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.     

Rates and impact of human antibody glycation in vivo

Glycation of immunoglobulin G (IgG) can result from incubation with a reducing sugar in vitro or during circulation in vivo. Upon injection of a recombinantly produced human therapeutic IgG into humans, changes in the glycation levels could be observed as a function of circulation time. Mass changes on the individual IgG polypeptide chains as the results of glycation were determined using reversed-phase liquid chromatography/mass spectrometry. Changes to the light and heavy chains were low but easily detectable at 0.00092 and 0.0021 glucose (Glc) additions per chain per day, respectively. Levels of glycation found on the Fc portion of IgG isolated from healthy subjects, using a similar analytical approach, were on average 0.045 Glc molecules per fragment. In vivo glycation rates could be approximated in vitro by modeling the physiological glycation reaction with a simplified incubation containing physiological Glc concentrations, pH and temperature but with a high concentration of a single purified IgG. To test the impact of glycation on IgG function, highly glycated IgG1 and IgG2 were prepared containing on average 42-49 Glc molecules per IgG. Binding to FcγIIIa receptors, neonatal Fc receptor or protein A was similar or identical to the non-glycated IgG controls. Although the modifications were well distributed throughout the protein sequence, and at high enough levels to affect the elution position by size-exclusion chromatography, no changes in the tested Fc functions were observed.     

Proteomic profiling of nonenzymatically glycated proteins in human plasma and erythrocyte membranes

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this work, we report the first proteomics-based characterization of nonenzymatically glycated proteins in human plasma and erythrocyte membranes from individuals with normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes mellitus. Phenylboronate affinity chromatography was used to enrich glycated proteins and glycated tryptic peptides from both human plasma and erythrocyte membranes. The enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation-tandem mass spectrometry, resulting in the confident identification of 76 and 31 proteins from human plasma and erythrocyte membranes, respectively. Although most of the glycated proteins could be identified in samples from individuals with normal glucose tolerance, slightly higher numbers of glycated proteins and more glycation sites were identified in samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus.     

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.     

Identification of the site of glycation of human insulin

This study evaluates the nature of glycated human insulin formed following exposure to hyperglycemic conditions in vitro. Glycated insulin was purified by RP-HPLC and its molecular mass (5971.3 Da) determined by plasma desorption mass spectrometry (MS). The difference in mass (163.7 Da) from nonglycated insulin (5807.6 Da) corresponds to a single reduced glucose (glucitol) residue. Following reduction of insulin disulfide bridges, MS confirmed that the B-chain was glycated. Enzymatic digestions with trypsin, endoproteinase Glu-C, and thermolysin, followed by RP-HPLC and identification of fragments by MS, localized glycation to the B-chain (1–5) region. Electrospray tandem MS identified the site of glycation as the B-chain NH₂-terminal Phe¹ residue. This was confirmed by automated Edman degradation with glycated human insulin.     

Relative quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESIMS) was used for relative quantification of glycated Cu-Zn superoxide dismutase (SOD-1) in human erythrocytes. SOD-1 samples were prepared from erythrocytes by removing hemoglobin using hemoglobind gel followed by ethanol and chloroform extraction. The reproducibility in measurement of the relative percentage of glycated protein was good, and the standard deviation of each measurement was 4.0%. From the mass spectral analysis of a mixture of commercial SOD-1 and in vitro partially glycated SOD-1 in several ratios, it was found that free and glycated SOD-1 have the same ionization efficiencies. The percentage of glycation on SOD-1 was measured in 30 individuals, including patients with diabetes mellitus. The glycation levels ranged from 4.5% to below the detection limit. The SOD-1 sample extracted from erythrocytes was fractionated by Glyco-Gel B chromatography, and the separated fractions were analyzed by MS. The mass spectra of absorbed fraction showed significant amounts of non-specific binding of non-glycated proteins to Glyco-Gel B.     

Characterization of bovine serum albumin glycated with glucose, galactose and lactose

The non-enzymatic reaction between reducing sugars and proteins, known as glycation, has received increased attention from nutritional and medical research. In addition, there is a large interest in obtaining glycoconjugates of pure well-characterized oligosaccharides for biological research. In this study, glycation of bovine serum albumin (BSA) by d-glucose, d-galactose and d-lactose under dry-heat at 60 degrees C for 30, 60, 120, 180 or 240 min was assessed and the glycated products studied in order to establish their biological recognition by lectins. BSA glycation was monitored using gel electrophoresis, determination of available amino groups and lectin binding assays. The BSA molecular mass increase and glycation sites were investigated by mass spectrometry and through digestion with trypsin and chymotrypsin. Depending on time and type of sugar, differences in BSA conjugation were achieved. Modified BSA revealed reduction of amino groups' availability and slower migration through SDS/PAGE. d-galactose was more reactive than d-glucose or d-lactose, leading to the coupling of 10, 3 and 1 sugar residues, respectively, after 120 minutes of reaction. BSA lysines (K) were the preferred modified amino acids; both K256 and K420 appeared the most available for conjugation. Only BSA-lactose showed biological recognition by specific lectins.     

