A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior

The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product.     

Quantitative analysis of glycation and its impact on antigen binding

Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs.     

Detection of dideoxyosone intermediates of glycation using a monoclonal antibody: characterization of major epitope structures

Glycation or the Maillard reaction in proteins forms advanced glycation end products (AGEs) that contribute to age- and diabetes-associated changes in tissues. Dideoxyosones, which are formed by the long-range carbonyl shift of the Amadori product, are newly discovered intermediates in the process of AGE formation in proteins. They react with o-phenylenediamine (OPD) to produce quinoxalines. We developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate coupled to keyhole limpet hemocyanin. The antibody reacted strongly with ribose and fructose (+OPD)-modified RNase A and weakly with glucose and ascorbate (+OPD)-modified RNase A. Reaction with substituted quinoxalines indicated that this antibody favored the 2-methyl group on the quinoxaline ring. We used high performance liquid chromatography to isolate and purify three antibody-reactive products from a reaction mixture of N alpha-hippuryl-L-lysine+ribose+OPD. The two most reactive products were identified as diastereoisomers of N1-benzoylglycyl-N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and the other less reactive product as N1-benzoylglycyl-N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine. Our study confirms that dideoxyosone intermediates form during glycation and offers a new tool for the study of this important pathway in diabetes and aging.     

3-hydroxykynurenine-mediated modification of human lens proteins: structure determination of a major modification using a monoclonal antibody

Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified N(alpha)-acetyl lysine, N(alpha)-acetyl histidine, N(alpha)-acetyl arginine, and N(alpha)-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and N(alpha)-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-alpha-((5S)-5-acetamino-5-carboxypentyl amino)-gamma-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation.     

Effect of Chemical Chaperones on Glucose-Induced Lysozyme Modifications

Nonenzymatic glycation of biomacromolecules occurs due to the diabetes mellitus and ageing. A number of small molecules, known as chemical chaperones, stabilize protein conformation against thermal and chemically induced denaturation. These compounds are including: polyamines (e.g. spermine and spermidine), amino acids (e.g. lysine) and polyols (e.g. glycerol). In this study the effect of spermidine (Spd), spermine (Spm), and glycerol on glycation, structure and function of lysozyme (LZ), as an extra-cellular protein, by different techniques is investigated. LZ is incubated with or without glucose (50 or 100 mM) in the absence or presence of Spd/Spm/glycerol at 37 °C up to 16 weeks. All the observed changes of glycated-LZ in comparison with the native protein, including: increased fluorescence emission, alteration in the secondary and tertiary structure, and reduced electrophoretic mobility- indicate its structural changes that are accompanied with its reduced activity. Glucose in the presence or absence of Spd induces the protein dimerization, but glucose plus Spm induces its trimmerization. In contrast, glycerol inhibits the LZ glycation and prevents the large changes on its structure and function. Glucose binds lysine residues, decreases the protein positive charges and induces some alterations in its structure and activity. Polyamines also directly bind to LZ, increase its positive charges and hence induce more glycation; more conformational changes, oligomerization and its inactivation in the presence of glucose, but glycerol affect the protein environment and preserve protein from these harmful effects.     

Targeting properties of an anti-CD16/anti-CD30 bispecific antibody in an in vivo system

Bispecific antibodies are currently being used in clinical trials in increasing numbers in the areas of breast cancer, prostate cancer, non-Hodgkin's lymphoma and Hodgkin's lymphoma. We have previously performed two clinical trials in patients with Hodgkin's disease with an anti-CD30/anti-CD16 bispecific antibody and demonstrated a 30% response rate in a cohort of patients otherwise resistant to standard therapeutic modalities. However, no surrogate marker could be defined in these trials indicative of optimal antibody dosing/scheduling or predictive for favorable response. In order to evaluate accurately the potential biodistribution properties of bispecific antibody in patients, we have performed a detailed analysis of the binding properties and animal model in vivo characteristics of these constructs. For this purpose, the parental antibodies (anti-CD30 and anti-CD16) and the bispecific antibody (anti-CD30/anti-CD16) were radiolabeled with either ¹²⁵I or ¹¹¹In. Antibody integrity and binding properties after labeling were confirmed by Scatchard plot and Lindmo analysis. ¹¹¹In-labeled antibodies revealed superior targeting properties in a standard SCID mouse tumor model. Both the bivalent parental anti-CD30 monoclonal antibody and the monovalent anti-CD30/anti-CD16 bispecific antibody showed excellent uptake in CD30⁺ tumors which did not differ significantly between the two (maximum uptake 16.5% ± 4.2% vs. 18.4% ± 3.8% injected dose/gram tissue). The equivalent targeting properties of the bispecific antibody compared with the parental anti-CD30 antibody encourages the further clinical development of this bispecific antibody, and might help to explain the clinical responses seen with this antibody so far in patients suffering from Hodgkin's disease. Received: 26 October 2000 / Accepted: 15 December 2000     

