mol. Pathol. 75 (2003) 95–108. [45] K.M. Reiser, Nonenzymatic glycation of collagen in aging and diabetes, Proc. Soc. Exp. Biol. Med. 218 (1998) 23–37. [46] J.J. Harding, Nonenzymatic covalent posttranslational modification of proteins in vivo, Adv. Protein Chem. 37 (1985) 247–334. [47] J. Hirsch, E. Petrakova, M.S. Feather, C.L. Barnes, The reaction of D-glucose with aminoguanidine, Carbohydr. Res. 267 (1995) 17–25. [48] R.G. Khalifah, J.W. Baynes, B.G. Hudson, Amadorins: novel postAmadori inhibitors of advanced glycation reactions, Biochem. Biophys. Res. Commun. 257 (1999) 251–258. [49] P.A. Voziyan, T.O. Metz, J.W. Baynes, B.G. Hudson, A post-Amadori inhibitor pyridoxamine also inhibits chemical modification of proteins by scavenging carbonyl intermediates of carbohydrate and lipid degradation, J. Biol. Chem. 277 (2002) 3397–3403. [50] M.M. Heath, K.C. Rixon, J.J. Harding, Glycation-induced inactivation of malate dehydrogenase protection by aspirin and a lens molecular chaperone, alpha-crystallin, Biochim. Biophys. Acta 1315 (1996) 176–184. [51] H. Yan, J.J. Harding, The molecular chaperone, alpha-crystallin, protects against loss of antigenicity and activity of esterase caused by sugars, sugar phosphate and a steroid, Biol. Chem. 384 (2003) 1185–1194. [52] E. Ganea, J.J. Harding, Molecular chaperones protect against glycation-induced inactivation of glucose-6-phosphate dehydrogenase, Eur. J. Biochem. 231 (1995) 181–185. [53] J.P. Hendrick, F.U. Hartl, Molecular chaperone functions of heat-shock proteins, Annu. Rev. Biochem. 62 (1993) 349–384. [54] J. Becker, E.A. Craig, Heat shock proteins as a molecular chaperones, Eur. J. Biochem. 219 (1994) 11–23. [55] J.A. McLaughlin, R. Pethig, A. Szent-Györgyi, Spectroscopic studies of the protein-methylglyoxal adduct, Proc. Natl. Acad. Sci. U. S. A. 77 (1980) 949–951. [56] J.A. Tainer, E.D. Getzoff, J.S. Richardson, D.C. Richardson, Structure and mechanism of copper, zinc superoxide dismutase, Nature 306 (1983) 284–287. [57] D.N. Ermolenko, A.V. Zherdev, B.B. Dzantiev, Antibodies as specific chaperones, Biochemistry 69 (2004) 1233–1238. [58] B.K. Derham, J.C. Ellory, A.J. Bron, J.J. Harding, The molecular chaperone alpha crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress, Eur. J. Biochem. 270 (2003) 2605–2611. [59] R. Ulbrich-Hofman, R. Golbik, W. Damerau, in: W.J.J. Vander Tweel, A. Harder, R.M. Buitelar (Eds.), Stability sand stabilization of enzymes, Elsevier, Amsterdam, 1993, pp. 497–504. [60] H. Younus, M. Owais, D.N. Rao, M. Saleemuddin, Stabilization of pancreatic ribonuclease A by immobilization on Sepharose-linked antibodies that recognize the labile region of the enzyme, Biochim. Biophys. Acta 36454 (2001) 1–7. [61] H. Younus, J. Koeditz, M. Saleemuddin, R. Ulbrich-Hofman, Selective stabilization of a labile mutant from bovine pancreatic ribonuclease A by antibodies, Biotechnol. Lett. 24 (2002) 1821–1826. [62] P. Gupta, R.H. Khan, M. Saleemuddin, Binding of antibromelain monomeric Fab' improves the stability of stem bromelain against inactivation, Biochim. Biophys. Acta 1646 (2003) 131–135. [63] P. Holliger, P.J. Hudson, Engineered antibody fragments and the rise of single domains, Nat. Biotechnol. 23 (2005) 1126–1136. [64] R.S. Clements Jr., W.G. Robison Jr., M.P. Cohen, Anti-glycated albumin therapy ameliorates early retinal microvascular pathology in db/db mice, J. Diabetes its Complicat. 12 (1998) 28–33. [65] M.P. Cohen, F.N. Ziyadeh, Role of Amadori modified non-enzymatically glycated serum proteins in the pathogenesis of diabetic retinopathy, J. Am. Soc. Nephrol. 7 (1996) 183–190.