Application of shielding boronate affinity chromatography in the study of the glycation pattern of haemoglobin

Human haemoglobin (Hb) may appear in a number of glycated species. The glycation pattern of Hb using shielding boronate affinity chromatography (SBAC) has been studied in the present work. SBAC is a novel separation technique, which eliminates nonspecific boronate-protein interactions by introducing a so-called shielding reagent. Two samples from Bio-Rad (Lyphochek)--one from normal persons' blood with relatively low HbA(1c) level (HbL) and the other from diabetic patients' blood with an elevated HbA(1c) level (HbH)--were used for the investigation. Glycated Hb (GHb) was separated from nonglycated Hb species using Tris as the shielding reagent. Two eluted peaks, eluted peak 1 (E1) and eluted peak 2 (E2), were obtained using a linear gradient elution with Tris. Several bands were observed on isoelectric focusing gel, which showed the same migration positions as Hb adducts, such as HbA(0), which is major Hb component containing two alpha chains and two beta chains; HbA(1c), which is post-translational glycation on the N-terminus of the beta chains of HbA(0); Foetal Hb (HbF), consisting of two alpha chains and two gamma chains; and glutathione Hb (also called HbSSG), which is the result from thiol-disulphide interchain exchange during oxidation of the thiol groups of Hb. In both HbL and HbH samples, E2 exhibited slightly higher amounts of HbF than E1. Electrospray-ionisation mass spectrometry showed that: (1) HbL-E1 was glycated with single glucose on both alpha and beta chains while no observable glycated chains were present in HbL-E2; (2) both HbH-E1 and HbH-E2 were glycated with single glucoses on both alpha and beta chains, however, compared with HbH-E1, HbH-E2 showed a higher relative intensity of the glycated beta chain and lower relative intensity of the glycated alpha chain; and (3) the degree of glycation increased with increasing glycation level of the sample. The amount of HbA(1c) presented in the eluted peaks was further determined using enzymatic digestion of glycated Hb by endoproteinase Glu-C and the subsequent separation and analysis of the digested peptides by reversed-phase high-performance liquid chromatography and capillary electrophoresis. The values of HbA(1c)/HbA(0) of the eluted peaks, i.e. HbL-E1, HbL-E2, HbH-E1 and HbH-E2, were 0.27, 0.19, 0.50 and 0.43, respectively. In both HbL and HbH samples, E1 contained higher amounts of HbA(1c) than E2. This study demonstrates the structural heterogeneity of GHb as well as the possibility of using SBAC to detect glycated species of Hb.     

The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization

We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.     

Characterization of the glycated human cerebrospinal fluid proteome

Protein glycation is a nonenzymatic modification that involves pathological functions in neurological diseases. Despite the high number of studies showing accumulation of advanced end glycation products (AGEs) at clinical stage, there is a lack of knowledge about which proteins are modified, where those modifications occur, and to what extent. The goal of this study was to achieve a comprehensive characterization of proteins modified by early glycation in human cerebrospinal fluid (CSF). Approaches based on glucose diferential labeling and mass spectrometry have been applied to evaluate the glycated CSF proteome at two physiological conditions: native glucose level and in vitro high glucose content. For both purposes, detection of glycated proteins was carried out by HCD-MS2 and CID-MS3 modes after endoproteinase Glu-C digestion and boronate affinity chromatography. The abundance of glycation was assessed by protein labeling with (13)C(6)-glucose incubation. The analysis of native glycated CSF identified 111 glycation sites corresponding to 48 glycated proteins. Additionally, the in vitro high glucose level approach detected 265 glycation sites and 101 glycated proteins. The comparison of glycation levels under native and 15 mM glucose conditions showed relative concentration increases up to ten folds for some glycated proteins. This report revealed for the first time a number of key glycated CSF proteins known to be involved in neuroinflammation and neurodegenerative disorders. Altogether, the present study contains valuable and unique information, which should further help to clarify the pathological role of glycation in central nervous system pathologies. This article is part of a Special Issue entitled: Translational Proteomics.     

Chromatographic analysis of acetohexamide binding to glycated human serum albumin

Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used high-performance affinity chromatography to examine the changes in acetohexamide–HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K_a) for the high affinity interactions was in the range of 1.2–2.0 × 10⁵ M⁻¹ and increased in moving from normal HSA to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K_a for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K_a for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.     