World Bispecific Antibody Summit,September 27–28, 2011, Boston,MA

With more than 30 therapeutic monoclonal antibodies (mAbs) approved and annual global sales of the products at ∼$50 billion in 2010, these products have proven to be successful in many ways. Nevertheless, there is room for improvement in performance, and substantial unmet medical needs remain. As a consequence, numerous organizations are devoting resources to engineering novel mAbs such as bispecific antibodies that have increased functionality compared with unmodified IgG molecules. The World Bispecific Antibody Summit, organized by Hanson Wade, drew over 100 participants to Boston to discuss engineering novel bispecific antibodies, generating lead candidates and clinical study and commercialization of the molecules. Approaches such as the trifunctional antibody (TRION), dual variable domain-Ig (Abbott), two-in-one (Genentech), dual affinity retargeting (MacroGenics), kappa-lambda body (NovImmune), bispecific T-cell engager (Micromet) and chemical generation (CovX/Pfizer) were discussed in detail. In addition, posters describing bispecific Affibody^® molecules for targeting of EGFR and HER2 (Affibody), T-cell receptor based bi-specifics that target HLA-peptides (Immunocore), a novel mAb-Fv bispecific antibody format utilizing Fc region (Xencore), generation of a tetravalent bispecific antibody against IL4 and IL13 for the treatment of idiopathic pulmonary fibrosis (Sanofi), Combining Affibody^® molecules and the AlbumodTM technology to create long acting multispecific protein therapeutics (Royal Institute of Technology, Affibody) and COVA301 as a highly potent bispecific inhibitor of IL-17A and TNFα (Covagen) were presented.Key words: bispecific antibodies, antibody engineering, therapeutic antibodies     

Mass spectrometric determination of early and advanced glycation in biology

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N^ε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.     

The effect of bispecific monoclonal antibody recognizing both hepatoma-specific membrane glycoprotein and anthracycline drugs on the metastatic growth of hepatoma AH66

A monoclonal mouse antibody (MoHG) was produced using in vitro cultured AH66R tumor cells treated with cholesteryl hemisuccinate as an immunogen. The antibody identified a 90 kd membrane glycoprotein (HG-90) which is expressed on in vitro cultured hepatoma cell lines AH66 and AH66R. A monoclonal antibody was prepared to the anthracycline drug daunomycin, and it also reacted with adriamycin. A fusion was made of the hybridoma HG-90 with the hybridoma which recognized daunomycin/adriamycin. This bispecific hybridoma A8C recognized both determinants. We studied the therapeutic effect of the A8C bispecific antibody with adriamycin treatment and compared it to the effect of the bispecific antibody to which adriamycin had been conjugated via an albumin (Alb) bridge. The therapy model used was the tumor AH66R in Donryu rats. Tumor bearing rats had their subcutaneous tumors resected on day 10, a time when distant metastases were present. After the surgical resection of the tumor the rats were injected intravenously for two cycles with the bispecific antibodies, followed by the administration of adriamycin (ADR) or MoHG.Alb.ADR conjugates. A slight therapeutic effect occurred with either MoHG or ADR alone but treatment with the bispecific antibody followed by the administration of ADR or with the MoHG.Alb.ADR conjugates significantly prolonged survival, with 60% of the treated animals being "tumor free" when sacrificed on day 80. Lower serum concentrations of alphafetoprotein were observed with the bispecific antibody and drug treatment. This suggests that the bispecific antibody/drug treatment is potentially more beneficial in the suppression of distant metastases than the MoHG.Alb.ADR conjugate. This may be due to an increase in the local drug concentration of unmodified adriamycin.     

Controlling glycation of recombinant antibody in fed-batch cell cultures

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in-house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post-translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose--the only source of reducing sugar in our fed-batch cultures--would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2-L and 400-L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second-order chemical reaction that is first order with respect to both glucose and non-glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture.     