Antibodies and Fab fragments protect Cu,Zn-SOD against methylglyoxal-induced inactivation Rukhsana Jabeen a,b , Amin A. Mohammad a , Elizabeth C. Elefano a , John R. Petersen a , Mohammed Saleemuddin b,⁎ b
a Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0551, USA Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India
Received 2 December 2005; received in revised form 10 April 2006; accepted 11 April 2006 Available online 19 April 2006
Abstract Methyl glyoxal (MG) is a highly reactive α-oxoaldehyde that plays an important role in non-enzymatic glycosylation reactions, formation of Advanced Glycation End products (AGEs) and other complications associated with hyperglycemia and related disorders. Unlike sugars, glycation by MG is predominantly arginine directed, which is particularly more damaging since arginine residues have a high-frequency occurrence in ligand and substrate recognition sites in receptor and enzyme active sites. Using bovine erythrocyte Cu,Zn-superoxide dismutase (SOD) as model enzyme, the potential of anti-enzyme antibodies in imparting protection against MG-induced inactivation was investigated. A concentration- and time-dependent inactivation of SOD was observed when the enzyme was incubated with MG. The enzyme lost over 80% activity on incubation with 5 mM MG for 5 days. More marked inactivation was observed in 24 h when the MG concentration was raised up to 30 mM. The SOD inactivation was accompanied by the formation of high molecular weight aggregates as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI/TOF mass spectrometry). Inclusion of specific anti-SOD antibodies raised in rabbits or monomeric Fab fragments derived thereof offered remarkable protection against MGinduced loss in enzyme activity. The protection, however, decreased with increase in the concentration of MG. SELDI/TOF mass spectrometry also revealed that the antibodies restricted the formation of high molecular weight aggregates. The results emphasize the potential of antibody based therapy in combating glycation and related complications. Published by Elsevier B.V. Keywords: Methyl glyoxal; Glycation; SOD; Polyclonal antibodies
1. Introduction Non-enzymatic glycosylation or glycation comprises a complex series of reactions between reducing sugars and amino groups of proteins, lipids and nucleic acids [1]. During glycation, the carbonyl groups of sugars react slowly with the free amino groups yielding Schiff base. The Schiff bases undergo Amadori rearrangement and through a series of further rearrangements, cyclizations, dehydrations, etc. form a variety of diverse compounds, collectively described as Advanced Glycation End products (AGEs) [2,3]. AGE formation is accompanied by the formation, among others of a number of reactive oxygen species (ROS), α-oxoaldehydes including ⁎ Corresponding author. Tel.: +91 5712720449; fax: +91 5712721776. E-mail address: [email protected] (M. Saleemuddin). 0304-4165/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.bbagen.2006.04.002
methyl glyoxal (MG), that further react and damage the proteins and other important biological molecules [4]. MG formation in vivo can also take place from glycolytic intermediates, ketone bodies, as a result of threonine catabolism, oxidative decomposition of polyunsaturated fatty acids and during autoxidation of sugars by transition metal ions [5– 7]. The glyoxalase system converts MG to D-lactate and ensures the maintenance of low in vivo levels of MG [8]. Increased levels of MG have been however reported in the blood of diabetic patients and in the lens of streptozotosin-induced diabetic rats [9,10]. MG has been shown to be highly reactive both towards proteins and nucleic acids [11–13]. Glycation of proteins by MG, unlike that by reducing sugars, is mainly arginine-directed while other amino acids such as lysine, and to a lesser extent histidine and cysteine are also modified during the reactions of proteins with MG [14–17].