SMART Digest™ compared with pellet digestion for analysis of human immunoglobulin G1 in rat serum by liquid chromatography tandem mass spectrometry

The newly developed SMART Digest™ kit was applied for the sample preparation of human immunoglobulin G1 (hIgG1) in rat serum prior to qualitative and quantitative analyses by liquid chromatography tandem mass spectrometry (LC–MS/MS). The sequence coverages obtained for the light and heavy chains of hIgG1A were 50 and 76%, respectively. The calibration curve was linear from 1.00 to 1000 μg/ml for three of four generic peptides. Overall, the SMART Digest™ kit resulted in similar quantitative data (linearity, sensitivity, accuracy, and precision) compared with the pellet digestion protocol. However, the SMART Digest™ required only 2 h of sample preparation with fewer reagents.     

Detection of oxidized and glycated proteins in clinical samples using mass spectrometry — A user's perspective

Background

Proteins in human tissues and body fluids continually undergo spontaneous oxidation and glycation reactions forming low levels of oxidation and glycation adduct residues. Proteolysis of oxidised and glycated proteins releases oxidised and glycated amino acids which, if they cannot be repaired, are excreted in urine.

Scope of Review

In this review we give a brief background to the classification, formation and processing of oxidised and glycated proteins in the clinical setting. We then describe the application of stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of oxidative and glycation damage to proteins in clinical studies, sources of error in pre-analytic processing, corroboration with other techniques – including how this may be improved – and a systems approach to protein damage analysis for improved surety of analyte estimations.

Major conclusions

Stable isotopic dilution analysis LC-MS/MS provides a robust reference method for measurement of protein oxidation and glycation adducts. Optimised pre-analytic processing of samples and LC-MS/MS analysis procedures are required to achieve this.

General significance

Quantitative measurement of protein oxidation and glycation adducts provides information on level of exposure to potentially damaging protein modifications, protein inactivation in ageing and disease, metabolic control, protein turnover, renal function and other aspects of body function. Reliable and clinically assessable analysis is required for translation of measurement to clinical diagnostic use. Stable isotopic dilution analysis LC-MS/MS provides a “gold standard” approach and reference methodology to which other higher throughput methods such as immunoassay and indirect methods are preferably corroborated by researchers and those commercialising diagnostic kits and reagents. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Identification of isomerization and racemization of aspartate in the Asp-Asp motifs of a therapeutic protein

A thermally stressed Fab molecule showed a significant increase of basic variants in imaged capillary isoelectric focusing (iCIEF) analysis. Mass analyses of the reduced protein found an increase in −18 Da species from both light chain and heavy chain. A tryptic peptide map identified two isoAsp-containing peptides, both containing Asp–Asp motifs and located in complementarity-determining regions (CDRs) of light chains and heavy chains, respectively. The approaches of hydrolyzing succinimide in H₂¹⁸O followed by tryptic digestion were used to label and identify the sites of isomerization. This method enabled identification of the isomerization site by comparing the MS/MS spectra of isomerized peptides with and without ¹⁸O incorporation. The light chain peptide L2 VTITCITSTDID₁₂DDMNWYQQKPGK underwent simultaneous isomerization and recemization at residue Asp-12 after thermal stress as evidenced by the coinjection of synthetic peptide L2 with l-Asp-12, l-isoAsp-12, d-Asp-12, and d-isoAsp-12, respectively. A thermal stress study of the synthetic peptide (l-)L2 showed that the isomerization and racemization did not occur, indicating that the Asp degradation in this Asp–Asp motif is more related to the protein conformation than the primary sequence. Another isomerization site was identified as Asp-24 in the heavy chain peptide H5 QAPGQGLEWMGWINTYTGETTYAD₂₄DFK. No other isomerizations were detected in CDR peptides containing either Asp–Ser or Asp–Thr motifs.     

Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry

We describe a fast and informative method to investigate the posttranslational modifications of monoclonal antibodies (MAbs). The MAb is first digested by a specific enzyme that cleaves heavy chains under the hinge domain. After reduction of disulfide bridges, three polypeptide chains of approximately 25 kDa are released and analyzed by liquid chromatography–mass spectrometry (LC–MS). By bisecting the heavy chains prior to MS analysis, this method provides a better MS resolution and facilitates the study of the N-linked glycans as well as of other modifications (loss of C-terminal lysine, pyroglutamination, and oxidation). The sample preparation and analysis can be performed within few hours.     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.     