World Bispecific Antibody Summit,September 27–28, 2011, Boston,MA

With more than 30 therapeutic monoclonal antibodies (mAbs) approved and annual global sales of the products at ~$50 billion in 2010, these products have proven to be successful in many ways. Nevertheless, there is room for improvement in performance, and substantial unmet medical needs remain. As a consequence, numerous organizations are devoting resources to engineering novel mAbs such as bispecific antibodies that have increased functionality compared with unmodified IgG molecules. The World Bispecific Antibody Summit, organized by Hanson Wade, drew over 100 participants to Boston to discuss engineering novel bispecific antibodies, generating lead candidates and clinical study and commercialization of the molecules. Approaches such as the trifunctional antibody (TRION), dual variable domain-Ig (Abbott), two-in-one (Genentech), dual affinity retargeting (MacroGenics), kappa-lambda body (NovImmune), bispecific T-cell engager (Micromet) and chemical generation (CovX/Pfizer) were discussed in detail. In addition, posters describing bispecific Affibody® molecules for targeting of EGFR and HER2 (Affibody), T-cell receptor based bi-specifics that target HLA-peptides (Immunocore), a novel mAb-Fv bispecific antibody format utilizing Fc region (Xencore), generation of a tetravalent bispecific antibody against IL4 and IL13 for the treatment of idiopathic pulmonary fibrosis (Sanofi), Combining Affibody® molecules and the Albumod? technology to create long acting multispecific protein therapeutics (Royal Institute of Technology, Affibody) and COVA301 as a highly potent bispecific inhibitor of IL-17A and TNF-α (Covagen) were presented.     

Glycation of lysozyme with galactose,galactooligosaccharides and potato galactan through the Maillard reaction and optimization of the production of prebiotic glycoproteins

The production of glycated lysozyme (LZM), with galactose, galactooligosaccharides (GOSs) and potato galactan through the Maillard reaction, was investigated. The percent blocked lysine, estimated from the furosine content, reached a maximum value of 11.2% for LZM:galactan conjugates after 1 day incubation at a a_w of 0.65. A maximum percent blocked lysine of 7.0 and 13.5% were obtained for LZM:galactose/GOS conjugates at a lower a_w of 0.45 after 3 and 7 days, respectively. However, the low percent blocked lysine and the high protein aggregation index of LZM:galactose/GOS conjugates at a_w 0.79 and 0.65 revealed the prevalence of the degradation of the Amadori compounds and the protein cross-linking. Mass spectrometry of LZM conjugates revealed the formation of different glycoforms. Glycated LZMs containing up to seven galactose moieties were formed; while only mono- and diglycated LZMs with GOSs were detected. 2–3 mol of galactan were conjugated to 1 mol of LZM. Response surface methodology, based on a 5-level and 3-factor central composite design, revealed that molar ratio and temperature were the most significant variables for the glycation of LZM with GOSs. The optimal conditions leading to a high percent blocked lysine (16.11%) with a low protein aggregation index (0.11) were identified: temperature of 49.5 °C, LZM:GOS molar ratio of 1:9 and a_w of 0.65. To the best of our knowledge, this is the first study on the optimization of LZM glycation with GOSs.     

双专－性抗体介导的纤溶作用

用双功能团试剂将抗尿激酶单克隆抗体Ｎ３４的ＩｇＧ和抗人活化血小板α－颗粒膜蛋白ＧＭＰ－１４０单克隆抗体ＳＺ－５１的Ｆａｂ片段通过二硫键共价偶联,偶联的抗体保留了对各自抗原的亲和性。这种对尿激酶和血栓同时具有亲和活性的双专一性抗体（Ｎ３４－ＳＺ－５１）提高低分子量尿激酶的溶栓效率３８倍,且对血浆中纤维蛋白原的含量基本上不影响。     

Local antitumour treatment in carcinoma patients with bispecific-monoclonal-antibody-redirected T cells

In a pilot clinical study carcinoma patients with malignant ascites or pleural exudates have been treated locally with autologous lymphocytes activated ex vivo and redirected towards tumour cells with bispecific monoclonal antibodies. BIS-1, the bispecific monoclonal antibody used in this study, combines specificity against a tumour-associated antigen, AMOC-31, present on carcinomas, with a specificity against the CD3 complex on T lymphocytes. Patients selected for treatment had malignant pleural or peritoneal effusions. Treatment consisted of isolating autologous peripheral blood lymphocytes, ex vivo activation, incubation with bispecific monoclonal antibodies and injection at the effusion site of these BIS-1-redirected lymphocytes. To evaluate the effects of the bispecific monoclonal antibody, five patients received treatments with activated lymphocytes without bispecific antibodies. Effusion samples taken before and at various times after treatment were analysed by immunocytology and for the presence of the soluble factors carcinoembryonic antigen (CEA), interleukin-6 (IL-6), tumour necrosis factor (TNF), C-reactive protein and soluble CD8. In this way both immune activation and anti-tumour activity could be monitored. Conjugate formation between tumour cells and activated lymphocytes was seen as soon as 4 h after injection of BIS-1-redirected activated lymphocytes, followed by a disappearance or reduction of tumour cells after 24–48 h. In parallel with this, the soluble tumour marker CEA decreased in the effusion fluid following injection with the BIS-1-redirected lymphocytes. Furthermore, a steep increase in local granulocyte numbers was observed in the effusion fluid, which reached a maximum 24–48 h after the start of the treatment. Also levels of IL-6 and TNF were greatly elevated. The data suggest tha the treatment induces both antitumour activity and a strong local inflammatory reaction. This is accompanied by no or only minor local and systemic toxicity, i.e. mild fever, which disappeared as the local inflammatory reaction diminished 48–72 h after treatment.     