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MG reacts irreversibly with amino groups in proteins, forming AGEs [18,19]. Nϵ-(carboxyethyl)lysine (CEL) and MG-derived lysine dimer (MOLD) are the main adducts of the reaction of MG with lysine residues, while with arginine it forms mainly arginine-derived hydroimidazolone AGE, Nδ-(5methyl-imidazolone-2-yl)-ornithine (MG-H1) and Nδ-(5-hydroxy-4,6-dimethyl pyrimidine-2-yl)-L-ornithine commonly known as argpyrimidine [20–25]. Argpyrimidine is considered as a specific marker of protein glycation by MG and has been detected in renal tissues, lens proteins from diabetic patients, in kidney mesangial cells from diabetic rats, human carcinoma cells and in neurodegenerative disorders [15,20,26,27]. A number of proteins including BSA, RNase A, collagen and lysozyme have been shown to react with MG, accompanying alterations in conformation, biological activity and modification in interaction with other molecules [28]. MG is also toxic to cultured cells and it is believed that ROS-mediated apoptosis is responsible for the observed cytotoxic effects [29–31]. It is now well recognized that ROS cause widespread damage to biological macromolecules leading to lipid peroxidation, protein oxidation, enzyme inactivation, DNA base modifications and strand breaks in the DNA [32]. The damage caused by oxidative stress is expected to be exacerbated if the antioxidant enzymes themselves are inactivated by such events. Elevated levels of MG have been reported to adversely effect SODs which constitute the first and the most important line of anti-oxidant defense, particularly against superoxide radicals [33]. Exposure of Cu,Zn-SOD to MG has been shown to cause its covalent crosslinking associated with loss of enzymatic activity and release of copper ions [34]. Our recent work revealed that anti-SOD antibodies provide remarkable protection against inactivation of SOD by various sugars [35]. The objective of the present study was to evaluate the possible protective role of anti-enzyme antibodies and the Fab fragments derived thereof against MG-induced inactivation of Cu,ZnSOD. The results suggest that the anti-enzyme antibodies and the Fab fragments provide appreciable protection against MGinduced inactivation of the enzyme. 2. Materials and methods 2.1. Materials Bovine erythrocyte SOD (EC 1.15.1.1.), Methylglyoxal (MG 40% aqueous solution), Phenazine methosulfate (PMS), NADH and Nitro Blue Tetrazolium (NBT) were purchased from Sigma (St. Louis, MO, USA). Fab preparation kit was purchased from Pierce, Rockford, USA. Colloidal Blue Staining Kit (catalog no. LC6025) was purchased from Invitrogen Corporation, CA, USA. All other chemicals used were of analytical grade.
preserved at −20 °C. Formation of SOD specific antibodies was monitored by Ouchterlony double immunodiffusion, ELISA and Western blotting.
2.3. Purification of IgG The specific antibodies were purified by affinity chromatography following the published procedure [36]. The anti-SOD antiserum was applied to SODSepharose 4B packed column and allowed to bind at pH 7.8 in the presence of 20 mM sodium phosphate buffered saline. Unbound protein was removed by thorough washings and bound IgGs were eluted with 0.1 M glycine/HCl buffer, pH 3.0. The eluate was neutralized immediately with 1 M Tris buffer, pH 8.8. IgG from the sera of non-immunized rabbits was isolated by applying serum to Protein-A Sepharose packed column.
2.4. Incubation of SOD with MG Cu,Zn-SOD (0.25 mg/ml) was incubated with different concentrations of MG in 20 mM sodium phosphate buffer, pH 7.4 in dark, under sterile conditions for various time periods. At the end of incubation period, incubation mixtures were placed in Microcon filters (molecular mass cut off 3 kDa) and centrifuged to remove unbound MG. Protection by specific polyclonal antibodies was measured by incubating the enzyme with MG in presence of antibodies/Fab. Protein concentration was determined using Bio- Rad DC protein assay kit (Hercules, CA, USA).
2.5. Activity measurement and Protein crosslinking After incubation with various concentrations of MG in presence and absence of specific polyclonal antibodies for various time intervals, activity of Cu,ZnSOD was measured spectrophotometrically by employing PMS–NADH–NBT system where one unit of activity is defined as the quantity of enzyme that inhibits the NBT reduction by 50% [37]. Cu,Zn-SOD samples incubated with 30 mM MG for varying time periods were separated by SDS/PAGE in a Miniprotean 3 (Bio-Rad), using a 15% polyacrylamide separation gel and 5% stacking gel, as described by Laemmli [38]. Unstained precision plus protein standards (Bio-Rad) were also loaded on the gel. The gels were stained using Colloidal Blue Staining Kit (Invitrogen).