Unbiased in-depth characterization of CEX fractions from a stressed monoclonal antibody by mass spectrometry

Characterization of charge-based variants by mass spectrometry (MS) is required for the analytical development of a new biologic entity and its marketing approval by health authorities. However, standard peak-based data analysis approaches are time-consuming and biased toward the detection, identification, and quantification of main variants only. The aim of this study was to characterize in-depth acidic and basic species of a stressed IgG1 monoclonal antibody using comprehensive and unbiased MS data evaluation tools. Fractions collected from cation ion exchange (CEX) chromatography were analyzed as intact, after reduction of disulfide bridges, and after proteolytic cleavage using Lys-C. Data of both intact and reduced samples were evaluated consistently using a time-resolved deconvolution algorithm. Peptide mapping data were processed simultaneously, quantified and compared in a systematic manner for all MS signals and fractions. Differences observed between the fractions were then further characterized and assigned. Time-resolved deconvolution enhanced pattern visualization and data interpretation of main and minor modifications in 3-dimensional maps across CEX fractions. Relative quantification of all MS signals across CEX fractions before peptide assignment enabled the detection of fraction-specific chemical modifications at abundances below 1%. Acidic fractions were shown to be heterogeneous, containing antibody fragments, glycated as well as deamidated forms of the heavy and light chains. In contrast, the basic fractions contained mainly modifications of the C-terminus and pyroglutamate formation at the N-terminus of the heavy chain. Systematic data evaluation was performed to investigate multiple data sets and comprehensively extract main and minor differences between each CEX fraction in an unbiased manner.     

A chemical and computational approach to comprehensive glycation characterization on antibodies

Non-enzymatic glycation is a challenging post-translational modification to characterize due to the structural heterogeneity it generates in proteins. Glycation has become increasingly recognized as an important product quality attribute to monitor, particularly for the biotechnology sector, which produces recombinant proteins under conditions that are amenable to protein glycation. The elucidation of sites of glycation can be problematic using conventional collision-induced dissociation (CID)-based mass spectrometry because of the predominance of neutral loss ions. A method to characterize glycation using an IgG1 monoclonal antibody (mAb) as a model is reported here. The sugars present on this mAb were derivatized using sodium borohydride chemistry to stabilize the linkage and identified using CID-based MS² mass spectrometry and spectral search engines. Quantification of specific glycation sites was then done using a targeted MS¹ based approach, which allowed the identification of a glycation hot spot in the heavy chain complementarity-determining region 3 of the mAb. This targeted approach provided a path forward to developing a structural understanding of the propensity of sites to become glycated on mAbs. Through structural analysis we propose a model in which the number and 3-dimensional distances of carboxylic acid amino acyl residues create a favorable environment for glycation to occur.     

Controversial behavior of aminoguanidine in the presence of either reducing sugars or soluble glycated bovine serum albumin

The elucidation of the controversial inhibitory effect of aminoguanidine (AG) on the cross-linking and fluorescent advanced glycation end products (AGEs) formation during long-term in vitro glycation of type I collagen with 250 mM reducing sugars or 0.5 mg/ml soluble glycated bovine serum albumin (AGE-BSA) was researched.Chromatographic and SDS–PAGE analyses revealed the formation of aggregates during collagen glycation. AG at all concentrations (5–80 mM) prevented the cross-linking of collagen peptides with monosaccharides but an increase in fluorescence with a maximum value at 10 mM AG was noticed. In the presence of AGE-BSA, AG prevented the cross-linking process and decreased the fluorescence levels in a concentration-dependent manner.Our results suggest that AG is an efficient inhibitor of collagen cross-linking and the highest increase in fluorescence due to reducing sugars and AG can be explained by the competition between guanidine group of AG and arginine residues of some protein-bound dideoxyosones, which could form fluorescent compounds.     

Glycation and inactivation of human Cu-Zn-superoxide dismutase. Identification of the in vitro glycated sites

The nonenzymatic glycosylation (glycation) of Cu-Zn-superoxide dismutase led to gradual inactivation of the enzyme (Arai, K. Iizuka, S., Tada, Y., Oikawa, K., and Taniguchi, N. (1987) Biochim. Biophys. Acta 924, 292-296). The purified superoxide dismutase from human erythrocytes comprises both glycated and nonglycated forms. The nonglycated Cu-Zn-superoxide dismutase was isolated by boronate affinity chromatography. Incubation of the nonglycated superoxide dismutase with D-[6-3H]glucose in vitro resulted in the gradual accumulation of radioactivity in the enzyme protein, and Schiff base adducts were trapped by NaBH4. The sites of glycation of the superoxide dismutase were identified by amino acid analysis after reverse-phase high performance liquid chromatography of the trypsin-treated peptides. Lysine residues, i.e. Lys3, Lys9, Lys30, Lys36, Lys122, and Lys128, were found to be glycated. Three of the glycated sites lie in Lys-Gly, two in Lys-Ala, and one in Lys-Val. The inactivation of the superoxide dismutase on the glycation is due mainly to the glycation of Lys122 and Lys128, which are supposed to be located in an active site liganding loop. The remaining five sites, such as Lys-Glu, Lys-Asp, Lys-His, and Lys-Thr are relatively inactive as to the formation of either a Schiff base or an Amadori adduct.