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.     

The effect of peptide glycation on local secondary structure

Protein glycation is a non-enzymatic reaction between reducing sugars and amino groups that occurs in vivo and has been implicated in a number of disease states and pathologies including Alzheimer's and diabetes. Although glycation is thought to alter protein structure and function, there is currently little information on the structural consequences of this modification. We have used a model alpha-helix and a model beta-hairpin peptide, and NMR analysis, to investigate the effects of glycation upon secondary structure. Glycation of the dilysine motif within the alpha-helix peptide occurred preferentially at one lysine residue and resulted in severe disruption to the local secondary structure. The area immediately around the site of modification was extremely flexible and the peptide did not adopt a preferred conformation in this area of the helix in 30% TFE. Significant glycation of the beta-hairpin peptide was not detected and the structure was unchanged. These results show that glycation results in local secondary structure distortion of alpha-helices and that preferential glycation occurs in a sequence specific manner. The findings will allow us to interrogate the local environment in other peptides/proteins to predict the likelihood of glycation, and to model the potential effects such modification might have upon structure/function.     

Biotinylation sites of tumor necrosis factor-alpha determined by liquid chromatography-mass spectrometry.

Tumor pretargeting with biotinylated antibody/avidin complexes improves the therapeutic index of systemically administered biotin-tumor necrosis factor (TNF) conjugates. Since the number of biotins in this conjugate is known to be critical for activity, we have characterized the structure of different biotin-TNF conjugates, prepared by reaction with d-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester and identified the biotinylation sites by trypsin digestion, reverse-phase chromatography, and electrospray mass spectrometry analyses. The results have shown that N-terminal valine is a preferential biotinylation site at pH 5.8, half of biotins being located on the alpha-amino group of this residue in a conjugate bearing one biotin/trimer (on average). Moreover, evidence has been obtained to suggest that the remaining part of biotins are linked to the epsilon-amino group of lysine 128, 112, and 65, while lysine 11, 90, and 98 were practically unmodified. No evidence of O-biotinylation of serine, threonine and tyrosine was obtained.     

Ferulic acid prevents methylglyoxal-induced protein glycation,DNA damage,and apoptosis in pancreatic β-cells

Methylglyoxal (MG) can react with amino acids of proteins to induce protein glycation and consequently the formation of advanced glycation end-products (AGEs). Previous studies reported that ferulic acid (FA) prevented glucose-, fructose-, and ribose-induced protein glycation. In this study, FA (0.1–1 mM) inhibited MG-induced protein glycation and oxidative protein damage in bovine serum albumin (BSA). Furthermore, FA (0.0125–0.2 mM) protected against lysine/MG-mediated oxidative DNA damage, thereby inhibiting superoxide anion and hydroxyl radical generation during lysine and MG reaction. In addition, FA did not have the ability to trap MG. Finally, FA (0.1 mM) pretreatment attenuated MG-induced decrease in cell viability and prevented MG-induced cell apoptosis in pancreatic β-cells. The results suggest that FA is capable of protecting β-cells from MG-induced cell damage during diabetes.     

Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose

Summary. We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N^ε-(carboxymethyl)lysine; CML) were analyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N^ε-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.     

Formation of Nepsilon-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.

The objectives of this study were to estimate the structure of the lipid hydroperoxide-modified lysine residue and to prove the presence of the adducts in vivo. The reaction of lipid hydroperoxide toward the lysine moiety was investigated employing N-benzoyl-glycyl-L-lysine (Bz-Gly-Lys) as a model compound of Lys residues in protein and 13-hydroperoxyoctadecadienoic acid (13-HPODE) as a model of the lipid hydroperoxides. One of the products, compound X, was isolated from the reaction mixture of 13-HPODE and Bz-Gly-Lys and was then identified as N-benzoyl-glycyl-Nepsilon-(hexanonyl)lysine. To prove the formation of Nepsilon-(hexanonyl)lysine, named HEL, in protein exposed to the lipid hydroperoxide, the antibody to the synthetic hexanonyl protein was prepared and then characterized in detail. Using the anti-HEL antibody, the presence of HEL in the lipid hydroperoxide-modified proteins and oxidized LDL was confirmed. Furthermore, the positive staining by anti-HEL antibody was observed in human atherosclerotic lesions using an immunohistochemical technique. The amide-type adduct may be a useful marker for the lipid hydroperoxide-derived modification of biomolecules.