2.6. Mass spectrometric analysis Cu,Zn-SOD samples incubated with various MG concentrations in presence and absence of anti-enzyme antibodies were analyzed by SELDI-TOF mass spectrometry using PBS-II mass reader (Ciphergen Biosystems, Fremont, CA). Samples were loaded on to NP20 Protein chip array (Ciphergen Biosystems). Prior to sample loading, chips were pre-rinsed with HPLC grade water. 2.0 μl sample was loaded per spot and the sample was allowed to air dry onto the surface. The chip was washed with binding buffer thrice with shaking in Micromix and the binding buffer was removed. Finally, the chips were air-dried and 0.5 μl of saturated sinapinic acid (Ciphergen Biosystem) solution in 50% acetonitrile in water containing 0.5% trifluoroacetic acid was applied twice with air-drying step in between. The protein bound chips were then transferred into the chip reader for analysis. Spectra were collected with a laser intensity of 170 and a detector sensitivity of 10. Mass accuracy was calibrated externally using the All-in-1 peptide and All-in-1 Protein molecular mass standards (Ciphergen Biosystems).
2.7. Preparation of Fab monomers 2.2. Immunization Antibodies were raised in the rabbits by injecting Cu,Zn-SOD emulsified in Freund's complete/incomplete adjuvant. Briefly, rabbits were immunized subcutaneously with SOD preparation (200 μg/0.4 ml) in 20 mM sodium phosphate buffered saline, pH 7.2 using Freund's complete adjuvant. The animals were rested for 21 days and then boosted weekly with antigen preparation (100 μg) in Freund's incomplete adjuvant. After the fourth booster, the rabbits were bled through a marginal ear vein and blood was allowed to clot at room temperature for 6–8 h. Serum was then collected by centrifugation and
Fab monomers were prepared using the kit supplied by Pierce Co following manufacturer's protocol. Briefly, the antibodies raised against Cu,Zn-SOD were extensively dialyzed against sodium phosphate buffer (20 mM, pH 7.0) and concentrated to approximately 20 mg/ml. Around 0.5 ml of the immobilized papain was taken in a glass tube and equilibrated with 4.0 ml digestion buffer (cysteine–HCl in phosphate buffer). Papain was separated from buffer using serum separator tubes and washed again with digestion buffer. After washings, immobilized papain was resuspended in 0.5 ml of digestion buffer. Then, 0.5 ml of IgG solution (∼10 mg) was mixed with 0.5 ml digestion buffer and finally
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added to the tube containing equilibrated immobilized papain solution. The mixture was incubated for 5 h in a shaking water bath at 37 °C. The solubilized Fab, Fc fragments and undigested IgG was recovered from immobilized papain using the separator tube and the crude digest was applied to a 1.0 ml protein A column. The immobilized protein A binds the Fc and undigested IgG, allowing the Fab fragments to flow through the column. Non-specific Fab were prepared from IgG, isolated from the sera of non-immunized rabbits following the above mentioned protocol.
3. Results 3.1. Effect of MG on the activity and aggregation of Cu,Zn-SOD The effect of MG concentration and exposure time on SOD activity was studied. The extent of enzyme inactivation was found to be both concentration- and time-dependent (Fig. 1). At the end of day 5, SOD retained 43, 25 and 12% activity upon incubation with 1, 3 and 5 mM MG, respectively (Fig. 1A). SOD inactivation was more rapid at higher MG concentrations and the enzyme retained less than 10% activity after incubation with 30 mM MG for 24 h (Fig. 1B). Fig. 1C and D show the protective effect of anti Cu,Zn-SOD antibodies or the Fab fragments derived thereof on the MG-induced inactivation of the enzyme. Protection was remarkable at low concentrations of MG and decreased with increasing concentration of the oxoaldehyde. It is also clear from the figures that comparable concentrations of non-specific antibodies/Fab monomers were ineffective in protecting the enzyme even at lower MG concentrations. Formation of high molecular weight cross-linked materials in the SOD is evident from Fig. 2. Higher molecular weight bands started appearing within 6 h of incubation and their intensity increased with time. The efficiency of methylglyoxal in crosslinking is thought to be due to its ability to form stable heterocyclic compounds that crosslink proteins. Fig. 2 provides strong evidence for the non-enzymatic crosslinking property of MG. 3.2. Mass spectral analysis The degradation and modification of Cu,Zn-SOD, on incubation with MG, was analyzed by SELDI-TOF-MS as a function of peak intensity and net gain in molecular weight of the enzyme upon its binding with MG. Fig. 3A represents the mass spectra of unmodified Cu,Zn-SOD (control). As conceived, the molecular ion of Cu,Zn-SOD produced a major peak at m/z ratio of 15,582.5 Da, which is indicative of the natural monomer. Another peak with lesser intensity at 31059.8 Da corresponds to the dimer. Minor peaks, corresponding to the masses of doubly charged trimers (46 kDa) and tetramers (64 kDa) of SOD are also present. The modified Cu,Zn-SOD, in contrast reveals the different mass profile. There is a clear broadening of peaks indicative of MG binding, accompanied by decreased peak intensity, reflecting the loss of the natural monomer by excessive crosslinking. There is a 3-fold decrease in the monomer peak intensity, compared to control, after 12 h incubation of SOD with 30 mM MG. The intensity of monomer peak continues to decrease in a time-dependent manner where as the dimer and
Fig. 1. Effect of MG on the activity of Cu,Zn-SOD. (A) Cu,Zn-SOD (0.25 mg/ ml) was incubated with indicated concentrations of MG in 20 mM phosphate buffer, pH 7.4 at 37 °C for 1, 3 and 5 days. Activity of untreated enzyme is expressed as 100%. Specific activity of the untreated enzyme (control) was 4371 units/mg protein. (B) Cu,Zn-SOD (0.25 mg/ml) was incubated with 5, 10, 20 or 30 mM MG and activity determined after incubation at 37 °C for 24 h. Values are the averages from three independent experiments ± S.D. (C) Cu,ZnSOD (0.25 mg/ml) was incubated with varying MG concentrations (0, 5, 10, 20 & 30 mM) for 24 h in presence of specific anti-SOD IgG and non-specific IgG. Specific activity of the control was 4371 units/mg protein whereas the specific activity of SOD incubated with 30 mM MG for 24 h in absence and presence of antibodies was 367.16 and 1424.94 units/mg protein, respectively. (D) Cu,Zn-SOD (0.25 mg/ml) was incubated indicated MG concentrations for 24 h in presence of specific and non-specific Fab monomers. Specific activity of the control was 4371 units/mg protein while the activities in Fab absence and presence were 367.164 and 3777 units/mg protein, respectively.
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The mass spectra of Cu,Zn-SOD incubated with 30 mM MG in presence of the antibodies showed little evidence of aggregation even after 24 h incubation (Fig. 3B). 4. Discussion
Fig. 2. SDS-PAGE of Cu,Zn-SOD incubated with MG. Cu,Zn-SOD (0.25 mg/ ml) was incubated with 30 mM MG in 20 mM phosphate buffer, pH 7.4 at 37 °C for various time intervals and aliquots were subjected to SDS-PAGE. 6 μg of protein was applied in each lane (Lane 1 contains molecular weight markers (kDa); Lanes 2–6 contain SOD samples incubated with 30 mM MG for 0, 3, 6, 12 and 24 h, respectively).
tetramer peak intensity increases with time thereby reflecting the formation of high molecular weight aggregates due to crosslinking. However, there was significant increase in m/z ratio with time and finally the m/z ratio reached as high as 16319.6 as compared to monomer peak of control after 24 h incubation with 30 mM MG implying modification of SOD by MG binding. 3.3. Effect of the anti-SOD antibodies on MG induced alteration in Cu,Zn-SOD Inclusion of specific polyclonal antibodies in the incubation mixtures containing MG resulted in remarkable protection of SOD as is evident from Fig. 4A–C. Cu,Zn-SOD retained an appreciable 93, 84 and 79% enzymatic activity after incubation with 5 mM MG for 1, 3 and 5 days, respectively (Fig. 4A–C). The antibodies exhibited significant protective effect even at higher MG concentrations. When Cu,Zn-SOD was treated with extremely high MG concentrations for 24 h (10, 20 and 30 mM MG), it still retained around 71, 50 and 33% activity, respectively, as compared to 35, 20 and 8% in absence of antibodies (Fig. 5A–D). 3.4. Effect of Fab fragments on MG induced modification/ inactivation As shown in Figs. 4 and 5, specific Fab monomers derived from polyclonal anti-SOD IgG protected SOD against MG induced inactivation. The Fig. 4 clearly depicts that in presence of Fab monomers, enzyme retained around 87, 80 and 72% of its initial activity after incubating with 5 mM MG for 1, 3 and 5 days. Fab monomers rendered significant protection to Cu,ZnSOD even at higher MG concentrations. The extent of protection is summarized in Fig. 5A–D. Mass spectral analysis of the incubation mixtures in presence of antibodies also revealed the striking protective effect of antibodies against aggregation/cross-linking. The molecular ion of SOD incubated with MG in presence of antibodies showed monomer major peak at m/z ratio close to that of the control.
Post-translational modifications of proteins due to glycation and oxidation play a significant role in the development of a variety of complications associated with diabetes and aging. The accumulation of glycation intermediates like MG, leads to modification of DNA causing mutagenesis and apoptosis [39,40]. Modification of proteins with MG may in turn result in protein degradation, enzyme inhibition and a cytokinemediated immune response [41]. In vitro reaction with MG has been shown to induce conformational changes in proteins that expose the hydrophobic core and facilitate the oxidation of newly exposed thiols resulting in protein crosslinking [42,43]. Currently, several strategies are being employed to control protein glycation. These include inhibition of AGE formation and conversion of post-Amadori intermediates to AGEs, use of crosslinking inhibitors and manipulation of AGE receptor pathways [44]. A number of AGE inhibitors have also been proposed. Inhibitors like aspirin or pyridoxal-5-phosphate act to modify the free amino groups, thereby preventing sugar attachment [45,46]. Inhibitors like aminoguanidine, on the other hand react with sugars, diverting them from Maillard reactions on proteins [47]. Penicillamine, DETAPAC, Vitamin C and E act as chelators of in vivo metal ions, while inhibitors like L-arginine trap dicarbonyl intermediates to form substituted triazenes. Amadorins, like pyridoxamine (vitamin B6 derivative), are the compounds with potential to scavenge postAmadori products, inhibiting the conversion of glycation intermediates to AGEs [48,49]. Crosslink breakers disrupt the crosslinks between the protein molecules [50]. Recently, researchers have been investigating chaperones for protection against glycation-induced inactivation and loss of antigenicity [51,52]. Molecular chaperones not only assist during protein folding but also stabilize proteins and prevent aggregation [53,54]. Substantiating the earlier observation [35], this work shows the potential of the antibodies and monomeric Fab fragments in protecting the inactivation of Cu,Zn-SOD by a far more reactive α-oxoaldehde MG. MG reacts with arginine, lysine and to a lesser extent to cysteine and histidine residues leading to crosslinking of the enzyme [55]. Cu,Zn-SOD contains copper and zinc ions at the active site, bridged by a common ligand histidine and treatment of SOD with MG leads to the release of the ions at the active site and hence enzyme inactivation [34,56]. Fig. 1 shows that inactivation of the Cu,Zn-SOD is rapid and is accompanied by marked aggregation, especially at higher MG concentrations (Figs. 2 and 3). Specific polyclonal antibodies/Fab monomers protected significantly Cu,Zn-SOD against MG induced inactivation (Figs. 4 and 5) and crosslink formation (Fig. 3B). When enzyme was incubated with lower doses of MG (1, 3 and 5 mM), the antibodies were found to be highly effective in preventing MGinduced inactivation of SOD. Antibodies and Fab monomer fragments protected the enzyme, although to a smaller extent,
R. Jabeen et al. / Biochimica et Biophysica Acta 1760 (2006) 1167–1174 Fig. 3. SELDI-TOF mass spectra of Bovine Cu,Zn-SOD from an SPA matrix. Cu,Zn-SOD (0.25 mg/ml) was incubated with 30 mM MG at 37 °C for 0, 3, 6, 12 and 24 h in 20 mM phosphate buffer pH 7.4 in absence (A) or presence of 5-fold molar excess of anti-SOD antibodies (B). 1171
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aggregation upon binding to the antigen molecules. Alternatively, binding of antibodies may shift equilibrium between unfolded and native conformations of the protein antigen towards the native form or lower the activation barrier through stabilization of the folded transition state [57]. We believe that binding of antibody restricts the unfolding of the protein and consequently the further
Fig. 4. Effect of inclusion of specific anti-SOD antibodies/monomeric Fab against the MG-induced Cu,Zn-SOD inactivation. After incubation of Cu,ZnSOD (0.25 mg/ml) with various indicated MG concentrations for 1, 3 or 5 days in presence and absence of 5-fold molar excess specific polyclonal antibodies/ Fab monomers at 37 °C in dark, under sterile conditions, its enzymatic activity was determined (A–C). Values are the averages from three independent experiments ± S.D.
against the MG-induced inactivation even at very high concentrations of the α-oxoaldehyde (up to 30 mM). This protection is specific, since non-specific IgG and Fab monomers derived thereof did not show any protection against the inactivation (Fig. 1C and D). The exact mechanism for the protective role of antibodies needs to be elucidated. It has been found that some antibodies act as chaperones under specific conditions and can facilitate folding and prevent aggregation of protein antigens [57]. α-crystallin, a molecular chaperone and lens structural protein, protects the membrane enzyme Na/K-ATPase and preserves its activity upon incubation with methylglyoxal, fructose and H2O2. It is believed that α-crystallin may act through dynamic interactions, such that the chaperone may prevent the further unfolding but not bind to the target protein [58]. Antibodies may sterically hinder the
Fig. 5. Effect of anti-SOD antibodies/monomeric Fab on the inactivation of SOD induced by higher MG concentrations. Cu,Zn-SOD (0.25 mg/ml) was incubated with indicated MG concentrations and activity was measured after 0, 3, 6, 12 and 24 h. Values are the averages from three independent experiments ± S.D.
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modification and inactivation of Cu,Zn-SOD. Since monomeric Fab also protected the enzyme, binding rather than crosslinking and aggregation by the bifunctional anti-enzyme IgG may play an important role in restricting the MG-induced damage. Inclusion of antibodies prevents dimer formation by MG, probably the antibodies inhibit the exposure of glycation susceptible functional groups by preventing enzyme unfolding (Fig. 3B). Ulbrich-Hoffman and co-workers proposed that enzymes may contain specific regions where unfolding of the molecule may begin and blocking of such regions may lead to restriction of unfolding and enhancement in the stabilization of enzymes [59]. It has been shown in earlier studies that antibodies recognizing the labile regions of RNase can impart stability to the enzyme on binding [60]. Binding of the antibody also improved the stability of a labile mutant RNase with variation of the labile region aminoacid sequence [61]. Polyclonal antibodies raised in rabbits have been used in this study and the preparation may contain the populations that bind to such crucial regions of SOD, preventing further unfolding of the enzyme. Since the protection by specific polyclonal antibodies and Fab monomers is remarkable, enzyme therapy may turn out to be a promising approach in the treatment of disorders associated with glycation. The Fab monomers can be advantageous owing to the fact that interaction of complement and other molecules with the Fc portion can be avoided. We have recently demonstrated that soluble yet stable enzyme preparations can be prepared by binding of bromelain with monomeric Fab fragment derived from anti-bromelain IgG [62]. Valency and halflife of the fragments can be tailored through protein engineering approaches to suit the desired mechanism of action. Furthermore, antibody fragments are better suited for expression in microbial systems, providing benefits in terms of scaling up and ease of production [63]. A few attempts of using antibodies to restrict AGE-mediated damage are also available. An antiglycated albumin antibody has been shown to prevent BM thickening in diabetic db/.db mice and a monoclonal antibody A 717 was also shown to be effective in retarding the development of diabetic retinopathy in diabetic animal models [64,65]. The present study has been carried out using affinity purified anti-enzyme polyclonal IgG. It is quite likely that few epitope specific monoclonal antibody populations may be far more protective and attempts are being made to isolate such antibodies. To conclude, this work suggests a remarkable potential of anti-enzyme antibodies and Fab fragments in the protection of SOD against inactivation induced by MG.
[2] [3]
[4]
[5]
[6]
[7] [8]
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16] [17]
[18]
Acknowledgments [19]
This work has been primarily done at Department of Pathology, University of Texas Medical Branch, Galveston, USA. Authors are highly thankful to Dr. M.A. Khan for his valuable suggestions.
[20]
[21]
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